TISSUE SPECIFIC REDUCTION OF LIGNIN

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 61/792,864, filed March 15, 2013, which is incorporated by reference herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with government support under Contract No. DE-AC02- 05CH1 1231 awarded by the U.S. Department of Energy. The governmen t has certain rights in this invention.

BACKGROUND OF THE INVENTION [0003J Plant iignoceilulosic biomass is used as a renewable feedstock for biofuel production and is a promising alternative to fossil fuel consumption. However, a major bottleneck in biofuel production is the quality of available feedstocks. Available feedstocks have a high resistance (recalcitrance) to being reduced into simple sugars that can in turn be converted into fuel. Therefore, improving the composi tion and/or digestibility of the raw biomass will have an important beneficial impact on Iignoceilulosic biofueis production.

[0004] Lignocellulosic biomass is mainly composed of secondary cell walls, which comprise polysaccharide polymers embedded in lignin. The embedding of the

polysaccharide polymers in lignin reduces their extractability and accessibility to hydrolytic enzymes, resulting in cell wall recalcitrance to enzymatic hydrolysis. Lignin content and saccharification efficiency of plant cell wall usually are highly negatively correlated. See, e.g., Chen and Dixon, Nat. Biotechnol 25:759-761 (2007); Jorgensen et al., Biofuel Bioprod. Bior. 1 : 1 19-134 (2007); and Vinzant et al, Appl Biochem. Biotechnol. 62:99-104 ( 1997), However, most attempts at reducing lignin content during plant development have resulted in severe biomass yield reduction (Franke et al, Plant J. 30:33-45 (2002); Shadle et al, Phytochemislry 68: 1 521 -1529 (2007); and Voelker et al, Plant Physiol 154:874-886 (2010)) and therefore, there are few crops having significant lignin reduction. Although silencing strategies have been used to reduce the amount of lignin in plants, there remains a need for methods of reducing lignin in specific cell and tissue types that reduce cell wall recalcitrance, thus improving the extractabiiity and hydrolysis of fermentable sugars from plant biomass. BRIEF S UMMARY OF THE INVENTION

[0005] Irs one aspect, the present invention provides methods of engineering a plant having reduced lignin content. In some embodiments, the method comprises: introducing into the plant an expression cassette comprising a polynucleotide that encodes a protein that diverts a monolignol precursor from a lignin biosynthesis pathway (e.g., a p-couniaryl alcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis pathway) in the plant, and wherein the polynucleotide is operabiy linked to a heterologous promoter and

culturing the plant under conditions in which the protein that diverts the monolignol precursor from the lignin biosynthesis pathway is expressed. [0006] in some embodiments, the protein reduces the amount of cytosolic and/or plastidial shikimate that is available for the lignin biosynthesis pathway. In some embodiments, the protein is shikimate kinase (AroK), pentafunctional AROM polypeptide (ARO S ), dehydroshikimate dehydratase (DsDH), or dehydroshikimate dehydratase (QsuB). In some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0007] in some embodiments, the protein reduces the amount of cytosolic and/or plastidial phenylalanine that is available for the lignin biosynthesis pathway. In some embodiments, wherein the protein is phenylacetaldehyde synthase (PAAS) or phenylalanine aminomutase (PAM). In some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:29.

[0Θ08] in some embodiments, the protein reduces the amount of cinnamate and/or eoumarate that is available for the lignin biosynthesis pathway. In some embodiments, the protein is p-coumarate/cinnamate carboxylmethltransferase (CCMTT) or phenylacrylic acid decarboxylase (PDC). In some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:30.

[0009] In some embodiments, the protein reduces the amount of coumaroyl-CoA, caffeoyl- CoA, and/or feruSoyl-CoA that is available for the lignin biosynthesis pathway. In some embodiments, the protein is 2-oxogluta rate-dependent dioxygenase (C2'H), cfaalcone synthase (CHS), stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone (BAS). In some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO: 14, SEQ ID NO:3 i, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO;35, or SEQ ID NO:36.

[0010] In some embodiments, the protein activates or potentiates a metabolic pathway thai competes with the lignin biosynthesis pathway for the use of monolignol precursors. In some embodiments, the metabolic pathway is a stilbene biosynthesis pathway, a flavonoid biosynthesis pathway, a curcuminoid biosynthesis pathway, or a bensalacetone biosynthesis pathway. In some embodiments, the protein is a transcription factor that activates or potentiates the flavonoid biosynthesis pathway. In some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45. fOOli] In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a secondary ceil wall-specific promoter or a fiber cell-specific promoter. In some embodiments, the promoter is an 1RX5 promoter. In some embodiments, the promoter is from a gene that is co-expressed in the lignin biosynthesis pathway

(phenyipropanoid pathway), e.g., a promoter from a gene expressed in the pathway shown in Figure I . in some embodiments, the promoter is a C4H, C3H, HCT, CCR1, CAD4, CADS, F5H, PALI, PAL2, 4CL1, or CCoAMT promoter. 00Ϊ2] In some embodiments, the protein that diverts a monolignol precursor from a lignin biosynthesis pathway is targeted to a plastid in the plant. In some embodiments, the polynucleotide comprises a plastid targeting signal that is substantially identical to the polynucleotide sequence of SEQ ID NO: 15.

[0013] in some embodiments, the protein diverts a monolignol precursor from a sinapyl alcohol and/or coniferyl alcohol biosynthesis pathway. In some embodiments, the plant has reduced content of guaiacyl (G) and syringyl (S) lignin units.

[0014] In some embodiments, the plant (or plant part, or seed, flower, leaf, or fruit from the plant) is selected from the group consisting of Arabidopsis, poplar, eucalyptus, rice, com, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium. [Θ015] In another aspect, the present invention provides a plant cell comprising a polynucleotide thai encodes a protein thai diverts a monolignoi precursor from a lignin biosynthesis pathway in the plant, wherein the polynucleotide is operably linked to a heterologous promoter. [0016] In some embodiments, the plant ceil comprises a polynucleotide that encodes a protein that reduces the amount of cytosolic and/or plastidial shikimate that is available for the lignin biosynthesis pathway. In some embodiments, the protein is shikimate kinase (AroK), pentafunctional AROM polypeptide (AROl), dehydroshikimate dehydratase (DsDH), or dehydroshikimate dehydratase (QsuB). in some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0017] in some embodiments, the plant cell comprises a polynucleotide that encodes a protein that reduces the amoimt of cytosolic and/or plastidial phenylalanine that is available for the lignin biosynthesis pathway. In some embodiments, wherein the protein is phenylacetaldehyde synthase (PAAS) or phenylalanine aminomutase (PAM). in some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:29.

[0018] In some embodiments, the plant cell comprises a polynucleotide that encodes a protein that reduces the amount of cinnamate and/or coumarafe that is available for the lignin biosynthesis pathway. In some embodiments, the protein is p-coumarate/cinnarnate carboxylmethltransferase (CCMT1) or phenylacrylic decarboxylase (PDC). In some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:30.

|0019J in some embodiments, the plant ceil comprises a polynucleotide thai encodes a protein that reduces the amount of coumaroyl-CoA, eaffeoyl-CoA, and/or feruloyl-CoA that is available for the lignin biosynthesis pathway. In some embodiments, the protein is 2- oxoglutarate-dependent dioxygenase (C2'H), chaicone synthase (CHS), stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone (BAS). in some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO; 14, SEQ ID NO:33 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO;35, or SEQ ID NO:36.

[0020] in some embodiments, the plant cell comprises a polynucleotide that encodes a protein activates or potentiates a metabolic pathway that competes with the lignin biosynthesis pathway for the use of monolignoi precursors. In some embodiments, the metabolic pathway is a stilbene biosynthesis pathway, a flavonoid biosynthesis pathway, a curcurninoid biosynthesis pathway, or a bensalacetone biosynthesis pathway. In some embodiments, the protein is a transcription factor that activates or potentiates the flavonoid biosynthesis pathway, in some embodiments, the protein is substantially identical to an amino acid sequence of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID O;43, SEQ ID NO:44, or SEQ ID NO:45.

[0021] In some embodiments, the plant cell comprises a tissue-specific promoter. In some embodiments, the promoter is a secondary cell wall-specific promoter or a fiber cell-specific promoter. In some embodiments, the promoter is an IRX5 promoter. In some embodiments, the plant cell comprises a promoter from a gene that is co-expressed in the lignin biosynthesis pathway (phenylpropanoid pathway), e.g., a promoter from a gene expressed in the pathway shown in Figure L In some embodiments, the promoter is a C4H, C3H, HCT, CCRI, CAD4, CADS, F5H, PALI , PAL2, 4CL1 , or CCoAMT promoter.

[0022] In some embodiments, the plant cell comprises a polynucleotide encoding a protein that diverts a monolignoi precursor from a lignin biosynthesis pathway that is targeted to a plastid in the plant. In some embodiments, the polynucleotide comprises a plastid targeting signal that is substantially identical to the polynucleotide sequence of SEQ ID NO: 15.

[0023] In another aspect, the present invention provides plants comprising a plant cell as described herein. In some embodiments, the plant has reduced lignin content that is substantially localized to secondary cell wall tissue or fiber cells of the plant.

[0024] in yet another aspect, the present invention provides methods of engineering a plant having reduced lignin content by expressing or overexpressing a competitive inhibitor of a lignin biosynthesis pathway enzyme. In some embodiments, the method comprises; introducing into the plant an expression cassette comprising a polynucleotide that encodes a protein that produces a competitive inhibitor of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) in the plant, wherein the

polynucleotide is operabiy jinked to a heterologous promoter; and

culturing the plant under conditions in which the protein that produces a competitive inhibitor of HCT is expressed. [0025] In some embodiments, the protein produces one or more of the competitive inhibitors protocatechuate, gentisate, catechol, 2,3-dihydroxybenzoate, 3,6- dihydroxybenzoate, or 3--hydroxy-2-aminobenzoaie. In some embodiments, the protein produces the competitive inhibitor of HCT protocatechuate. In some embodiments, the protein is dehydroshikimate dehydratase (QsuB), dehydroshikimate dehydratase (DsDH), isochorismate synthase (ICS), salicylic acid 3-hydroxyiase (S3H), salicylate hydroxylase (nahG), or salicylate 5-hydroxylase (nagGH). [0Θ26] in some embodiments, the polynucleotide that encodes a protein thai produces a competitive inhibitor of HCT is operably linked to a tissue-specific promoter. In some embodiments, the promoter is a secondary cell wall-specific promoter or a fiber cell-specific promoter. In some embodiments, the promoter is an IRX5 promoter, in some embodiments, the promoter is from a gene that is expressed in the lignin biosynthesis pathway

(phenylpropanoid pathway), e.g., a promoter from a gene expressed in the pathway shown in Figure 1 . in some embodiments, the promoter is a C4H, C3H, HCT, CCR1, CAD4, CADS, F5H, PALI , PAL2, 4CL1, or CCoAMT promoter.

[0027] in still another aspect, the present invention provides a plant, plant part, or seed, flower, leaf, or fruit from the plant, or a plant cell comprising a polynucleotide that encodes a protein that produces a competiti ve inhibitor of HCT in the plant, wherein the polynucleotide is operably linked to a heterologous promoter.

[0028] in still another aspect, the present invention provides biomass comprising plant tissue from a plant or part of a plant as described herein,

[0029] In yet another aspect, the present invention provides methods of obtaining an increased amount of soluble sugars from a plant in a saccharification reaction. In some embodiments, the method comprises subjecting a plant as described herein to a

saccharification reaction, thereby increasing the amount of soluble sugars that can be obtained from the plant as compared to a wild-type plant.

[0030] In still another aspect, the present invention provides methods of increasing the digestibility of the biomass for ruminants. In some embodiments, the method comprises introducing an expression cassette as described herein into a plant; culturing the plant under conditions in which the protein that diverts the monolignol precursor from the lignin biosynthesis pathway, or the protein that produces a competitive inhibitor of HCT, is expressed; and obtaining biomass from the plan thereby increasing the digestibility of the biornass for ruminants. BRIEF DESCRIPTION OF THE DRAWINGS

[0Θ31] Figure L Representation of the lignin biosynthesis pathway. Modified Hgnin biosynthesis pathway from Fraser and Chappie (201 1 ). Enzyme descriptions: PAL:

phenylalanine ammonia-lyase; C4H: cinnamate-4-hydroxylase; 4CL: 4-bydroxycinnamate CoA-Iigase; HCT: hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase; C3'H: 4-hydroxycinnamate 3-hydroxylase; CCoAOMT: eaffeoyl-CoA O-methyltransferase; CCR: hydroxycinnamoyl-CoA NADPH oxidoreductase; COMT: caffeate O- methyltransferase; CAD: hydroxycinnamyl alcohol dehydrogenase; F5H: ferulate 5- hydroxylase. Name of the lignin precursors: 1 , phenylalanine; 2, cinnamate; 3, p-coumarate; 4, p-coumaroyl-CoA; 5, ?-coumaroyl-shikimate/quinate (R = shikimate/quinate); 6, caffeoyl- shikimate/quinate; 7, caffeoyl-CoA; 8, feruloyl-CoA; 9, p-coumaraldehyde; 10,

coniferaldehyde; 1 1, 5-hydroxy- coniferaldehyde; 12, sinapaldehyde; 13, p-coumaryl alcohol; 14, coniferyl alcohol; 15, sinapyi alcohol.

[0032] Figure 2. Lignin reduction via depletion of shikimate (HCT co-substrate),

Strategies for reducing or depleting the amount of shikimate that is available for the lignin biosynthesis pathway are shown. (1 ) The amount of cytosoHc shikimate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a shikimate kinase such as M tuberculosis shikimate kinase ("MtAroK"). (2) The amo unt of plastidial shikimate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a pentafunciional arom protein such as S, cerevisiae pentafunctional arom protein

("ScArol "). Plastidial expression of the protein can be accomplished via a plastid targeting signal, e.g., as described herein.

[0033] Figure 3, Lignin reduction via depletion of shikimate and production of new stoppers. Strategies for reducing or depleting the amount of shikimate that is available for the lignin biosynthesis pathway are shown. For example, the amount of plastidial shikimate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a dehydroshikimate dehydratase such as C. glutamicum dehydroshikimate dehydratase ("CgQsuB") o " P. anserina dehydroshikimate dehydratase ("PaDsDH"). Plastidial expression of the protein can be accomplished via a plastid targeting signal, e.g., as described herein. [0034] Figure 4, Lignin reduction via depletion of phenylalanine (PAL substrate).

Strategies for reducing or depleting the amount of phenylalanine that is available for the lignin biosynthesis pathway are shown. For example, the amount of (1 ) cytosolic and/or (2) plastidial phenylalanine that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a phenylacetaldehyde such as P.hybrida phenylacetaldehyde synthase ("PhPAAS"). Plastidial expression of the protein can be accomplished via a plastid targeting signal, e.g., as described herein.

{0035] Figure 5, Lignin reduction via depletion of phenylalanine (PAL substrate).

Strategies for reducing or depleting the amount of phenylalanine that is available for the lignin biosynthesis pathway are shown. For example, the amount of (1 ) cytosolic and/or (2) plastidial phenylalanine that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a phenylalanine aminomutase such as T. canadensis phenylalanine aminomutase ("TcPAM"). Plastidial expression of the protein can be accomplished via a plastid targeting signal, e.g., as described herein.

[0036] Figure 6, Lignin reduction via depletion of cinnamate (C4H substrate) and coumarate (4CL substrate). Strategies for reducing or depleting the amount of cinnamate and/or p-coumarate that is available for the lignin biosynthesis pathway are shown. For example, the amount of cytosolic cinnamate and/or p-coumarate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a einnamate/p- coumarate carboxyl methyltransferase such as O. basilicum cinnarnate/p-coumarate carboxyl methyltransferase ("ObCCMTl ").

[0037} Figure 7. Lignin reduction via depletion of cinnamate (C4H substrate) and coumarate (4CL substrate). Strategies for reducing or depleting the amount of cinnamate and/or p-coumarate that is available for the lignin biosynthesis pathway are shown. For example, the amount of cytosolic cinnamate and/or p-coumarate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a phenylacrylic decarboxylase (PDC or PAD).

[0038] Figure 8, Lignin reduction via depletion of coumaroyl-CoA (HCT substrate). Strategies for reducing or depleting the amount of coumaroyl-CoA that is available for the lignin biosynthesis pathway are shown. For example, the amount of cytosolic coumaroyl- CoA that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a 2-oxoglutarate-dependent dioxygenase such as R. graveolens C2'H (2- oxoglutarate-dependent dioxygenase) ("RbC2'H"). [0039] Figure 9. Lignin reduction via depletion of coumaroyl-CoA (HCT substrate).

Strategies for reducing or depleting the amount of coumaroyl-CoA that is available for the lignin biosynthesis pathway are shown. For example, the amount of cytosolic coumaroyl- CoA that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a chalcone synthase (CHS), stilbene synthase (SPS), cucuminoid synthase (CUS), or ben zal acetone (BAS).

[0040] Figure 10. Lignin reduction via depletion of feruloyl-CoA (CCR substrate).

Strategies for reducing or depleting the amount of feru!oyl-CoA that is available for the lignin biosynthesis pathway are shown. For example, the amount of cytosolic feruloyl-CoA that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a 2- oxoglutarate-dependent dioxygenase such as R, graveolens C2'H (2-oxoglutarate-dependent dioxygenase) ("Rc 'l S";.

[00413 Figure 11. Lignin reduction via depletion of caffeoyi-CoA feruloyl-CoA (CCR substrate). Strategies for reducing or depleting the amount of caf eoyl-CoA and/or feruloyl- CoA that is available for the lignin biosynthesis pathway are shown. For example, the amount of cytosolic eaffeoyl-CoA and/or feruloyl-CoA that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a chalcone synthase (CHS), synthase (SPS), cucuminoid synthase (CUS), or benzalacetone (BAS). [0042] Figure 12. Growth phenofype analysis of S-QsuB lines. Picture of 3 weeks-old plants at rosette stage. No phenotypie differences could be observed between S-QsuB lines and WT plants at tfie rosette stage.

[0043] Figure 13, Total reducing-sugars released from stem biomass of S-QsuB lines and WT plants after 72h incubation with a cellufo lytic enzyme cocktail Total reducing-sugars released from biomass after hot-water pretreatnient (1 h at 120C) and incubation with a celluiolytic engine cocktail (Novozymes Cellic® CTec2) at a loading of 0.88% (g enzyme / g biomass) were measured using the 3,5-Dinitrosalicylic acid assay as described in Eudes et al. 2012 {Plant Biotech Journal 10(5):609-620).

[0044] Figure 14, Time course for total reducing-sugars released from stem biomass of S- QsuB lines and WT plants after incubation with different loadings of a celluiolytic enzyme cocktail. Time course for total reducing-sugars released from biomass after hot-water pretreatment (lh at 120C) and incubation with different loadings (0.88%, 0.176% or 0.088%; g of enzyme / g of biomass) of a celluiolytic enzyme cocktail (Novozymes Cellic® CTec2) .

Measurements were performed as described in (Eudes et al. 2012 Plant Biotech Journal 10(5):609-620).

[0045] Figure 15. Total reducing-sugars released from stem biomass of S-DsDH lines after 72h incubation with a celluiolytic enzyme cocktail. Time course for total reducing-sugar released from biomass after hot- water pretreatment ( 1 h at 120C) and incubation with a ceilulolytic enzyme cocktail (Novozymes Cellic® CTec2) at a loading of 0.88% (g enzyme / g biomass). Measurements were performed as described in (Eudes et ai. 2012 Plant Biotech Journal 10(5):609-620). [0046] Figure 16, QsuB expression in Arabidopsis stems. Detection by Western blot of QsuB tagged with the At.tB2 peptide (approximate size 70 kDa) using the "universal antibody" and stem proteins from nine independent 6-wk-old pC4H::schl::qsuB (C4H:;qsuB) T2 transformants. A stem protein extract from wild type was used as a negative control (WT) and a Ponceau staining of Rubiseo large subunit (rbcL) is shown as a loading control [0047] Figure 17. Partial short-range ^U C- ^! H (HSQC) spectra (aromatic region) of cell-wall material from mature senesced stems of wild-type (WT), pC4H::schl::qsuB-l (C4H::qsuB~l) and pC4H::schl::qsuB-9 (C4H::qsuB-9) plants. Lign i monomer ratios are provided on the figures.

|0048] Figure 18. Polydispersity of ceilulolytic enzyme lignins from wild-type and C4H::qsuB lines. Ceilulolytic enzyme lignins were purified from mature senesced stems of wild-type (WT, black Wm), pC4H::schl::qsuB-l (C4H::qsuB~l„ red line) and

pC4H::schl::qsuB-9 (C4H::qsuB~9, purple line) plants and analyzed for polydispersity by size-exclusion chromatography (SEC). SEC chromatograms were obtained using UV-F fluorescence (E 25o Em. 5o). m, molecular weight. [0049] Figure 19. Saccharification of biomass from mature senesced stems of wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) lines. (A) Amounts of sugars released from biomass after various pretreatments and 72-h enzymatic digestion with ceilulase (1% w/w). Values are means ± SE of four biological replicates (» = 4). Asterisks indicate significant differences from the wild type using the unpaired Student's t-test (*P < 0,05; **P < 0.005). (B) Amounts of sugars released from biomass after hot water pretreatment and 72-h enzymatic digestion using two different cel!ulase loadings (1% or 0.2% w/w). Values are means ± SE of four biological replicates (» = 4), Asterisks indicate significant differences from the wild type at 1 % ceilulase loading using the unpaired Student's t-test (*P < 0.05; **P < 0.005). [0Θ5Θ] Figure 20. The lignin biosynthetic pathway. Abbreviations: DAMPS, 3-deoxy-D- arabino-heptulosonate 7-phosphate synthase; DHQS, 3-dehydroquinate synthase; DHQD/SD, 3-dehydroquinate dehydratase; SK, shikimate kinase; ESPS, 3-phosphoshikimate 1 - carboxyvinyltransferase; CS, chorismate synthase; CM, chorismate mutase; PAT, prephenate aminotransferase; ADT, arogenate dehydratase; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; CSE, eaffeoyl shikimate esterase; 4CL, 4-coumarate CoA ligase; CAD, cinnamyl alcohol dehydrogenase; F5H, ferulate 5-hydroxylase; C3H, coumarate 3- hydroxylase; COMT, caffeic acid 3-O-methylfransferase; CC , cinnamoyi-CoA reductase; HCT, hydroxycinnamoyl-Coenzyme A shikitnate/quinate hydroxycinnanioy trarssferase; CCoAOMT, caffeoyl/CoA-3-O-methyitransferase; qsuB, 3-dehydroshikimate dehydratase from Coryne bacterium glutamicum.

{0Θ51 j Figure 21. Subcellular localization of SCHL-QsuB. The left panel displays the transient expression of SCHL-QsuB-YFP fusion protein expressed under the contro! of the 35S promoter in epidermal cells of N. benihamicma and imaged by confoca! laser scanning microscopy. The central panel displays fluorescing ehloroplasts and the right panel shows the merged images (c localizations are visible as yellow dots). Scale bars = 20 μτη.

[0Θ52] Figure 22. Summary of the fold changes observed for the methanol-soluble metabolites extracted from plants expressing QsuB. [0053] Figure 23, Partial short-range ^{! j} C- ^! H (FiSQC) spectra (aliphatic region) of cell wall material from mature senesced stems of wild-type (WT), pC4H: :schl: :qs B-l {C4H::qsiiB~i) and pC4H::schl::qsuB-9 ⁽ C4H::qsuB~9) plants.

[0054] Figure 24. Lignin staining by phloroglueinol-HCi of stem sections from 5-wk-old wild-type (WT) and pC4H::sch!::qsuB (C4H::q$uB) plants. [0055] Figure 25. LC-MS chromatograms from A HCT in-vivo activity assays, LC-MS chromatograms of coumarate conjugates produced by AtHCT after feeding a recombinant yeast strain co-expressing At4CL5 and AtHCT with / xmmarate and (A) shikimate, (B) 3,6- dihydroxybenzoate, (C) 3-hydroxy-2-amino benzoate, (D) 2,3-dibydroxybenzoate, (E) catechol, or (F) protocatechuate are presented. Structures of coumarate-dihydroxybenzoate esters are arbitrary shown with an ester linkage at the 3-hydroxy position of the

dihydroxybenzoate ring. The structure of coumaroyl-3-hydroxyanthranilate ( ) is represented as determined in Moglia et al. (34).

[0056] Figure 26. LC-MS chromatogram ofp-coumaraldehyde detected in methanol- soluble extracts of stems from lines expressing QsuB. [0057] Figure 27. Competiti ve inhibitor pathways.

[0058] Figure 28. Characteristics and relative molar abundances (%) of the compounds released after pyro-GC/MS of extracti ve-free senesced mature stems from wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) plants. Values in brackets are the SE from duplicate analyses, nd, not detected.

DETAILED DESCRIPTION OF THE INVENTION L Definitions

[0059] As used herein, the term "lignin biosynthesis pathway" refers to an enzymatic pathway (the phenylpropanoid pathway) in plants in which the lignin monomers (p-coumaryl (4-hydroxycinnamyi) alcohol, coniferyi (3-methoxy 4-hydroxycinnamyi) alcohol, and sinapyl (3,5-dimethoxy 4-hydroxycinnamyl) alcohol) are synthesized from phenylalanine. The lignin biosynthesis pathway and enzymatic components of the pathway are depicted, for example, in Figure 1.

[0060] As used herein, the term "monolignoi precursor" refers to a substrate of the lignin biosynthesis pathway that is directly or indirectly synthesized into a lignin monomer. In some embodiments, a monolignoi precursor is a substrate of the lignin biosynthesis pathway that is identified in any of Figures 1-1 1.

[0061] As used herein, the term "protein that diverts a monolignoi precursor from a lignin biosynthesis pathway" refers to a protein that activates, promotes, potentiates, or enhances expression of an enzymatic reaction or metabolic pathway that decreases the amount of monolignoi precursor that is available for the synthesis of a lignin monomer. The term includes polymorphic variants, alleles, mutants, and interspecies homologs to the specific proteins (e.g., enzymes) described herein. A nucleic acid that encodes a protein that diverts a monolignoi precursor from a lignin biosynthesis pathway (or a nucleic acid that encodes a protein that diverts a monolignoi precursor from a p-coumaryl alcohol, sinapyl alcohol, and/or coniferyi alcohol pathway) refers to a gene, pre-mRNA, raRN A, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants, and interspecies homologs of the particular proteins (e.g., enzymes) described herein. In some embodiments, a nucleic acid that encodes a protein that diverts a monolignoi precursor from a lignin biosynthesis pathway (1 ) has a nucleic acid sequence that has greater than about. 50% nucleotide sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or higher nucleotide sequence identity, preferably over a region of at least about 10, 15, 20, 25, 50, 100, 200, 500 or more nucleotides or over the length of the entire polynucleotide, to a nucleic acid sequence of any of SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1, or 13; or (2) encodes a polypeptide having an amino acid sequence that has greater than about 50% amino acid sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 300, 200 or more amino acids or over the length of the entire polypeptide, to a polypeptide encoded by a nucleic acid sequence of any of SEQ ID NOs: l, 3, 5, 7, 9, 11 , or 13, or to an amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 43, 44, or 45. In some embodiments, a protein that diverts a mono!ignol precursor from a lignin biosynthesis pathway has an amino acid sequence having greater than about 50% amino acid sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200 or more amino acids or over the length of the entire polypeptide, to an amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 42, 43, 44, or 45. [0062] The term "protein that produces a competitive inhibitor of HCT" refers to a protein that, directly or indirectly produces a molecule that can compete with p-coumaroy!-CoA and/or shikimate as a substrate for hydroxycinnamoyl-CoA shikiraate/quinate

hydroxyciimamoyltrasisferase (HCT), thereby acting as a competitive irshibitor of HCT. Non- limiting examples of molecules (e.g., metabolites) that can act as competitive inhibitors of HCT are shown in Figure 27. In some embodiments, the competitive inhibitor of HCT is protocatechuate, catechol, 3,6-dihydroxybenzoate, 3-hydroxy-2-arainobenzoate, or 2,3- dihydroxybenzoate. Thus, in some embodiments, the protein that produces a competitive inhibitor of HCT is a protein (e.g., an enzyme) that directly or indirectly produces protocatechuate, catechol, 3,6-dihydroxybenzoate, 3-hydroxy-2-aminobenzoate, or 2,3- dihydroxybenzoate, including but not limited to the enzymes dehydroshikimate dehydratase (QsuB), dehydroshikimate dehydratase (DsDH), isochorismate synthase (ICS), salicylic acid 3-hydroxyIase (S3H), salicylate hydroxylase (naliG), and salicylate 5-hydroxylase (nagGH). In some embodiments, an in vivo enzymatic assay, for example as described in the Examples section below, can be used to determine whether a molecule can compete with _p-coumaroyl- CoA and/or shikimate as a substrate for HCT.

[0063] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a single or double-stranded polymer of deoxy ribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A nucleic acid of the present invention will generally con tain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate,

phosphorodithioate, or O-methylphopboroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones: non- ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase,

"Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may- contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

[0Θ64] The term "substantially identical," used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. For example, a first polynucleotide is substantially identical to a second polynucleotide sequence if the first polynucleotide sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the second polynucleotide sequence.

[0065] Two nucleic acid sequences or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g. , charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are weii known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1 , The scoring of conservative substitutions is calculated according to, e.g. , the algorithm of Meyers & Miller, Computer AppUc. Biol. Sci. 4: 1 1- 17 (1988) e.g. , as implemented in the program PC/GENE (Inielligenetics, Mountain View, California, USA). [0066] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0067} A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about. 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optima] alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. App!. Main. 2:482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. Moi. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection. [0068] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol Biol 215: 403-410 and Altschul et al (1977) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value: the cumulative score goes to zero or below, due to the

accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M ^~ 1 , N ^::;: -2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix {see HenikofF & Henikoff, Proc. Nail Acad. Sci USA 89: 10915 (1989)). (0069] The BL ST algorithm also performs a statistical analysis of the similarity between two sequences {see, e.g., Karlin & Altschul, Proc. Nat'i. Acad. Sci. USA. 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(NY), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smalles sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10°, and most preferably less than about 10 ^"20 .

[0070] Nucleic acid or protein sequences that are substantially identical to a reference sequence include "conservatively modified variants." With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule.

Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. [0071] As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "consen'atively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0072] The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F). Tyrosine (Y), Tryptophan (W).

{see, e.g., Creighton, Proteins ( 1984)). [ΘΘ73] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions.

Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at oil 7 and the temperature is at least about 60°C. For example, stringent conditions for hybridization, such as R A-DM A hybridizations in a blotting technique are those which include at least one wash in 0.2X SSC at 55°C for 20 minutes, or equivalent conditions.

[0074] As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the

polynucleotide constructs of the invention include cis- and trans- acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start cod on (i.e., including the 5' untranslated region of the mR A, typically comprising 100-200 bp). Most often the core promoter sequences lie within 1-5 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls.

[0075] A "constitutive promoter" is one that is capable of initiating transcription in nearly all cell types, whereas a "cell type-specific promoter" initiates transcription only in one or a few particular cell types or groups of cells forming a tissue, in some embodiments, the promoter is secondary cell wall-specific and/or fiber cell-specific. A "fiber cell-specific promoter" refers to a promoter that initiates substantially higher levels of transcription in fiber cells as compared to other non-fiber cells of the plant. A "secondary cell wall-specific promoter" refers to a promoter that initiates substantially higher levels of transcription in cell types that have secondary cell walls, e.g., lignified tissues such as vessels and fibers, which may be found in wood and bark cells of a tree, as well as other parts of plants such as the leaf stalk. In some embodiments, a promoter is fiber cell-specific or secondary cell wall-specific if the transcription levels initiated b the promoter in fiber cells or secondary cell walls, respectively, are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher or more as compared to the transcription levels initiated by the promoter in other tissues, resulting in the encoded protein substantially localized in plant cells that possess fiber cells or secondary cell wall, e.g., the stem of a plant. Non- limiting examples of fiber cell and/or secondary cell wall specific promoters include the promoters directing expression of the genes IRX1 , IRX3, IRX5, IRX7, IRX8, IRX9, IRX10, IRX14, NST1 , NST2, NST3, MYB46, MYB58, MYB63, MYB83, MYB85, MYB 103, PALI, PAL2, C3H, CcOAMT, CCR1 , F5H, LAC4, LAC 17, CADc, and CADd. See, e.g., Turner et ai 1997; Meyer et al 1998; Jones et al 2001; Franke et al 2002; Ha et al 2002;Robde et ai 2004; Chen et al 2005; Stobout et al 2005; Brown et al 2005; Mitsuda et al 2005; Zhong et al 2006; Mitsuda et ai 2007; Zhong et al 2007a, 2007b; Zhou et al 2009; Brown et al 2009; McCarthy et a! 2009; Ko et al 2009; Wu et al 2010; Berthet et al 201 1. In some

embodiments, a promoter is substantially identical to a promoter from the lignin biosynthesis pathway (e.g., a promoter for a gene encoding a protein shown in Figure 1). Non-limiting examples of promoter sequences are provided herein as SEQ ID NOs: 17-28. A promoter originated from one plant species may be used to direct gene expression in another plant species.

[0076] A polynucleotide is "heterologous" to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety, or a gene that is not naturally expressed in the target tissue).

[0077] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or

modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0078] The term "expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an R A or polypeptide, respectively, Antisense or sense constructs that are not or cannot be translated are expressly included by this definition, in the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense, RNAi, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence.

{0079] The term "plant," as used herein, refers to whole plants and includes plants of a variety of a ploidy levels, including aneuploid, polyploid, diploid, and haploid. The term "plant part," as used herein, refers to shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), branches, roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), frail (e.g., the mature ovary), seedlings, and plant tissue (e.g., vascular tissue, ground tissue, and the like), as well as individual plant cells, groups of plant cells (e.g., cultured plant ceils), protoplasts, plant extracts, and seeds. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnospemis, ferns, and multicellular algae.

[0080] The term "biomass," as used herein, refers to plant material that is processed to provide a product, e.g., a biofuel such as ethanoi, or livestock feed, or a cellulose for paper and pulp industry products. Such plant material can include whole plants, or parts of plants, e.g., stems, leaves, branches, shoots, roots, tubers, and the like.

[0081] The term "reduced lignin content" encompasses reduced amount of lignin polymer, reduced amount of either or both of the guaiacyl (G) and/or syringyl (8) lignin units, reduced size of a lignin polymer, e.g., a shorter lignin polymer chain due to a smaller number of monolignols being incorporated into the polymer, a reduced degree of branching of the lignin polymer, or a reduced space filling (also called a reduced pervaded volume). In some embodiments, a reduced lignin polymer can be shown by detecting a decrease in the molecular weight of the polymer or a decrease in the number of monolignols by a t least 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, or more, when compared to the average lignin molecule in a control plant (e.g., a non-transgenic plant). In some embodiments, reduced lignin content can be shown by detecting a decrease in the number or amount of guaiacyl (G) and/or syringyi (S) lignin units in the plant as compared to a control plant (e.g.. a non- transgenic plant). In some embodiments, a plant as described herein has reduced lignin content if the amount of guaiacyl (G) and/or syringyi (S) lignin units in the plant is decreased by at least about 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50% or more, as compared to a control plant. Methods for detecting reduced lignin content are described in detail below.

Ii. Introduction

[0082] Plant cell walls constitute a polysaccharide network of cellulose microfibrils and hemicelhilose embedded in an aromatic polymer known as lignin. This ramified polymer is mainly composed of three phenylpropanoid-den ^' ved phenolics (i.e., mono!ignols) named p- coumaryl, conifety!, and sinapyl alcohols which represent the ?-hydroxyphenyl (H), guaiacyl (G) and syringyi (8) lignin units (Boerjan et al, 2003). Monolignols have a G5C3 carbon skeleton which consists of a phenyl ring (C _& ) and a propane (C ₃ ) side chain. Lignin is crucial for the development of terrestrial plants as it confers recalcitrance to plant cell wails, it also provides mechanical strength for upright growth, confers hydrophobicity io vessels that transport water, and acts as a physical barrier against pathogens that degrade cell walls (Boudet, 2007). Notably, lignin content and composition are finely regulated in response to environmental biotic and abiotic stresses (Moura et al., 2010).

[0083] Economically, lignocellulosic biomass from plant cell walls is widely used as raw material for the production of pulp in paper industry and as ruminant livestock feed. Plant feedstocks also represent a source of fermentable sugars for the production of synthetic molecules such as pharmaceuticals and transportation fuels using engineered microorganisms (Keasling, 2010). However, negative correlations exist between lignin content in plant biomass and pulp yield, forage digestibility, or polysaccharides enzymatic hydrolysis (de Vrije et al, 2002; Reddy et ah, 2005; Dien et al, 2006; Chen and Dixon, 2007; Dien et al, 2009; Taboada et al , 2010: Elissetche et al, 201 1 ; Studer et al, 201 1 ). Consequently, reducing lignin recalcitrance in plant feedstocks is a major focus of interest, especially in the lignocellulosic biofuels field for which efficient enzymatic conversion of polysaccharides into monosaccharides is crucial to achieve economically and environmentally sustainable production (Carroll and Somerville, 2009). [0084] Lignin biosynthesis is well characterized and well conserved across land plants (Weng and Chappie 2010). Genetic modifications such as silencing of genes involved in particular steps of this pathway or its regulation have been employed to reduce lignin content (Simmons el al, 2010; Umezawa, 2010) but this approach often results in undesired phenotypes such as dwarfism, steriiity, reduction of plant biomass, and increased susceptih!y to environmental stress and pathogens (Bonawitz and Chappie, 2010). These pleiotropic effects are generally the consequences of a loss of secondary cell wall integrity, accumulation of toxic intermediates, constitutive activa tion of defense responses, or depletion of other phenylpropanoid-derived metabolites which are essential for plant development and defense (Li et a!., 2008; Naoumkina et ah, 2010, Gallego-Giraldo et al, 201 1 ). Alternatively, chaoging the recalcitrant structure and physico-chemical propertses of lignin can be achieved by modifying its monomer composition. For example, incorporation of coniferyl ferulate into lignin improves enzymatic degradation of cell wail polysaccharides (Grabber et al., 2008). Recently, it has been demonstrated that enrichment in 5-hydroxy-G units and reduction in S units in lignin contribute to enhanced saccharification efficiencies without affecting drastically biomass yields and lignin content (Weng et al., 2010; Dien et al, 201 1 ; Fu et al., 201 1).

[0085] The present invention provides an alternative strategy to reduce lignin content (e.g., reducing the amount of j-hydroxyphenyl (H), guaiacyl (G) and/or syringyl (S) lignin units, or any combination of H-3ignin, G-lignin, and S- lignin units). In this strategy, the plant is engineered to express one or more proteins that diverts or shunts a monolignoi precursor from a lignin biosynthesis pathway (e.g., a p-coumaryi alcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis pathway) into a competitive pathway. By diverting or shunting the production of monolignoi precursors from p-hydroxyphenyl (H), guaiacyl (G) and/or syringyl (S) lignin unit production to the production of alternative products (e.g., stilbenes, flavonoids, curcuminoids, or bensalacetones, protocatechuates, aromatic amino acids, vitamins, quinones, or volatile compounds) as described herein, the amount of lignin content or its composition, e.g., in specific cell or tissue types such as in secondary cell wall, can be altered in order to enhance saccharification efficiencies without dramatically affecting biomass yield. The present invention also provides plants that are engineered by the method described herein, as well as a plant cell from such a plant, a seed, flower, leaf, or fruit from such a plant, a plant cell that contains an expression cassette described herein for expressing a protein diverts or shunts a monolignoi precursor from a lignin biosynthesis pathway into a competitive pathway, and biomass comprising plan t tissue from the plant or part of the plant described herein.

III. Plants Having Reduced Lignin Content

A. Expression of a Protein That Diverts a MoHoligrtoi Precursor From a Lignin Biosynthesis Pathway

[0086 j in one aspect, the present invention provides a method of engineering a plant having reduced lignin content (e.g., reduced amount of lignin polymers, reduced size of lignin polymers, reduced degree of branching of lignin polymers, or reduced space filling), ϊη some embodiments, the plant has reduced lignin content that is substantially localized to specific cell and/or tissue types in the plant, For example, in some embodiments the plant has reduced lignin content that is substantially localized to secondary cell walls and/or fiber cells, in some embodiments, the method comprises: introducing into the plant an expression cassette comprising a polynucleotide that encodes a protein that diverts a monolignol precursor from a lignin biosynthesis pathway (e.g., a p-coumaryl alcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis pathway) in the plant, and wherein the polynucleotide is operably linked to a heterologous tissue- specific promoter; and

culturing the plant under conditions in which the protein that diverts the monolignol precursor from the lignin biosynthesis pathway (e.g., the p-coumaryl alcohol, sinapyl aicohoi, or coniferyl alcohol biosynthesis pathway) is expressed.

[0087] In some embodiments, the gene that encodes a protein that diverts a monolignol precursor from a lignin biosynthesis pathway (e.g., a p-coumaryl alcohol, sinapyl alcohol, and/or coniferyl aicohoi biosynthesis pathway) reduces the amount of cytosolic and/or plastidial shikimate that is available for the p-coumary! alcohol, sinapyl alcohol, or coniferyl alcohol biosynthesis pathway; reduces the amount of cytosolic and/or plastidial phenylalanine that is available for the p-coumaryl alcohol, sinapyl aicohoi, or coniferyl alcohol biosynthesis pathway; reduces the amount of cinnamate and/or coumarate that is available for the p- coumary! alcohol, sinapyl alcohol, or coniferyl alcohol biosynthesis pathway; and/or reduces the amount of coumaroy!-CoA, caffeoyl-CoA, and/or feraloyl-CoA that, is available for the p- coumaryl aicohoi, sinapyl alcohol, or coniferyl alcohol biosynthesis pathway. In some embodiments, the gene that encodes a protein that diverts a monolignol precursor from a lignin biosynthesis pathway (e.g., a p-coumaryl alcohol, sinapyl alcohol, and/or coniferyl alcohol biosynthesis pathway) activates or potentiates a metabolic pathway that competes with the p-coumaryf alcohol, sinapyl alcohol, or coniferyl alcohol biosynthesis pathway biosynthesis pathway for the use of monoiignol precursors, including but not limited to a metabolic pathway selected from a stilbene biosynthesis pathway, a flavonoid biosynthesis pathway, and an anthocyanin biosynthesis pathway. [0088] An expression cassette as described herein, when introduced into a plant, results in the plant having reduced lignin content (e.g., reduced amount of lignin polymers, reduced size of lignin polymers, reduced degree of branching of lignin polymers, or reduced space filling) that is specifically localized to certain ceil and/or tissue types (e.g., specifically localized to secondary cell walls and/or fiber cells), thus reducing cell wall recalcitrance to enzymatic hydrolysis while avoiding defects in plant growth or reductions in biomass yield.

[0089] One of skill in the art will understand that the protein that diverts a monoiignol precursor from a lignin biosynthesis pathway that is introduced into the plant by an expression cassette described herein does not have to be identical to the protein sequences described herein (e.g., the protein sequences of SEQ ID NOs:2, 4, 6. 8, 10, 12, or 14). in some embodiments, the protein that is introduced into the plant by an expression cassette is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to a protein sequence described herein (e.g., a protein sequence of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14). In some embodiments, the protein that is introduced into the plant by an expression cassette is a homo!og, ortholog, or paralog of a protein that diverts a monoiignol precursor from a lignin biosynthesis pathway as described herein (e.g., a protein sequence of SEQ ID Os:2, 4, 6, 8, 10, 12, or 14).

[0090] Gene and protein sequences for enzymes that divert a monoiignol precursor from a lignin biosynthesis pathway are described in the Sequence Listing herein. Additionally, gene and protein sequences for these proteins, and methods for obtaining the genes or proteins, are known and described in the art. One of skill in the art will recognize that these gene or protein sequences known in the art and/or as described herein can be modified to make substantially identical enzymes, e.g., by making conservative substitutions at one or more amino acid residues. One of skill will also recognize that the known sequences provide guidance as to what amino acids may be varied to make a substantially identical enzyme, For example, using an amino acid sequence alignment between two or more protein sequences, one of skill will recognize which amino acid residues are not highly conserved and thus can likely be changed without resul ting in a significant effect on the function of the enzyme. Proteins that Reduce the Amo nt of Sliikimate

[0091] In some embodiments, a protein that diverts a monolignol precursor from a lignin biosynthesis pathway reduces the amount of cytosolic and/or piastidiai shikimate that is available for the lignin biosynthesis pathway. Examples of such a protein are shown in Figures 2 and 3, In some embodiments, the protein is an enzyme that modifies a shikimate substrate, e.g., a shikimate kinase or a pentafunctional arom protein. In some embodiments, the protein is an enzyme that utilizes shikimate in the synthesis of another compound (e.g., a protocateehuate, an aromatic amino acid, a vitamin, or a quinone), e.g., a dehydryoshikimate dehydratase. [0092] Non-limiting examples of a shikimate kinase enzyme are described in Gu ei al, J. Mol. Biol. 319:779-789 (2002). In some embodiments, the protein is a Mycobacterium tuberculosis sliikimate kinase (AroK) having the amino acid sequence set forth in SEQ ID NO:2. in some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at feast 85%, at least 90%, at least 91 , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO:2. in some embodiments, the protein is a hornoiog of a Mycobacterium tuberculosis shikimate kinase (AroK) having the amino acid sequence set forth in SEQ ID NQ:2. in some embodiments, a polynucleotide encoding the shikimate kinase comprises a polynucleotide sequence that is identical or substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 1.

[Θ093] Non-limiting examples of a pentafunctional arom protein are described in Duncan et al, Biochem. J. 246:375-386 (3987). In some embodiments, the protein is & Saccharomyces cerevisiae. pentafunctional arom enzyme (Arol) having the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO:4. In some embodiments, the protein is a hornoiog of a Saccharomyces cerevisiae pentafunctional arom enzyme (Arol) having the amino acid sequence set forth in SEQ 3D NO:4. In some embodiments, a polynucleotide encoding the pentafunctional arom protein comprises a polynucleotide sequence that is identical or substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:3.

[0094] Non-limiting examples of a dehydryoshikimate dehydratase are described in Teramoto et al, Appl Environ, Microbiol. 75:3461-3468 (2009) and Hansen et al, Appl Environ. Microbiol. 75:2765-2774 (2009). In some embodiments, the protein is a

Corynebacterium glutamicum dehydryoshikimate dehydratase (QsuB) having the amino acid sequence set forth in SEQ ID NO:6 or a Podospora anserina dehydryoshikimate dehydratase (DsDH) having the amino acid sequence set forth in SEQ ID NO:8. in some embodiments, the protein is substantially identical (e.g.. at least 50%, at least 55%, at least 60%, at least

65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical) to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8. In some embodiments, the protein is a homolog of a Corynebacterium glutamicum dehydryoshikimate dehydratase (QsuB) having the amino acid sequence set forth in SEQ) ID NO:6 or a homolog of the Podospora anserina dehydryoshikimate dehydratase (DsDH) having the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, a polynucleotide encoding the dehydryoshikimate dehydratase comprises a polynucleotide sequence that is identical or substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:5 or SEQ ID NO:7.

Proteins that Reduce the Amount of Phenylalanine

[0095] In some embodiments, a protein that diverts a monoiignoJ precursor from a lignin biosynthesis pathway reduces the amount of cytosolic and/or plastidial phenylalanine that is available for the lignin biosynthesis pathway. Examples of such a protein are shown in Figures 4 and 5. in some embodiments, the protein is an enzyme that modifies a

phenylalanine substrate. In some embodiments, the protein is an enzyme that utilizes phenyial anjne in the synthesis of another compound ( .g., a volatile compound), e.g., a phenylacetaldehyde synthase or a phenylalanine aminomutase.

[0096] Non-limiting examples of a phenylacetaldehyde synthase are described in Kaminaga et al, J. Biol Chem. 281 :23357-23366 (2006) and in Farhi et al, Plant Mol. Biol 72:235-245 (2010). In some embodiments, the protein is a Petunia hybrida phenylacetaldehyde synthase (PAAS) having the amino acid sequence set forth in SEQ ID NO: 10, i some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the protein is a homolog of a Petunia hybrid phenylacetaidehyde synthase (PAAS) having the amino acid sequence set forth in SEQ ID NO: 30. in some embodiments, a polynucleotide encoding the phenylacetaidehyde synthase comprises a polynucleotide sequence that is identical or substantially identical {e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:9.

[0097] Non-limiting examples of a phenylalanine aminorautase are described in Feng et al, Biochemistry 50:2919-2930 (201 1). in some embodiments, the protein is a T. canadensis phenylalanine aminorautase (PAM) having the amino acid sequence set forth in SEQ ID NO:29. in some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO:29. In some embodiments, the protein is a homolog of a T. canadensis phenylalanine aminomutase (PAM) having the amino acid sequence set forth in SEQ ID NG:29.

Proteins that Reduce the Amount _, of Cinnamate and/or Cournarate

[0098] In some embodiments, a protein that diverts a monolignol precursor from a lignin biosynthesis pathway reduces the amount of cinnamate and/or cournarate that is available for the lignin biosynthesis pathway. Examples of such a protein are shown in Figures 6 and 7. in some embodiments, the protein is an enzyme that modifies a cinnamate and/or cournarate substrate, e.g., a cinnamate/p-coumarate carboxyl methyltransferase. In some embodiments, the protein is an enzyme that utilizes cinnaniate and/or cournarate in the synthesis of another compound (e.g., a volatile compound, e.g., styrene or p-hydroxystyrene), e.g., phenylacrylic acid decarboxylase or feruiic acid decarboxylase.

[0099] Non-limiting examples of a cinnamate/p-coumarate carboxyl methyltransferase enzyme are described in apteyn et al, Plant Cell 19:3212-3229 (2007). Irs some embodiments, the protein is a Ocimiim basiliciim cinnamate/p-coumarate carboxyl methyltransferase (CCMT) having the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO: 12. in some embodiments, the protein is a homolog of a Ocimum basilicum cinnamate/p-coumarate carboxyl methyltransferase (CCMT) having the amino acid sequence set forth in SEQ ID NO: 12, In some embodiments, a polynucleotide encoding the cinnamate/p-coumarate carboxyl methyltransferase comprises a polynucleotide sequence that is identical or substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least

70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 1 1.

[0100] Non-l imiting examples of a phenylacrylic acid decarboxylase are described in McKenna et al, Me tab Eng 13 :544-554 (201 1 ). in some embodiments, the protein is a P. penosaceus phenylacrylic aicd decarboxylase (PDC) having the amino acid sequence set forth in SEQ ID NO:30. in some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO:30. In some embodiments, the protein is a homolog of a P. penosaceus phenylacrylic acid decarboxylase (PDC) having the amino acid sequence set forth in SEQ ID NO:30.

Proteins that Reduce the Amount of ( ' o s troyl-CoA, ( ^' affe vS-COA, and/or FeruIoyS- CoA

[010.1] In some embodiments, a protein that diverts a monolignol precursor from a lignin biosynthesis pathway reduces the amount of coumaroyl-CoA and/or feruloyl-CoA that is available for the lignin biosynthesis pathway. Examples of such a protein are shown in Figures 8-3 3 , In some embodiments, the protein is an enzyme that modifies a coumaroyl- CoA and/or feruloyi-CoA substrate. In some embodiments, the protein is an enzyme that utilizes coumaroyl-CoA and/or feruloyl-CoA in the synthesis of another compound (e.g., urnbellsferone, a volatile compound, scopoSetin, chalcone, trihydroxychalcone, stilbene, curuminoid, or benzylacetone), e.g., 2-oxoglutarase-dependent dioxygenase, chalcone synthase, stilbene synthase, ciicuminoid synthase, or benzalacetone synthase. [0.102] A non-limiting example of a 2-oxoglutarase-dependent dioxygenase enzyme is described in Vialart et al, Plant J. 70:460-470 (2012), In some embodiments, the protein is a Ruta grave.ole.ns 2-oxoglutarase-dependent dioxygenase (C2'H) having the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the protein is substantially identical {e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the protein is a homolog of a Ruia graveoiens 2-oxoglutarase-dependent dioxygenase (C2'H) having the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, a polynucleotide encoding the oxoglutarase-dependent dioxygenase comprises a polynucleotide sequence that is identical or substantially identical {e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 13.

[0103] Other non-limiting examples of proteins that reduce the amount of coumaroyl-CoA, eaffeoyl-CoA, and/or feruIoyl-CoA that is available for the !ignin biosynthesis pathway chalcone synthase (CHS), stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone synthase (BAS), described in Katsuyama et a!., J. Biol. Chem. 282:37702- 37709 (2007); Sydor et al, App!. Environ. Microbiol. 76:3361-3363 (2010); Jiang et al,

Ph iochemistry 67:2531 -2540 (2006); Abe and Morita, Nat. Prod. Rep. 27:809 (2010); Dao et al, Phytoche . Rev. 10:397- 12 (201 1 ); Suh et al., Biochem J. 350:229-235 (2000); Tropf et al, J. Biol. Chem. 270:7922-7928 ( 1995); Knogge et al.. Arch. Biochem. Biophys.

250:364-372 (1986); Ferrer el al., Nat. Struct. Biol. 6:775-784 (1999); Miyazono et al., Proteins 79:669-673 (2010): and Abe et al, Eur. J. Biochem.. 268:3354-3359 (2001 ). In some embodiments, the protein is a PhyscomitreUa patens CHS having the amino acid sequence set forth in SEQ ID NO:31; an Arahidopsi thaliana CHS having the amino acid sequence set forth in SEQ ID NO:32; a Viiis vinifera SPS having the amino acid sequence set forth in SEQ ID NO:33; an Oryza saliva COS having the amino acid sequence set forth in SEQ ID NO:34 or SEQ ID NO:35; or a Rheum palmatum BAS having the amino acid sequence set forth in SEQ ID NO:36; or a homolog thereof. In some embodiments, the protein is substantially identical {e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of any of SEQ ID NOs:31 , 32, 33, 34, 35, or 36.

Proteins that Activate a Competitive Metabolic Patlwsty

[0104] In some embodiments, a protein that diverts a monolignol precursor from a lignin biosynthesis pathway activates, upreguiates, or potentiates a metabolic pathway that competes with the lignin biosynthesis pathway biosynthesis pathway for the use of monolignol precursors. Non-limiting examples of metabolic pathways that are competitive with the lignin biosynthesis pathway include the stiibene biosynthesis pathway, the flavonoid biosynthesis pathway, the curcuminoid biosynthesis pathway, and the bensaiacetone biosynthesis pathway. Thus, in some embodiments, the protein that diverts a monolignol precursor from a lignin biosynthesis pathway is a protein (e.g., a transcription factor, a TALE-based artificial transcription factor (see Zhang el al, Nat. Biolechnol. 29: 149-153 (201 1)), or an enzyme) that activates, upreguiates, induces, or potentiates a stiibene biosynthesis pathway, a flavonoid biosynthesis pathway, a curcuminoid biosynthesis pathway, or a bensaiacetone biosynthesis pathway

[0105] As one non-limiting example, a protein can be expressed that activates, upreguiates, induces, or potentiates a flavonoid biosynthesis pathway. The flavonoid biosynthesis pathway utilizes monolignol precursors such as couraaroyi-CoA, caffeoyl-CoA, and feruloyl- CoA from the lignin biosynthesis pathway for the synthesis of flavonoids such as chaicones, fiavonones, dihydroflavonols, fiavonols, and anthocyanins. See Figures 9 and 11. In some embodiments, the protein that diverts a monolignol precursor from a lignin biosynthesis pathway is a protein that activates, upreguiates, induces, or potentiates the expression and/or activity of an enzyme in the flavonoid biosynthesis pathway (e.g., an enzyme such as chalcone synthase or flavonol synthase). In some embodiments, the protein that diverts a monolignol precursor from a lignin biosynthesis pathway is a transcription factor.

Transcription factors in the flavonoid biosynthesis pathway are known in the art. See, e.g., Bovy et al, Plant Cell 14:2509-2526 (2002); Tohge et a!., Plant ./. 42:218-235 (2005); Peel el al, Plant ! 59: 136-149 (2009); Pattanaik et al, Planta 231 : 1061 - 1076 (2010); and Hichri el al., J Exp Botany 62:2465-2483 (201 1 ); incorporated by reference herein. Non-limiting examples of transcription factors in the flavonoid biosynthesis pathway include MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 transcription factors. In some embodiments, the protein is an Arabidopsis thaliana PAP1 R2R3 MYB transcription factor having the amino acid sequence set forth in SEQ ID NO:37; an Arabidopsis thaliana PAP2 R2R3 MYB transcription factor having the amino acid sequence set forth in SEQ ID NO:38: an Arabidopsis thaliana TT2 R2R3 MYB transcription factor having the amino acid sequence set forth in SEQ ID NO:39; a Nicotiana iabacum

An2 R2R3 MYB transcription factor having the amino acid sequence set forth in SEQ ID NO:40; a Medicago t uncat la LAP 1 R2R3 MYB transcription factor having the amino acid sequence set forth in SEQ ID NO:41 ; a Zea mays MYB--C R2R3 transcription factor having the amino acid sequence set forth in SEQ ID NO:42; a Zea mays MYC-Lc BHLH

transcription factor having the amino acid sequence set forth in SEQ ID NO:43; an

Arabidopsis thaliana TT8 BHLH transcription factor having the amino acid sequence set forth in SEQ ID NO:44; or a Vitis vinifera Myc l BHLH transcription factor having the amino acid sequence set forth in SEQ ID NO:45; or a homolog thereof. In some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of any of SEQ ID NOs:37, 38, 39, 40, 41 , 42, 43, 44, or 45.

[0106] In some embodiments, a plant is engineered to express two, three, four or more proteins as described herein. In some embodiments, the plant expresses two or more proteins, each of which is identical or substantially identical to SEQ ID NOs:2. 4, 6, 8, 10, 12, 14, 29, 30, 3 1 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 42, 43, 44, or 45. In some embodiments, the two or more proteins utilize different substrates or activate different pathways; for example, in some embodiments the plant expresses a first protein that reduces the amount of shikimate that is available for the Hgnin biosynthesis pathway and a second protein that reduces the amount of phenylalanine that is available for the lignin biosynthesis pathway, in some embodiments, the two or more proteins potentiate or activate the same pathway: for example, in some embodiments the plant expresses a first transcription factor and a second transcription factor that function cooperatively to induce the flavonoid biosynthesis pathway.

Proteins that Produce a Competitive Inhibitor of HCT

[0107] In some embodiments, a plant having reduced lignin content is engineered by expressing or overexpressing a competitive inhibitor of a lignin biosynthesis pathway enzyme (e.g., a molecule that competes with /?-coumaroyl-CoA and/or shikimate as a substrate for hydroxycinnarrtoyl-CoA shikimate/quinate hydroxycinnamoyJtransferase (HCT)). In some embodiments, the method comprises: introducing into the plant an expression cassette comprising a polynucleotide that encodes a protein that produces a competitive inhibitor of hydroxycinnamoyl-CoA shikirnate/quinate hydroxycinnamoyltransferase (HCT) in the plant, wherein the

polynucleotide is operably linked to a heterologous promoter; and

cuituring the plant under conditions in which the protein that produces a competitive inhibitor of HCT is expressed.

[0108j In some embodiments, the protein directly or indirectly produces one or more of the competitive inhibitors protocatechuate, gentisate, catechol, 2,3-dihydroxybenzoaie, 3,6- dihydroxybenzoate, or 3-hydroxy-2-aminobenzoate (e.g., by ca talyzing the formation of the competitive inhibitor or by catalyzing the formation of a precursor to the competitive inhibitor). Examples of pathways to produce competitive inhibitors of HCT are shown in Figure 27.

[0109J As a non-limiting example, in some embodiments, the competitive inhibitor of HCT is protocatechuate. As shown in Figure 27, protocatechuate can be produced by the enzyme dehydroshikimate dehydratase (QsuB) or by the enzyme dehydroshikimate dehydratase

(DsDH). In some embodiments, the protein that produces a competitive inhibitor of HCT is a Corynebacterium glutamicum dehydryoshikimate dehydratase (QsuB) having the amino acid sequence set forth in SEQ ID NO:6 or a Podospora anserina dehydryoshikimate dehydratase (DsDH) having the amino acid sequence set forth in SEQ ID NO:8. in some embodiments, the protein is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least

65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence of SEQ ID NO:6 or SEQ ID NQ:8. In some embodiments, the protein is a homolog of a Corynebacterium glutamicum dehydryoshikimate dehydratase (QsuB) having the amino acid sequence set forth in SEQ ID NO:6 or a homolog of the Podospora anserina dehydryoshikimate dehydratase (DsDH) having the amino acid sequence set forth in SEQ ID NO:8, In some embodiments, a polynucleotide encoding the dehydryoshikimate dehydratase comprises a polynucleotide sequence that is identical or substantially identical (e.g., at least 50%. at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at feast 98%, or at least 99% identical) to SEQ ID NO:5 or SEQ ID NO:7. B. Plastidial Expression of Proteins

[0110} In some embodiments, the protein thai diverts a monolignoi precursor from a lignin biosynthesis pathway as described herein is expressed in one or more specific organelles of the plant, e.g. , in the plastid of the plant. The polynucleotide sequence encoding the protein that diverts a monoiignoJ precursor from a lignin biosynthesis pathway (e.g., a polynucleotide encoding sbikimate kinase (AroK), pentafunctional AROM polypeptide (AROl), dehydroshikimaie dehydratase (DsDH), debydroshikimate dehydratase (QsuR),

phenylacetaldehyde synthase (PAAS), or phenylalanine aminomutase (PAM), e.g., a polynucleotide comprising a sequence that is identical or substantially identical to a polynucleotide sequence of SEQ ID O: l , 3, 5, 7, or 9, or a polynucleotide comprising a sequence that encodes a polypeptide is identical or substantially identical to an amino acid sequence of SEQ ID NO:2, 4, 6, 8. 10, or 29) can be engineered to include a sequence that encodes a targeting or transit signal for the organelle, e.g., a targeting or transit signal for the plastid. Targeting or transit signals act by facilitating transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid, and mitochondrial membranes.

[0111} In some embodiments, the plastid targeting signal is a targeting signal described in US Patent No. 5, 510,471 , incorporated by reference herein . in some embodiments, the plastid targeting signal is identical or substantially identical (e.g., at least 50%, at least 55 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to an amino acid sequence of SEQ ID NO: 16. In some embodiments, the plastid targeting signal is identical or substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least p%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to a polynucleotide sequence of SEQ ID NO: 15. in some embodiments, the organelle targeting signal (e.g., the plastid targeting signal) is linked in-frame with the coding sequence for the protein that diverts a monolignoi precursor from a l ignin biosynthesis pathway.

C. Promoters

01I2] In some embodiments, the polynucleotide encoding the protein that diverts a monolignoi precursor from the lignin biosynthesis pathway, or the protein that produces a competitive inhibitor of HCT, is operably linked to a heterologous promoter, in some embodiments, the promoter is a cell- or tissue-specific promoter as described below. In some embodiments, the promoter is from a gene in the lignin biosynthesis pathway (e.g., a promoter from a gene expressed in the pathway shown in Figure i). In some embodiments, the promoter is from a gene in the iignin biosynthesis pathway, with the proviso that the promoter is not the native promoter of the polynucleotide encoding the protein that diverts a monolignol precursor from the Iignin biosynthesis pathway or the native promoter of the polynucleotide encoding the protein that produces a competitive inhibitor of HCT to be expressed in the plant. In some embodiments, the promoter is a C4H, C3H, HCT, CCR1, CAD4, CADS, F5H, PALI , PAL2, 4CL1 , or CCoAMT promoter. In some embodiments, the promoter is identical or substantially identical to a polynucleotide sequence of any of SEQ ID Os: 1 , 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28.

Cell- or Tissue-Speciilc Promoters

[0113] In some embodiments, the polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway, or the protein that produces a competitive inhibitor of HCT, is operably linked to a tissue-specific or cell-specific promoter. In some embodiments, the promoter is a secondary cell wall-specific promoter or a fiber cell- specific promoter. The secondary cell wall-specific promoter is heterologous to the polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway, e.g., the promoter and the promoter coding sequence are derived from two different species. A promoter is suitable for use as a secondary cell wall-specific promoter if the promoter is expressed strongly in the secondary cell wall, e.g. , in vessel and fiber ceils of the plant, but is expressed at a much lower level or not expressed in cells without the secondary ceil wall. A promoter is suitable for use as a fiber cell-specific promoter if the promoter is expressed strongly in fiber cells as compared to other non-fiber cells of the plant. [0114] In some embodiments, the promoter is an IRX5 promoter, IRX5 is a gene encoding a secondary cell wall cellulose synthase Cesa4 1 IRX5, (Genbank Accession No.

AF458083_1 ). In some embodiments, the promoter is identical or substantially identical to the pIRX5 polynucleotide sequence of SEQ ID NO: 17.

[0115] Secondary cell wall-specific promoters are also described in the ait. See, for example, Mitsuda et al, Plant Ceil 17:2993-3006 (2005); Mitsuda et al, Plant Cell 19:270- 280 (2007); and Ohtani et al, Plant Journal 67:499-512 (203 i).

[0116] It will be appreciated by one of skill in the art that a promoter region can tolerate considerable variation without diminution of activity. Thus, in some embodiments, a promoter (e.g., a promoter from the lignin biosynthesis pathway, a secondary cell wall- specific promoter, or a fiber cell-specific promoter) is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to a polynucleotide sequence of any of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. The effectiveness of a promoter may be confirmed using a reporter gene (e.g., β-glueuronidase or GUS) assay known in the art.

D. Preparation of Recombinant Expression Vectors

[0117] Once the promoter sequence and the coding sequence for the gene of interest (e.g., coding for a protein that diverts a monolignol precursor from the lignin biosynthesis pathway) are obtained, the sequences can be used to prepare an expression cassette for expressing the gene of interest in a transgenic plant. Typically, plant transformation vectors include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant transformation vectors may also contain a promoter (e.g., a secondary cell wall- specific promoter or fiber cell-specific promoter as described herein), a transcription initiation start site, an RNA processing signal (such as intrors splice sites), a transcription termination site, and/or a polyadenylation signal, [0118] The plant expression vectors may include RNA processing signals that may be positioned within, upstream, or downstream of the coding sequence. In addition, the expression vectors may include regulatory sequences from the 3'-untranslated region of plant genes, e.g., a 3' terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3' terminator regions. [0119] Plant expression vectors routinely also include dominant selectable marker genes to allow for the ready selection of transform ants. Such genes include those encoding antibiotic resistance genes (e.g. , resistance to hygroraycin, kanamycin, bleomycin, G41 8, streptomycin or spectinomycin), herbicide resistance genes (e.g., phosphinothricin acetyitransierase), and genes encoding positive selection enzymes (e.g. mannose isomerase). [0120] Once an expression cassette comprising a polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway and operably linked to a promoter as described herein has been constructed, standard techniques may be used to introduce the polynucleotide into a plant in order to modify gene expression. See, e.g., protocols described in Ammtrato et al, ( 1984) Handbook of Plant Cell Cuiture-Crop Species. Macmillan Publ. Co. Shimamoto et ai. (1989) Nature 338:274-276; Froram et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.

[0121] Transformation and regeneration of plants are known in the art, and the selection of the most appropriate transformation technique will be determined by the practitioner.

Suitable methods may include, hut are not limited to: eiectroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacteriwn titmeficiem mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence. Examples of these methods in various plants include: U.S. Pat. Nos. 5,571,706; 5,677,1 75; 5,510,471; 5,750,386; 5,597,945; 5,589,61 5; 5,750,871 ; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

[0122] Following transformation, plants can be selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants or the ability to grow on a specific substrate, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic, herbicide, or substrate.

[0123] The polynucleotides coding for a protein that diverts a monolignol precursor from the lignin biosynthesis pathway, as well as the polynucleotides comprising promoter sequences for secondary cell wall-specific promoters or fiber cell-specific promoters, can be obtained according to any method known in the art. Such methods can involve amplification reactions such as PGR and other hybridization-based reactions or can be directly synthesized.

E. Plants m Which Lignin Content Can Be Reduced

[0124] An expression cassette comprising a polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway and operably linked to a promoter, or comprising a polynucleotide encoding the protein that produces a competitive inhibitor of HCT and operably linked to a promoter, as described herein, can be expressed in various kinds of plants. The plant may be a m onocotyledonous plant or a dicotyledonous plant. In some embodiments of the invention, the plant is a green field plant. In some embodiments, the plant is a gyrnnosperm or conifer.

[0125] In some embodiments, the plant is a plant that is suitable for generating faiomass. Examples of suitable plants include, but are not limited to, Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, Jatropha, and

Brachypodium.

[0126] In some embodiments, the plant into which the expression cassette is introduced is the same species of plant as the promoter and/or as the polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway or encoding the protein that produces a competitive inhibitor of HCT (e.g., a polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway and a secondary ceil wall-specific or fiber cell-specific promoter from Arabidopsis is expressed in an Arabidopsis plant), in some embodiments, the plant into which the expression cassette is introduced is a different species of plant than the promoter and/or than the polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway (e.g., a polynucleotide encoding the protein that diverts a monolignol precursor from the lignin biosynthesis pathway and/or a secondary cell wall-specific or fiber eeiS-specific promoter from Arabidopsis is expressed in a poplar plant). See, e.g., McCarthy et ah, Plant Cell Physiol 51 : 1084-90 (2010); and Zhong et ah, Plant Physiol. 152: 1044-55 (2010).

F. Screening for Plants Having Reduced Lignin Content

[0127] After transformed plants are selected, the plants or parts of the plants can be evaluated to determine whether expression of the protein that diverts a monolignol precursor from the lignin biosynthesis pathway, or expression of the protein that produces a competitive inhibitor of HC T, e.g., under the control of a secondary ceil wall-specific promoter or a fiber cell-specific promoter, can be detected, e.g., by evaluating the level ofRNA or protein, by measuring enzymatic activity of the protein, and/or by evaluating the size, molecular weight, content, or degree of branching in the lignin molecules found in the plants. These analyses can be performed using any number of methods known in the art.

[0128] In some embodiments, plants are screened by evaluating the level of RNA or protein. Methods of measuring RNA expression are kno wn in the art and include, for example, PGR, northern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), and microarrays. Methods of measuring protein levels are also known in the art and include, for example, mass spectroscopy or antibody-based techniques such as ELISA, Western blotting, ^" flow cytometry, immunofluorescence, and immunohistochemistry.

[0129] In some embodiments, plants are screened by assessing for activity of the protein being expressed, and also by evaluating lignin size and composition. Enzymatic assays for the proteins described herein (e.g., shikimate kinase (AroK), pentafunctionai AROM polypeptide (AROl ), dehydroshikimate dehydratase (DsDH), dehydroshikimate dehydratase (QsuB), phenylacetaldehyde synthase (PAAS), phenylalanine aminomutase (PAM), p- coumarate/cinnamate carboxy!methitransferase (CCMTl ), ferulic acid decarboxylase (FDC 1 ), phenylacrylic acid decarboxylase (PDC 1 ), 2-oxoglutarate-dependent dioxygenase (C2'H), chalcone synthase (CHS), stilbene synthase (SPS), cucuminoid synthase (CUS), or benzalacetone (BAS)) are well known in the art, Lignin molecules can be assessed, for example, by nuclear magnetic resonance (NMR), spectrophotometry, microscopy, klason lignin assays, thioacidolysis, acetyl-bromide reagent or by histochemicai staining (e.g., with phloroglucinol).

[0130| As a non-limiting example, any of several methods known in the art can be used for quantification and/or composition analysis of lignin in a plant or plant part as described herein. Lignin content can be determined from extract free cell wall residues using acetyl bromide or Klason methods. See, e.g., Eudes et al, Plant Biotech. J. 1 :609-620 (2012); Yang et al, Plant Biotech J. (2013) (in press); and Dence et al (eds) Lignin determination. Berlin: SpringerVerlag (1992); each of which is incorporated by reference herein. Extract free cell wall residues correspond to raw biomass, which has been extensively washed to remove the ethanol soluble component. Eudes et al, Plant Biotech. J. } 0:609-620 (2012); Yang et al, Plant Biotech. J. (2013) (in press); Sluiter et al, Determination of structural carbohydrates and lignin in biomass. in: Laboratory Analytical Procedure. National

Renewable Energy Laboratory, Golden, CO, USA; and Kim et al, Bio. Res. 1 :56-66 (2008). Lignin composition analysis and G/S lignin subunit determination can be performed using any of various techniques known in the art such as 2D 13C-H1 HSQC NMR spectroscopy (Kim and Ralph, Org. Biomol Chens. 8:576-591 (2010); Kim et al., Bio. Res. i :56-66 (2008)); thioacidolysis method (Lapierre et al., Plant Physiol 1 19: 153- 164 (1999); Lapierre et aL Res. Chem. intermed. 21 :397-412 (3995); Eudes et al, Plant Biotech. J. 30:609-620 (2012)): derivaiization followed by reductive cleavage method (DFRC method; Lu and Ralph, J. Agr. Food Chem 46:547-552 (1 98) and Lu and Ralph, J. Agr. Food Chem

45:2590-2592 (1 97)) and pyrolysis-gas chromatograph method (Py-GC method; Sonoda et al, Anal Chem. 73 :5429-5435 (2001)) directly from extract free cell wall residues or from cellislolytic enzyme lignin (CEL lignin). CEL lignin derives from cell wall residues, which were hydrolyzed with crude cellulases to deplete the polysaccharide fraction and enrich the lignin one (Eudes et al, Plant Biotech. J. 10:609-620 (2012)). IV. Methods of Using Plants Having Reduced Ligsihi Content

[0131] Plants, parts of plants, or plant biomass material from plants having reduced Signification due to the expression of a protein that diverts a monolignol precursor from the !ignin biosynthesis pathway or due to the expression of a protein that produces a competitive inhibitor of HCT, e.g., under the control of a secondary cell wall-specific promoter or a fiber cell-specific promoter, can be used for a variety of methods. In some embodiments, the plants, parts of plants, or plant biomass material generate less recalcitrant biomass for use in a conversion reaction as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used in a saccharification reaction, e.g., enzymatic saccharification, to generate soluble sugars at an increased level of efficiency as compared to wild-type plants, in some embodiments, the plants, parts of plants, or plant biomass material are used to increase biomass yield or simplify downstream processing for wood industries (such as paper, pulping, and construction) as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase the quality of wood for construction purposes. In some embodiments the plants, parts of plants, or plant biomass material can be used in a combustion reaction, gasification, pyrolysis, or polysaccharide hydrolysis (enzymatic or chemical). In some embodiments, the plants, parts of plants, or plant biomass material are used as feed for animals (e.g., ruminants),

[0132] Methods of conversion, for example biomass gasification, are known in the art. Briefly, in gasification plants or plant biomass material (e.g., leaves and stems) are ground into small particles and enter the gasiiler along with a controlled amount of air or oxygen and steam. The heat and pressure of the reaction break apart the chemical bonds of the biomass, forming syngas, which is subsequently cleaned to remove impurities such as sulfur, mercury, particulates, and trace materials. Syngas can then be converted to products such as ethanol or other biofuels.

[0133] Methods of enzymatic saccharification are also known in the art. Briefly, plants or plant biomass material (e.g., leaves and stems) are optionally pre-treated with hot water, dilute alkaline, AFEX (Ammonia Fiber Explosion), ionic liquid or dilute acid, followed by enzymatic saccharification using a mixture of cell wall hydro!ytic enzymes (such as hemicel!ulases, cellulases and beta-glucosidases) in buffer and incubation of the plants or plant biomass material with the enzymatic mixture. Following incubation, the yield of the saccharification reaction can be readily determined by measuring the amount of reducing sugar released, using a standard method for sugar detection, e.g. the dinttrosalicylic acid method well known to those skilled in the art. Plants engineered in accordance with the invention provide a higher saccharificaton efficiency as compared to wild-type plants, while the plants' growth, development, or disease resistance is not negatively impacted.

EXAMPLES [0134] The following examples are provided to illustrate, but not limited the claimed invention.

Example 1: Strategies for Diverting a Monojignol Precursor from the Lignin

Biosynthesis Pathway

[0135] The engineered plants of the present invention express one or more genes encoding a protein that di verts a precursor component from the lignin biosynthesis pathway (Figure 1 ) to a competitive pathway. This diversion reduces the amount of lignin that is produced and increases the amount of product produced by the competitive pathway.

[0136] Figures 2-1 1 provide exemplary strategies for diverting a precursor component from the lignin biosynthesis pathway, in one strategy (Figures 2 and 3), the monolignol precursor shikimate can be reduced or depleted. For example, the amount of cytosolic and/or plastidial shikimate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a shikimate kinase such as M. tuberculosis shikimate kinase ("MtAro "), a pentafunctional arom protein such as S. cerevisiae pentafunctional arom protein ("ScArol "), a dehydroshikimate dehydratase such as C. glutamicum dehydroshikimate dehydratase ("CgQsuB"), or a P. anserina dehydroshikimate dehydratase ("PaDsDH").

[0137] In another strategy (Figures 4 and 5), the monolignol precursor phenylalanine can be reduced or depleted. For example, the amount of cytosolic and/or plastidial phenylalanine that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a phenylacetaldehyde such as P.hybrida phenylacetaldehyde synthase ("PhPAAS") or a phenylalanine aminomutase such as 7 ^" . canadensis phenylalanine aminomutase ("TcPAM").

[0138] In another strategy (Figures 6 and 7), the monolignol precursors cinnamate and/or p- coumarate are reduced or depleted. For example, the amount of cytosolic cinnamate and/or p- coumarate that is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a cinnamate/p-coumarate carboxyl methyltransferase such as O. basilicum cinnamate/p-coumarate carboxyl methyltransferase ("ObCCMTl ") or a phenylacrylic acid decarboxylase such as P. penlosaceus phenylacrylic decarboxylase ("PDC"). [0139] In another strategy (Figures 8-1 1 ), the monolignol precursors coumaroyl-CoA, caffeoyl-CoA, and/or feruloyl-CoA are reduced or depleted. For example, the amount of cytosoHc coumaroyl-CoA, caffeoyl-CoA, and/or feruloyl-CoA thai is available for the lignin biosynthesis pathway can be reduced or depleted by expressing a 2-oxoglutarate-dependent dioxygenase such as R, graveolens C2'H (2-oxoglutarate-dependent dioxygenase)

("RbC2'H"), a chalcone synthase (CHS), a stilhene synthase (SPS), a cucuminoid synthase (CUS), or a benzalacetone (BAS).

Example 2: Generation οί Tnuaxt enie Limes Expressing Qs¾B or DsDH in pSastids |0140] The promoter (pC4H) of the lignin C4H gene from Arabidopsis was synthesized with flanking Smal and Avril restriction sites at the 3 ^f and 5' ends respectively (Genscript), The encoding sequence of the ehloroplastie targeting signal peptide sequence (ctss; Patent US 5510471 ) was codon optimized and synthesized (Genscript), then amplified by PGR and inserted into the Avril restriction site iocated at the 5' end of pC4H using ln-Fusion cloning (Clontech). The pC4Hctss DNA fusion was then used to replace the IRX5 promoter from pTKan-plRX5 (Eudes et al. Plant Biotechnol J 10, 609-620 (2012)) using Gateway technology (Invitrogen) and to generate a new p ^' Tkan-pC4Hctss-GWR3R2 vector. This vector is designed to clone in-frame with the ctss sequence any gene of interest previously cloned into a pDONR221.P3-P2 vector according to the manufacturer instruction

(Invitrogen). [0141] Codon-optimized nucleotide sequences encoding for the dehydroshikimate dehydratases QsuB from Corynebacterium glutamic m (accession number A4QB63) and DsDH from Podospora anserina (accession number CAD60599) were synthesized for expression in Arabidopsis (Genescript), cloned in pDONR22!.P3~P2 gateway vector according the manufacturer instruction (invitrogen), and transferred into pTkan-pC4Hctss- GWR3R2 by LR clonase reaction (Invitrogen) to generate the pTKan-pC4Hctss-QsuB and pTKan-pC4Hetss-DsDH binary vectors respectively. The in-frame fusions of cttss with QsuB and DsDH encoding sequences were verified by sequencing.

[0142] Both constructs were introduced independently into WT Arabidopsis plants (ecotype ColO) via Agrobacterium iumefaciens-medmted transformation (Bechtold and

Pelletier, Methods Mol Biol 82:259-266 (1998)) and several independent S-QsuB and S-

DsDH lines harboring ctss::QsuB and ctss:;DsDH gene fusions respectively were generated.

Residts [0143] Nine independent lines resistant to kanamycin and therefore harboring the pTKan- pC4Hctss-QsuB construct (S-QsuB lines) were selected and analyzed at the T2 generation. These lines express the dehydroshikimate dehydratase QsuB protein from Corynehacterium glutamicum fused to a piastid targeting signal peptide to address the QsuB protein in their plastids. At the rosette stage (3-week-old), S-QsuB lines were phenotypically

indistinguishable from wild-type (WT) plants {Figure 1 1 ). The biomass from dried senesced stems collected from S-QsuB lines and WT plants was used to perform saccharification analysis. As shown on Figure 12, the amount of reducing sugars released from the biomass of all the S-QsuB lines was higher compared to the amount released from WT plants. In particular, using similar amount of cellulolytic enzyme, the S-QsuB lines #1 , 4, and 9 showed improved saccharification efficiencies of up to 3.0 fold compared to WT plants (Figure 12). Moreover, the amount of reducing sugars released from the biomass of S-QsuB lines (#1, #4, #9) and WT plants using different loadings of cellulolytic enzyme cocktail was investigated. As shown on Figure 13, the saccharification efficiency was on average 75% higher for the three S-QsuB lines although 10 times less enzyme was used compared to WT biomass. This result shows that much less cellulolytic enzyme is required to release similar amount of sugars from the biomass of S-QsuB lines compared to that of WT plants.

[0144] Alternatively, five independent lines resistant to kanamycin and therefore harboring the pTKan-pC4Hctss-DsDH construct (S-DsDH lines) were selected and analyzed at the T2 generation. These lines express the dehydroshikimate dehydratase DsDH protein from

Podospora anserine fused to a piastid targeting signal peptide to address the QsuB protein in their plastids. The biomass from dried senesced stems collected from S-DsDH lines and WT plants was used to perform saccharification analysis. As shown on Figure 14, using identical amount of cellulolytic enzyme, the amount of reducing sugars released over time from the biomass of all the S-DsDH lines was higher compared to the amount released from WT plants, representing an improvement of up to 1.4 fold after 72 h of hydrolysis, Similarly to the S-QsuB lines, this result indicates that the biomass of S-DsDH lines is less recalcitrant to polysaccharide enzymatic digestion compared to WT piants.

Example 3: _. Expression of a bacterial 3-dehydroshildmate dehydratase reduces lignin content and improves biomass saccharification efficiency

ABSTRACT

[0145] Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as a source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene-knockout or gene-silencing techniques to constitutive])' repress one of these metabolic pathways. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynehacterhim glutamicum) reduces iignin deposition in

Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate an intermediate of the shikimate pathway— into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, cinnamate, />- coumarate, 7-coumaraldehyde, and couraaryl alcohol. 2D-NMR spectroscopy, thioacidoiysis, and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase ofp- hydroxyphenyi units and a reduction of guaiacyi units in the lignin of QsuB lines, while size- exclusion chromatography indicates a lower degree of lignin polymerization. Our data show that the expression of QsuB primarily affects one of the key enzymatic steps within the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of- function strategy for spatio-temporal reduction of lignin in plant biomass.

SIGNIFICANCE

[0146] Lignin is a complex aromatic polymer found in plant cells walls that is largely- responsible for the strength and toughness of wood. These properties also confer

"recalcitrance" to biomass, so materials high in lignin content are more difficult to break down in processes such as production of biofuels. Efforts to reduce lignin content through altering plant gene expression often result in reduced biomass yield and compromise plant fitness, in this study, we present an effective alternative strategy: reducing lignin content and biomass recalcitrance through expression of a bacterial 3-dehydroshikimate dehydratase in plants. We demonstrate that this strategy achieved dramatic changes in the lignin composition and structure in transgenic plants, as well as improved conversion of biomass into fermentable sugars.

INTRODUCTION

[0147] Plant cells walls are the primary source of terrestrial biomass and mainly consist of cellulosic and hemiceilulosic polysaccharides impregnated with lignins. Lignins are polymers of p-hydroxycinnamyl alcohols {i. e., monolignols), which are synthesized inside the ceils, exported to the cell wall, and ultimately undergo oxidative polymerization via laccase and peroxidase activities. The main monolignols p-coumaryl, coniferyl, and sinapyl alcohols — give rise to the p~hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units, respectively (1 ). Lignification generally confers mechanical strength and hydrophobic ity in tissues that develop secondary cell walls, such as sclerenchyma (i.e., fibers) and xyiem vessels. In addition to its essential role for upright growth, lignin also serves as a physical barrier against pathogens that degrade cell walls (2).

[0148] Lignocellulosic biomass is used for pulp and paper manufacture, ruminant livestock feeding, and more recently has been considered an important source of simple sugars for fermentative production of intermediate or specialty chemicals and biofuels (3). It is well- documented that lignin in plant biomass negatively affects pu!p yield, forage digestibility, and polysaccharide saccharificatton (4-6). This has prompted major interest in developing a better understanding of lignin biosynthesis to reduce biomass recalcitrance by modifying lignin content and/or composition.

10149] The shikimate pathway, which is located in plastids in plants, provides a carbon skeleton for the synthesis of phenylalanine, the precursor of the cytosolic phenylpropanoid pathway responsible for the biosynthesis of monolignols (Fig. 20). Ail the metabolic steps and corresponding enzymes for both pathways are known and well-conserved across land plants (7-10). Classic approaches to lignin reduction have relied on genetic modifications, such as transcript reduction and allelic variation of specific genes from the phenylpropanoid pathway (1 1 , 12). However, these strategies often result in undesired phenotypes— including dwarfism, sterility, and increased susceptibly to environmental stresses due to loss of cell- wall integrity, depletion of other phenylpropanoid-related metabolites, accumulation of pathway intermediates, or the constitutive activation of defense responses (13, 14). Such negative effects are unfortunately difficult to avoid because of the non-tissue specificity of the strategies employed: allelic variations are transmitted to every cell of the plant during cell divisions, and small interfering RNAs generated for gene silencing generally move from cell- to-cell and over long distance in vegetative tissues (15).

[0150] Alternatively, there are novel and promising gain-of-function strategies that involve expression of specific proteins to reduce the production of the three main monolignols or change their ratios. Using specific promoters with restricted expression patterns, these strategies would enable the alteration of lignin at later developmental stages or, for example, only in certain tissues such as fibers— without compromising the functionality of conductive vessels for the transport of water (14). Examples of such expressed proteins are transcription factors that act as negative regulators of lignin biosynthesis (16- 19); enzymes that use intermediates of the lignin pathway for the synthesis of derived metabolites (20-22); engineered enzymes that modify monolignols into their non-oxidizable forms (23): or proteins that mediate the post-transcriptional degradation of enzymes from the Hgnin biosynthetic pathway (24).

[01511 in this study, we report for the first time on the expression of a bacterial 3- dehydroshikimate dehydratase in Arabidopsis (25). We selected QsuB from C. glutamicum and targeted it to the piastids to convert the shikimate precursor 3-dehydroshikiraate into protocatechuate, with the aim of reducing iignin content and modifying its composition and structure in the biomass of transgenic lines. Metabolomic analysis of plants expressing QsuB revealed higher amounts of cinnamate, >-coumarate, and of the two direct precursors of H- Iignin units: j-couniaraidehyde and j?-coumaryl alcohol. Conversely, the direct precursors of G and S units— coniferaldehyde, coniferyl alcohol, sinapaldehyde, and sinapyl alcohol— were reduced, Lignin content was severely reduced in these transgenic lines and exhibited an enrichment of H units at the expense of G units and a lower polymerization degree.

Compared to those of wild-type plants, cell walls from lines expressing QsuB released significantly higher amounts of simple sugars after ceiiulase treatment and required less enzyme for saccharifi cation. Collectively, these results support the hypothesis that expression of a plastidic QsuB affects one of the enzymatic steps within the lignin biosynthetic pathway.

RESULTS

Targeted expression of QsuB in Arabidopsis

[0152] A sequence encoding QsuB was cloned downstream of the sequence encoding for a plastid-targeting signal peptide (SCHL) for expression in piastids. Using transient expression in tobacco, we first confirmed that QsuB was correctly targeted to the piastids by analyzing its subcellular localization when fused at the C-terminus to a YFP marker (Fig. 21). The schl- qsuB sequence was cloned downstream of the Arabidopsis C4H promo ter for expression in lignifying tissues of Arabidopsis . Western blot analysis confirmed that QsuB was expressed in stems of several T2 plants homozygous for the pC4H::schl::qsuB construct (Fig. 16 ). Based on the migration of molecular weight markers, QsuB was detected at around 70 kDa, which corresponds to the theoretical size of its native sequence after cleavage of the chloroplast transit peptide (Fig. 16). Five lines with different QsuB expression levels (C4H::qsuB~l , -3, -6, - 7, and -9) were selected for biomass measurement. Although a height reduction was observed for these Sines, only two of them C4H::qs B-l and -9) showed a slight decrease of biomass yield (stem dry weight) by 18% and 21 %, respectively (Table I ). Table 1. Height and dry weight of the main inflorescence stens of ^' senesced mature type (WT) and pC4H::schl::q$uB (C4H::qsuB) plants.

Metabolite analysis of C4H::qsuB lines

[0153] Methanol soluble metabolites from stems of the C4H::qs B~l and C4H::qsuB-9 lines were extracted for analysis (Table 2, Fig. 22). Compared to wild-type plants, protocatechuate content was increased 53- and 485-fold in those two transgenic lines, respectively. However, except for tyrosine in line C4H::qsuB-9, no significant reduction was observed for the content of several metabolites derived from the shikimate pathway in piastids such as salicylate and aromatic amino acids. Instead, salicylate was slightly increased, 1.3-1.4-fold, in both lines and phenylalanine was 1 .6-fold higher in line

C4H::qsuB-L Interestingly, several metabolites from the phenylpropanoid pathway were increased in the transgenic lines. Cinnamate and -coumaraldehyde were detected only in transgenic lines; while j-cournaraie and j-coumaryl alcohol contents were increased, compared to those of wild type, 14-18-fold and 3.5-30- fold, respectively. Kaempferol and qnercetin, two flavonols derived from ju-eoumaroyl-CoA, were also found in higher amounts in both C4H;:qsuB lines. The direct precursors of G- and S-lignin units were negatively altered; coniferaldehyde was reduced -40% in both transgenic lines, while coniferyl alcohol, sinapaldehyde, and sinapyl alcohol were decreased twofold in C4H::qsuB-9 (Table 2),

[0154] Cell wall-bound metabolites released from cell wail residues by mild alkaline hydrolysis were also analyzed (Table 3). Protocatechuate was found in cell walls of the C4H::qsv.B lines but not in those from wild-type plants. The content of >-coumarate was significantly increased in line C4H::qsuB~l, whereas ferulate was reduced in both transgenic lines.

Table 2. Quantitative analysis of methanol-soluhle metabolites in stems from 6-wk-old wild-type (WT) and pC4H::schl::qsuB (C4H::qsuB) plants.

I ! Mean ± SE

a (}ig/g fresh weight)

β (ug g fresh weight)

φ Using a detection limit of 34 ng/g fresh weight

Values are means of four biological replicates (n = 4). nd, not detected. Asterisks indicate significant differences from the wild type using the unpaired Student's t-test (*P < 0.1 : **P < 0.05; ***P < 0.005; ****P < 0.001).

Table 3. Quantitative analysis of cell wall-bound aroinatks in stems from extractive-free senesced mature wild-type (WT) and pC4H::schI;:qsuB {C4H::qsuB) plants.

Values are means of four biological replicates (« = 3). nd, not detected. Asterisks significant differences from the wild type using the unpaired Student's t-test (*P < 0.05; **P < 0.005; * * * P < 0.001).

Compositional analysis of cell wall from C4H::qsuB lines

[0155] Using the Klason method, the lignin content measured in the stem of lines

C4H::qsuB-l and C4H::qsuB-9 was reduced 50% and 64%, respectively, compared to that of wild type (Table 4). Analysis of the cell-wall monosaccharide composition showed higher amounts of glucose (+ 4-10%), xylose (+ 13- 19%), and other less abundant sugars in the transgenic lines, resulting in 8% increase in total cell-wall sugars for the C4H::qsuB-l line and an 1 1% increase for C4H::qs B-9 line (Table 4). Table 4. Chemical composition of senesced mature stems from wild-type (WT) and pC4H::$chl:;qsuB (C4H::qsuB) plants.

Values are means ± SE of triplicate analyses (n = 3). Asterisks indicate significant differences from the wild type using the unpaired Student's t-test (*P < 0.05; **P < 0.005).

Lignin menomeric composition and structure in C4H: qsuB lines

Θ156] Determination of the lignin monomer composition, using thioacidolysis, indicated an increase in the relative amount of H units in transgenic lines. H units represented 12.7% and 27.9% of the total lignin monomers in lines C4H::qsuB~l and C4H::qsuB-9, which corresponds to 21 - and 46-fold increases compared to that of wild type, respectively (Table 5). The relative amount of G units in transgenics (-45%) was also reduced compared to wild type (~64%), whereas 8 units were higher in C4H::qsiiB~l and lower in C4H::qsuB-9 (Table 5).

[0157] NMR (2D ^- H-correiated, HSQC) spectra of cell-wall material from C4H::qsuB- 1 and C4H::qsuB-9 lines were also obtained for determination of lignin composition and structure. Analysis of the aromatic region of the spectra confirmed the higher relative amount of H units in both C4H::qsuB lines (29% and 64.4% respectively) compared to that in wild type (3.6%), as well as a reduction of G units (Fig. 17). Moreover, analysis of the aliphatic region of the spectra indicated a strong reduction of phenylcoumaran (β-5) and resinol (β-β) linkages in the lignin of the transgenic lines (Fig. 23).

[0158] Finally, cell-wall material from stems of wild-type and C4H::qsuB lines were analyzed by pyro-GC/MS. For each line, identification and relative quantification of the pyrolysis products derived from H. G, or S units allowed determination of H/G/S ratios (Figure 28). Compared to wild type, H units were increased 3.5- and 10- fold, and G units were reduced 1.4- and 2.2-fold, in lines C4H::qsuB-l and C4H::qsuB~9, respectively. Table 5. Main H, G, and S !ignin-derived monomers obtained by thioacidoiysis of extractive-free senesced mature stems from wild-type (WT) and pC4H::schl::q$uB {C4H::qs E) plants.

differences from the wild type using the unpaired Student's t-test (*P < 0.05; **P < 0.01).

Ligniiis from C4H::qsuB lines have a lower polymerization degree

[0159] Lignin fractions were isolated from wild-type and C4H::qsnB lines for analysis of their polydispersity using size-exclusion chromatography (SEC). Elution profiles acquired by monitoring UV-F fluorescence of the dissolved iignin revealed differences between wild-type and transgenic lines (Fig. 18). The total area of the three mass peaks, corresponding to the largest lignin fragments detected between 7.8 min and 12.5 min, was significantly reduced in C4H::qsuB lines compared to wild type. Similarly, intermediate molecular mass material, which eiutes in a fourth peak between 12.5 min and 1 8 min, was also less abundant in C4H::qsuB lines. Conversely, the area corresponding to the smallest lignin fragments.

detected between 18 min and 23.5 min, was increased in the transgenic lines. These results demonstrate a reduction in the degree of polymerization of lignins purified from plants expressing QsuB compared to that of wild type.

Biomass from C4H::qsuB lines shows improved saccharification

[0160] Saccharifieation assays on stem material were conducted to evaluate the cell-wall recalcitrance of the C4H::qsuB lines. As shown in Fig. 19 A, higher amounts of sugars were released after 72 hr enzymatic hydrolysis of biomass from the C4H::qsuB lines (-1 , -3, -6, -7 and -9) compared to those of wild type in all pretreatments tested. Saccharifieation improvements ranged between 79-330% after hot water; 63-104% after dilute alkali; and 26- 40% after dilute acid pretreatments (Fig. 19A). Moreover, similar saccharifieation experiments using hot water pretreated biomass, at 5x lower cellulase loadings, revealed that biomass from ail C4H::qsuB lines releases more sugar than that of wild type hydro!yzed with a typical enzyme loading (Fig, 19B). Taken together, these data demonstrate that cellulose from the C4H::qsvB lines is less recalcitrant to cellulase digestion and requires a lower amount of enzyme to be converted into high yields of fermentable sugars. DISCUSSION

[0161] Gain-of-function strategies have several advantages for the manipulation of metabo!ic pathways, For example, they can be used to bioengineer lignin deposition in plants via better spatio-temporal control of monolignol production in iignifying cells, and to adjust lignin composition and its biophysical properties (26). Therefore, identification of proteins in which in planta-expr&ssioti results in modifications of lignin content or composition is of particular interest and presents novel opportunities. In this work, we demonstrate that expression of the 3-de3iydroshikimate dehydratase QsuB in piastids leads to drastic reduction and compositional changes of lignin in Arabidopsis (Table 4). As a result, biomass from these transgenic plants exhibits much higher saecharification efficiency after pretreatment (Fig. 19A), which is a highly desired trait for several agro-industries and the bioenergy sector. Moreover, the efficiency of this approach to decrease lignin content in plant biomass allows a reduction of hydrolytic enzyme loadings by at least five-fold, while retaining greater saecharification potential than control plants hydrolyzed at standard enzyme loading (Fig. 19B). Consequently, the transfer of this technology to energy crops should have a great impact on the cost-effectiveness of eeliulosic biofuels production, since enzyme cost is the major barrier in this process (27).

[01621 In this study, as a proof of concept, we used the promoter of the AtC4H gene to ensure strong QsuB expression in all iignifying tissues of the plant. This resulted in a slight decrease of plant height for all the lines; but no significant reductions in biomass yield except for that of two transgenic lines, which expressed QsuB very strongly (Table 1 ; Fig. 16) and exhibited— in some stem transverse sections (Fig. 24)— evidence of vessel collapse that could impair xylem conductivity (14). Nevertheless, our strategy offers the potential to overcome these defects by selecting more stringent promoters (e.g., fiber-specific) that would exclude QsuB expression from xylem-conductive elements (26, 28). Moreover, translation of our technology from model plant to crops is expected to be straightforward: it is based solely on the expression of QsuB, does not require any particular genetic backgrounds, and the lignin and shikimate pathways are well-conserved among vascular plants.

{0163] A direct consequence of QsuB expression is the accumulation of protocatechuate in the biomass of transgenic plants (~I % dry weight in line C4H::qsuB-9; Table 2). Considering the beneficial properties of protocatechuate in the bio-based polymer industry and human health sector, such de novo production adds extra commercial value to the biomass of plants expressing QsuB (29, 30). Much higher amounts of protocatechuate were recovered after acid treatment of the methanoi-soluble extracts from transgenic plants (data not shown), which suggests its conjugation in the cytosol after export from the plastids. interestingly. QsuB expression did not affect substantially the level of metabolites derived from the shikimate pathway, such as aromatic amino acids and salicylate, suggesting that p!astidic 3- dehydroshikimate is not limiting (Table 2). On the other hand, a buildup of cinnamate and p- coumarate was observed in these lines, accompanied by an accumulation ofp- coumaraldehyde and />-coumaryl alcohol pools (Table 2 and Fig, 22).

[0164] Analysis of the lignin monomeric composition— using 2D NMR spectroscopy, thioacidolysis, and ym-GC/MS— unequivocally demonstrated an increase in H units in plants expressing QsuB (Fig. 17 and Fig. 28; Table 5). These data could explain the reduced degree of polymerization of these lignins, which has been previously observed in various lignin mutants that exhibit high content of H units, incorporation of which typically slows or stops iignin-ehain elongation (3 1 , 32: Fig. 18). Therefore, reduced lignin-polysaccharide crosslinking within the biornass of the transgenic lines is expected, and this could contribute to its superior enzymatic digestibility. [0165] A low lignin content rich in H-units corresponds to a phenotype previously characterized in plants down-regulated for hydroxyciniiamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), /?- coumarate 3-hydroxylase (C3H), or caffeoyl shikimate esterase (CSE). This suggests that an alteration of these biosynthetic steps has occurred in the C4H::qsuB lines (10, 32, 33). A possible explanation is that QsuB activity in plastids affects the export of shikimate from the plastids to the cytosol. This would indirectly limit the availability of cytosolic shikimate used for the enzymatic step catalyzed by HCT. The distribution of shikimate between plastids and the cytosol is still poorly understood, and shikimate levels were below the detection limit in our stem extracts from wild-type and transgenic plants. Alternatively, because previous studies reported a substrate flexibility- of HCTs (34, 35), the large accumulation of protocbatechuate could act as inhibitor of AtHCT, which couples >-coumaroyl-CoA and shikimate. Using an in vivo enzymatic assay to determine the substrate preference of AtHCT, we confirmed its affinity for/>~coumaroyl-CoA and shikimate, but also demonstrated its capacity to accept protocatechuate and several other substrates such as catechol, 3,6-dihydroxybenzoate, 3-hydroxy-2-aminobenzoate, and 2,3- dihydroxybenzoate (Fig. 25). Therefore, we cannot exclude the possibility that the protocatechuate pool accumulated in C4H::qs B plants exerts a competitive inhibition of HCT and limits the synthesis of coumaroyl shikimate required for the production of G- and S-Hgnin units. MA TE RIALS AND METHODS

Plant material and growth conditions

[0166] Arahidopsis thaiiana (ecotype Columbia, Col-0) seeds were germinated directly on soil. Growing conditions were 150 ,umol/m ² /s, 22 °C, 60% humidity, and 30 h of light per day for the first 4-5 wk, followed by 14 h of light per day until senescence. Selection of Tl and T2 transgenic plants was made on Murashige and Skoog vitamin medium

(Phyto Technology Laboratories, Shawnee Mission, KS), supplemented with 1 % sucrose, 1.5% agar, and 50 fig/niL kanamycin.

Generation of binary vectors

[0167] The promoter p35S, with a single enhancer, was amplified by PGR from p ^' RTl OO with phosphorylated primers F-p35S (5 '-GTCA ACATGGTGGAGCACGACAC-3 ') and R- p35S (5 '-CGAGAATCTAGATTGTCCTCTCCAA ATGAAATGAACTTC-3 '), and cloned into a S/wal-digested dephosphorylated pTkan vector (36) to generate a pTKan-pJ5,S' vector. Subsequently, a GW-YFP cassette was extracted from the pX-YFP vector (37) hy XhoU ^' Spel digestion, and ligated into a pTKm-p35S vector to generate the pTkan- p 35S-G WR 3 R2-YFP vector.

[0168] A chimeric DNA construct was synthesized (GenScript, Piscatway, Ν.Ϊ): it was flanked by the gateway sequences attB4r (5 '-end) and attB3r (3 '-end), and contained, in the following order, the iG7 terminator; the restriction sites Stria), Kpnl, Hind]]] and Xho a 2.9- Kb sequence corresponding to the Arahidopsis C4H promoter (pC4H); and a sequence encoding a piastid targeting signal (SCHL; 38). This attB4r-/G7-pC H-sc ?/-attB3r construct was then subcloned into the Gateway pDONR221 -P4rP3r entry vector by BP recombination (Life technologies, Foster City, CA, USA) to generate pENTR-L4-tG7- ? 4'//-sc,¾/-L3. An LR recombination reaction was performed with pTkan-p/ ? J-GW (21 ), pENTR-Ll- »Xac- lacZalpha-1,4 (Life technologies, Foster City, CA, USA), pENTR-L3-p/,<ac-Tet-L2 (Life technologies, Foster City, CA, USA), and pENTR-L4-/G 7-pC4H: :schl-L3. The obtained construct was subsequently digested by Smal to remove the /?Z c-lacZalpha and tG7 fragments. The pLac-Tet fragment was replaced by the gateway cassette using BP recombination to generate the pTKm-pC4H: :J?CM-GWR3R2 vector. Generation of a pTkim-pC4ii :schi~qsuB p!asmid and plant transformation

[0169] A gene sequence encoding QsuB from C. ghUamicum (GenBank accession number YP_001 137362.1 ) without stop codon and flanked with the Gateway attB3 (5'-end) and attBl (3 '-end) recombination sites was synthesized for expression in Arahidopsis (GenScript, Piseatway, NJ) and cloned into the Gateway pDONR221 -P3P2 entry vector by BP

recombination (Life technologies, Foster City, CA, USA), A sequence-verified entry clone was LR recombined with the pTKan-/>C¥H:, ^* scA/-GWR3R2 vector to generate the pTKan- pC4H::schl-qsuB construct, which was introduced into wild-type Arabidopsis plants (ecotype Col-0) via Agrobacterium-medlated transformation (39).

Westers blot analysis

[0170] Proteins from Arabidopsis stems were extracted using a buffer containing 250 mM Tris-HCl pH 8.5, 25 mM EDTA, 2 mM DTT, 5 mM β-mercaptoethanol, and 10% sucrose; and were quantified using the Bradford method (40). Proteins (15 μg) were separated by SDS-PAGE, blotted, and immunodetected using a universal antibody, as previously described (41 ).

Methanol-soluble metabolites extraction

[0171] Arabidopsis stems of 6-wk-old wild-type and transgenic lines were collected in liquid nitrogen and stored at -80 °C until further utilization. Prior the metabolite extraction, collected stems were pulverized in liquid nitrogen. For extraction of methanol-soluble metabolites, 700-1 ,000 mg of frozen stem powder was mixed with 2 ml of 80% (v/v) methanol-water and mixed (1 ,400 rpm) for 15 min at 70 °C. This step was repeated four times. Pooled extracts were cleared by centrifugation (5 min, 20,000 x g, at room

temperature), mixed with 4 mL of analytical grade water and filtered using Amicon Ultra centrifugal filters (10,000 Da MW cutoff regenerated cellulose membrane; EMD Millipore, Billerica, MA). Filtered extracts were lyophiiized and the resulting pellets dissolved in 50% (v/v) methanol-water prior to LC-MS ana lysis. An acid-hydrolysis of the samples was performed for the quantification of protocatechuate, salicylate, and flavonols; an aliquot of the filtered extracts was dried under vacuum, resuspended with 1 N HCI and incubated at 95 °C for 3 h. The mixture was subjected to three ethyl acetate partitioning steps, Ethyl acetate fractions were pooled, dried in vacuo, and resuspended in 50% (v/v) methanol-water prior to LC-MS analysis.

Cell-wall bound aromatics extraction

[0172] Senesced stems were ball-milled using a Mixer Mill MM 400 (Retsch Inc., Newtown, PA) and stainless steel balls for 2 min at 30 s ^{' !} . Extractive-free cell-wall residues (C WR) were obtained by sequentially washing 60 mg of ball-milled stems with 1 mL of 96% ethanol at 95 °C twice for 30 min and mixing with 1 mL of 70% ethanol twice for 30 sec. The resulting CWR were dried in vacuo overnight at 30 °C. The C WR (6 mg) were mixed with 500 μΡ of 2 M NaOH and shaken at 1 ,400 rpm for 24 h at 30 °C. The mixture was acidified with 1 00 μί., of concentrated HCl, and subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo, and suspended in 50% (v/v) methanol- water prior to LC-MS analysis. LC-MS analysis

[0173] As previously described in Bokinsky et al. (42) and Eudes et al. (43)— aromatic amino acids, and aromatic acids and aldehydes, respectively— were analyzed using high- performance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of- flight (TOP) mass spectrometry (MS). Aromatic alcohols were analyzed by HPLC— atmospheric pressure chemical ionization (APCI)— TOP MS. Their separation was conducted on an

Agilent 1200 Series Rapid Resolution HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) using a Phenomenex Kinetex XB-C 18 (100 mm length, 2.1 mm interna! diameter, and 2.6 μπι particle size: Phenomenex, T orrance, CA, USA). The mobile phase was composed of 0.1% formic acid in water (solvent A) and methanol (solvent B). The elution gradient was as follows: from 5%B to 25%B for 6 min, 25%B to 5%B for 1 min, and held at 5%B for a further 3 min. A flow rate of 0.5 mL/min was used throughout. The column compartment and sample tray were set to 50 °C and 4 °C, respectively. The HPLC system was coupled to an Agilent Technologies 6210 LC/TOF mass spectrometer with a 1 :4 post- column split. Mass spectrometrie detection was conducted using APCI in the positive ion mode. MS experiments were carried out in the full scan mode, at 0.86 spectra second, for the detection of ions. Drying and nebulizing gases were set to 10 L/min and 25 psi, respectively, and a drying gas temperature of 330 °C was used throughout. The vaporizer and corona were set to 350 °C and 4 μΑ respectively, and a capillary voltage of 3,500 V was also used. Fragroentor and OCT 1 RF voltages were each set to 135 V, while the skimmer voltage was set to 50 V. Data acquisition and processing were performed by the MassHunter software package (Agilent Technologies Inc., Santa Clara, CA, USA). Metabolites were quantified via 10-point calibration curves of authentic standard compounds for which the coefficients were > 0.99. The ?-couroaraldehyde content was estimated by integrating the area of the mass peak eluting at Rt = 8.6 min ([M-H] ^" 13 .050238) and for which the ratio [theoretical mass/observed mass] was less than ±5 ppm (Fig. 26).

Carbohydrate and Isgnin assays

[0174] For each genotype (wild type, C4H::qs B-l, and C4H::qsuB~9), samples consisted of equal mixtures of stem material from three independent cultures, Biomass was extracted sequentially by sonication (20 min) with 80% ethanol (three times), acetone (one time), chloroform-metbanol (1 : 1, v/v, one time) and acetone (one time). For determination of carbohydrate composition, the biomass was acid-hydrolyzed as previously described (44). After CaC0 ₃ neutralization, monomelic sugars from the biomass hydro iyzates were separated by high-performance anion exchange chromatography with pulsed amperiometric detection using a PA20 column (Dionex, Simnyvale, CA, USA) and quantified as previousiy described (45). A calibration curve of monosaccharide standards was run for verification of response factors. The standard NREL biomass protocol was used to measure lignin and ash (46). All carbohydrate and lignin assays were conducted in triplicate. The thioacidolysis procedure was carried out as described (47, 8) and the lignin-derived monomers were identified by GC-MS as their trimethyi-silylated derivatives.

2D ^*3 C- ⁵ H heteronuclear single quantum coherence (HSQC) NMR spectroscopy

[0175] For each genotype (wild type, C4H::qsuB-l and C4H::qsuB-9) _> samples consisted of equal mixtures of stem material from three independent cultures. Samples were extracted and ball milled as previously described (49, 50), The gels were formed using DMSO- dg/pyridine-ds (4: 1 ) and sonicated until homogenous in a Branson 2510 table-top cleaner (Branson Ultrasonic Corporation, Danbury, CT). The temperature of the bath was closely monitored and maintained below 55 °C. The homogeneous solutions were transferred to NMR tubes. HSQC spectra were acquired at 25 °C using a Bruker Avaoce-600 MHz instrument equipped with a 5 mm inverse-gradient ^! H/ ¹³ C cryoprobe using a hsqcetgpsisp2.2 pulse program (ns = 400, ds = 16, number of increments = 256, d, = 1.0 s) (53). Chemical shifts were referenced to the central DMSO peak (5C/5H 39.5/2.5 ppm). Assignment of the HSQC spectra was described elsewhere (51 , 54), A semi-quantitati ve analysis of the volume integrals of the HSQC correlation peaks was performed using Bruker's Topspin 3.1

(Windows) processing software. A Guassian apodization in F? (LB = -0.50, GB = 0.001 ) and squared cosine-bell in Fj (LB = -0.10, GB = 0.001 ) were applied prior to 2D Fourier

Transformation. isolation of cel!ul lytic enzyme lignin

[0176] For each genotype (wild type, C4H::qsvB-l and C4H::qsuB-9), samples consisted of equal mixtures of stem material from three independent cultures. The extracted biomass was bail-milled for 3 h per 500 mg of sample (in 10 min on/10 min off cycles) using a PM 100 ball mil! (Retsch, Newtown, PA) vibrating at 600 rpm in zirconium dioxide vessels (50 mL) containing Zr0 ₂ ball bearings (10 * 10 mm). Bali-milled walls were digested four times over 3 d at 50 °C with the polysaceharidases Ce!Hc CTecl and HTec2 (Novozymes, Davis, CA) and pectinase from Aspergillus niger (Sigma-AIdrich, St. Louis, MO) in sodium citrate buffer (pH 5.0). The obtained celiuloiytic lignin was washed with deionized water and lyophilized overnight.

Size exclusion chromatography

[0177] Lignin solutions, 1% (w/v), were prepared in analytical-grade l -methyl-2- pyrrolidinone (NMP). The polydispersity of dissolved lignin was determined using analytical techniques involving SEC UV-F250/400 as previously described (53). An Agilent 1200 series binary LC system (G1312B) equipped with diode-array (G 1315D) and fluorescence

(G1321 A) detectors was used. Separation was achieved with a Mixed-D column (5 jam particle size, 300 mm x 7.5 mm i.d., linear molecular mass range of 200 to 400,000 u, Agilent Technologies inc.) at 80 °C using a mobile phase of NMP at a flow rate of 0.5 ml/min.

Absorhance of materials eluting from the column was detected using UV-F fluorescence (Ex25o/Eni45o). Spectral intensities were area-normalized and molecular mass estimates were determined after calibration of the system with polystyrene standards.

Cell wall pretreatments and saccharification

[0178] Ball-milled senesced stems (10 mg) were mixed with 340 qL of water, 340 q.L of H ₂ SO ₄ (1.2%, w/v), or 340 qL of NaOH (0.25%, w/v) for hot water, dilute acid, or dilute alkali pretreatments, respectively; shaken at 1 ,400 rpm (30 °C, 30 min), and autoclaved at 120 ^C 'C for 1 h. Samples pretreated with dilute acid were neutralized with 5 N NaOH (25 qL). Saccharification was initiated by adding 650 qL of 100 mM sodium citrate buffer pH 5 (for hot water- and dilute alkali-pretreated samples) or 625 qL of 80 mM sodium citrate buffer pH 6.2 (for dilute acid-pretreated samples) containing 80 qg/ml. tetracycline and 1 % w/w or 0.2% w/w Celiic CTecl cellulase (Novozymes, Davis, CA). After 72 h of incubation at 50 °C with shaking (800 rpm), samples were centrifuged (20,000 x g, 3 min) and 10 qL of the supernatant was collected for measurement of reducing sugars using the 3,5-dinitrosalicylic acid assay and glucose solutions as standards (54).

Subcellular localization of QsuB

[0179] The schl-qsuB nucleotide sequence from the pTkm-pC4H: :scM-qsuB construct was amplified using oligonucleotides 5 ^! ~

GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCTTCGATCTCCTCCT-3' (attB l site underlined) and 5'-

GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTTGGGATACCTCTCTCTAAATCT C-3' (attB2 site underlined) and cloned into the Gateway pDONR22I-fl entry vector (Lalonde S, et ai . (2010) Front Physio! 1 :24). A sequence-verified entry clone was LR recorabined with the pTKan-/?5JS-G WR 1R2-YFP vector to generate the pTK -p35S-scM- qsuB-YFP construct. Infiltration of 4- wo N. benthamiana leaves was done using the

Agrobaclerium strain GV3101 , following the method described by Sparkes et al. (Nat Protoc ] (4):2G 19-2025). Plants transiently expressing the SCHL-QsuB-YFP fusion protein were analyzed by confocal laser scanning microscopy 2 d after the infiltration. The microscopy was performed using a Zeiss LSM 710 device (Carl Zeiss Microscopy, Jena, Germany) equipped with an argon laser (excitation at 514 nm and emission collected at 510 to 545 nrn), Lignirt histochemical staining

[01801 Histochemical staining was performed as described by Pradhan-Miira and Loque ("Histochemical staining of Arabidopsis thaliana secondary cell wall elements," JoVE {in press)). Basal stem transverse sections ( 100 μιη thick) were obtained using a vibratome. Sections were incubated for 3 min in phloroglueinol-HCi reagent (VWR International, Brisbane, CA), rinsed with water, and observed using bright field light microscopy (Leica Microsystems inc., Buffalo Grove, IL).

Pyrolysis-gas chromatography mass spectrometry

[0181] Chemical composition of lignin in plant cell-wall samples were analyzed by pyrolysis-gas chromatography (GC)/mass spectrometry (MS) using a previously described method with some modifications (Del Rio iC, et al. (2012) J AgricFood Chem 60(23);5922- 5935). Pyrolysis of plant cell walls was performed with a Pyroprobe 5200 (CDS Analytical, inc.) connected with GC/MS (Thermo Electron Corporation with Trace GC Ultra and Pofaris- Q MS) equipped with an Agilent HP-5MS column (30 m x 0.25 mm i.d., 0.25 μτη film thickness). The pyrolysis was carried out at 550 °C. The chromatograph was programmed · from 50 °C (1 msn) to 300 °C at a rate of 30 °C/min; the final temperature was held for 10 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The mass spectrometer was operated in scan mode and the ion source was maintained at 300 °C. The compounds were identified by comparing their mass spectra with those of the NIST library and those previously reported (Del Rio JC, Gutierrez A. (2006) JAgric Food Chem

54(13):4600-4610; Ralph J, Hatfield RD (1991) J Agric Food Chem 39(8): 1426-1437). Peak molar areas were calculated for the lignin degradation products, the summed areas were normalized. Analyses on all samples were conducted in duplicate and data were averaged and expressed as percentages. In vivo HCT activity assay

[0182] For the cloning of AtHCT, total Arabidopsis RNA (I g) were extracted using the Plant R easy extraction kit (Qiagen, Valencia, CA) and reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN). The obtained cDNA preparation was used to amplify AtHCT (GenBank accession number NP J 99704. I) using the following oligonucleotides 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT C ATGAAAATTA ACATCAGAGA TTCC-3' (attBl site underlined) and 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG

GTCTCATATCTCAAACAAAAACTTCTCAAAC-3' (attB2 site underlined) for cloning into the Gateway pDQNR221-fl entry vector by BP recombination (Life Technologies, Foster City, CA). A sequence-verified AtHCT entry clone was LR recombined with the pDRf!-^CXJ-GW vector (41 to generate the pDRfl -4CL5-AtHCT construct.

[0183] For For HCT activity assays, the pDRfl. -4CL5-AtHCT and pDRfl -4CL5 vectors were transformed into the S. cerevisiae pad! knockout (MAT& his3A! leu2 0 mellSAO ura3A0 Apadl, ATCC 4005833) as previously described (41 ). Overnight cultures from single colonies harboring the pDRfl -4CL5-AtHCT and pDRfl -4CL5 vectors were grown in 2X yeast nitrogen base medium without amino acids (Difco, Detroit, MI) supplemented with 6% glucose and 2X dropout mix without uracil (Sunrise Science Products, San Diego, CA). Overnight cultures were used to inoculated 10 mL of fresh minimal medium at an ODsoo ~ 0.1. Substrates (/j-coumarate, catechol or benzoates) were added to the medium 4 h later at a final concentration of 1 mM and the cultures were gro wn for 22 h. For the detection of the eoumarate conjugate products, an aliquot of the culture medium was collected, cleared by centrifugation (20,000 x g for 5 min at 4 °C), mixed with an equal volume of 50% (v/v) methanol water and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore, Billerica, MA) prior to HPLC-ESLTOF MS analysis.

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ILLUSTRATIVE SEQUENCES

SEQ ID NO: 1 - MtAroK polynucleotide sequence

ATGGCACCAAAAGCTG TTTAGTGGGACTTCCTGG-^AGTGGAAAG CCAC ATCGGTAGAAG GTTGGCTAAAGCATTAGGAGTTGGTTTGTTAGACACTGATGTGGCTATAGAACAAAGGAC AG GAAGATCAATAGCAGACATTTTTGCTACAGATGGTGAACAGGAGT CAGAAGGATAGAAGAG GATGTTGTGAGAGCTGCATTGGCTGACCATGATGGTGTTCTTAGTTTGGGTGGAGGTGCA GT TACTTCCCCAGGAGTGAGAGCTGCACTTGCTGGTCACACAGTTGTGTATTTGGAAATCTC AG CTGCAGAGGGAGTGAGAAGGACAGGTGGTAACACCGTGAGACCACTTTTGGCAGGTCC GAT AGGGCTGAAAAGTATAGAGCTTTGATGGCAJ AAAGGGCTCCTTTATACAGAAGGGTTGC C TATGAGAGTGGATACAAATAGAAGGA/vCCCAGGTGCAGTTGTTAGGCACATTTTATCCA GGT TGCAGGTTCCATCTCCTTCTGAGGCAGCTACT SEQ ID NO:2 - MtAroK amino acid sequence (Mycobacterium tuberculosis shikimate kinase; NP 217055)

MAPKAVLVGLPGSGKS'T I GRRLAKALGVGLL D DVAI EQRTGRS I DI FA DGEQEFRRI EE DVVRAALADHDGVLSLGGGAVTS PGVRAALAGHTVVYLE I SAAEGVRRTGG TVRPLLAG PD RAEKYRALMAKRAPLYRRVATMRVDTNRR PGAVVRHT LSRLQVPS PSEAAT

SEQ ID O:3 - ScArol polynucleotide sequence

ATGGTTCAGCTTGCTAAGGTGCCTATTTTGGGTAACGACATCATTCACGTTGGA TAACAT TCACGATCATTTGGTTGAGACTATTATCAAGCATTGTCCATCTTCTACTTATGTTATTTG TA ACGATACCAACCTTTCTAAGGTTCCTTA TACCAACAGTTAGTGCTTGAGTTTAAGGCTTCT TTGCCAGAAGGAAGTAGATTGTTAACTTATGTTGTGAAACCTGGAGAGACTTCTAAGTCA AG GGAAACAAAAGCTCAATTGGAGGACTACCTTTTGGTTGAAGGATGTACCAGAGATACTGT GA TGGTTGCTATTGGTGGAGGTGT ATAGGTGATATGATTGGATTTGTGGCATCAACTTTCATG AGAGGTGTTAGGGTTGTGCAAGTGCCAACAAGTTTACTTGCTATGGTTGACAGTTCCATC GG AGGAAAGACAGCAATAGATACCCCATTGGGAAAAAACTTTATTGGTGCTTTCTGGCAGCC TA AGTTCGTGCTTGTTGATATCAAGTGGCTTGAGACATTGGCTAAGAG GAATTTA.T CAACGGA ATGGCAGAAGTTA CAAGACAGCTTGTATTTGGAACGCAGATGAGTTTACCAGATTGGAATC AAATGCTAGTTTGTTCTTAAACGTTGTGAACGGTGCAAAGAoACGTGAAGG ACTAACCAAC TTACAAACGAGATCGATGAAATCTCAAATACCGACATCGAAGCTATGCTTGATCACACTT AC AAACTTGTTTTGGAGTCTATCAAGGTGAAAGCAGAAGTTGTGTCTTCAGATGAGAGAGAA A.G TTCCTTGAGGAACTTGCTTAAC TCGGTCATTCAATCGGACACGCTTACGAAGCAATCTTAA CTCCACAAGCTCTTCATGGAGAATGTGTTTCTATTGGTATGGTGAAGGAGGCAGAA'TTG TCA AGATACTTCGGAA ATT AGTCCTACACAGGTTGCAAGGTTGTCCAAAATTTT'GGTTGCTTA CGGTTTGCCAGTGTCTCCTGA GAGAAGTGGTTCAAGGAATTAACACTTCATAAAAAGACCC C T T T AGAC AT C C T T T G AA AG GT C CAT C G T AAAAAG AAT G G G G T T C T AAAAAG AAA GTTGTGATCTTAGAATCTATCGGAAAGTGC TGGAGACTCCGCTCAATTTGTTTCTGATGA GGACCTTAGATTCATTTTGACAGATGAAACCCTTGTTTACCCATTTAAAGATATACCTGC TG ACCAACAGAAGGTTGTGATTCCACCTGGTAGTAAATCCATTTCTAACAGAGCATTGATCT TA GCTGCAT'TGGGTGAAGGACAGTGTAAGATAAAGAACCTTCTTCATTCAGATGACACTAA GCA C GCTTACA _* GCAGTTCATGAATTGAAAGGTGCTACAATCT'CTTGGGAGGATAACGGAGAAA CCGTTGTGGTTGAAGGTCATGGAGGTTCCACTTTGTCTGCTTGCGCAGATCCACTTTATT TG GGTAATGCTGGAACCGCATCAAGATTTTTAACTAGTCTTGCTGCTTTGGTTAACTCAACT TC TTCACAAAAGTACATTGTGTTAACTGGTAATGCAAGAATGCAACAGAGGCCAATCGCTCC TT TAGTTGATTCTCTTAGAGCAAACGGAACAAAGATCGAGTACCTTAACAACGAAGGTTCAC TT CCTATCAAGGTTTACACTGATAGTGTGTTCAAAGGAGGTAGAATAGAATTAGCTGCAACA GT TAGTTCCCAATATGTGTCTTCAATTCTTATGTGTGCTCCATACGCAGAAGAGCCTGTTAC TT TAGCTCTTGTGGGAGGAAAGCCAATCTCAAAATTGTACGTTGATATGACAATCAAGATGA TG GAAAAGTTCGGAATCAACGTTGAGACTTCTACTACAGAACCATACACATACTACATCCCT AA GGGTCATTACATCAACCCTTCAGAGTACGTTATCGAAAGTGATGCTAGTTCCGCAACTTA TC CATTAGCTTTCGCTGCAATGACCGGAACCACTGTGACTGTTCCTAATATTGGATTTGAAT CT CTTCAAGGTGACGCTAGATTCGCAAGGGATGTTTTGAAGCCAATGGGTTGTAAAATCACT CA GACAGCTACCTCAACAACCGTTAGTGGTCCACCTGTGGGAACATTAAAGCCACTTAAACA CG TTGACATGGAACCTATGACAGATGCTTTCTTGACCGCATGTGTGGTTGCTGCAATTTCAC AT GATAGTGACCCAAATTCTGCTAACACTACAACCATAGAGGGAATAGCAAACCAAAGAGTT AA GGAATGCAACAGGATCTTGGCTATGGCAACT GAGTTAGCTAAATTTGGTGTTAAAACTACAG AATTACCTGATGGAATCCAGGTGCACGGTCTTAATTCAATCAAGGACTTGAAAGTTCCAA GT GATTCTTCAGGTCCTGTGGGAGTTTGTACTTATGATGACCATAGAGTGGCAATGTCATTC AG TTTGTTAGCTGGTATGGTTAATTCTCAAAACGAGAGGGATGAAGTGGCTAACCCAGTTAG AA TTTTGGAAAGGCACTGCACTGGAAAGACATGGCCTGGTTGGTGGGACGTTTTGCATAGTG AA TTAGGAGCTAAACTTGATGGTGCAGAGCCTTTAGAA GTACTTCTA.AGAAAAATTCCAAGAA JiTCTGTGGTTATTATCGGAATGAGAGCTGCAGGTAAAACCACTATTTCCAA-ATGGTGC GCTT CTGCATTGGGATACAAATTGGTTGATTTAGACGAGCTTTTTGAACAACAGCAJAATAACC AA TCAGTTAAGCAGTTCGTGGT GAGAACGGTTGGGAAAAATTTAGAGAAGAGGAAACTAGGAT CTTCAAGGAAGTTATCCAAAACTACGGTGA GACGGATACGTTTTCTCTACAGGAGGTGGAA TTGTGGAGTCAGCTGAAAGTAGAAAGGCACTTAAAGATTTCGCTAGTTCCGGTGGATATG TG TTGCATTTACACAGGGACATTGAGGAAACTATCGTTTTCTTGCAALTCTGATCC TCAAGACC AGCTTATGTTGAGGAAATTAGAGAAGTGTGGAACAGAAGGGAGGGTTGGTAxCAAGGAAT GTT CAAACTTCTCTTTCTTTGCTCCACACTGCTCTGCTGAGGCAGAATTTCAAGCTCTTAGAA GG TCCTTCTCTAAATACATCGCAACTATAACAGGAGT AGAGAGATCGAAATACCATCCGGTAG GTCTGCTTTTGTTTGTTTGACCTTCGATGACTTAACCGAGCAGACTGAAAACTTAACTCC TA TTTGTTATGGTTGCGAGGCAGTGGAAGTTAGAGTGGACCATCTTGCTAATTACTCAGCAG AT TTCGTTTCCAAGCAATTGTCTATCCTTAGAAAGGCTACTGATAGTATCCCAATAA TTTCAC AGTTAGGACCATGAAACAGGGTGGi^CTTTCCTGACGAGGi TTTAAGACACTTAGAGAAT TGTACGATATAGCTCTTAAGAATGGTGTTGAGTTTCTTGACTTGGAATTAACTCTTCCTA CA G TATCCAATACGAAGTTATCAACAAGAGAGGAAACACTAAGATC TAGGTTCCCATCACGA TTTTCAAGGATTATACTCTTGGGATGACGCTGAGTGGGAAAATAGATTCAACCAGGCATT GA CCTTAGATGTTGACGTGGTTAAGTTTGTGGGT C GCTGTΤΑΆΤΐTC.GAGGACAACCT GA TTGGAACATTTTAGGGATACACACAAGAACAAGCCACTTATCGCAGTTAACATGACCTCA AA AGGATCAATCAGTAGAGTGTTGAATAACGTTTTAACCCCTGTGACTTCCGATCTTTTGCC AA ACTCTGCTGCACCTGGTCAACTTACCGTTGCTCAGATCAACAAGATGTACACTTCTATGG GT GGAATTG GCCAAAAG AGTTTTCGTGGTTGGAAAGCCAATCGGACATTCAAGATCACCT T CTTGCATAACACTGGATACGAAATTTTAGGTCTTCCTCATAAGTTCGATAAATTCGAGAC AG AATCTGCTCAATTGGTTAAGGAAAAATTACTTGATGGTAACAAGAACTTTGGTGGAGCTG CA GTTACTATCCCATTGAAATTGGATATCATGCAGTACATGGATGAATTGACAGACGCTGCA AA GGTTATTGGTGCTGTGAATACCGTTATCCCACTTGGAAACAAGAAGTTCAAGGGTGATAA CA CAGACTGGCTTGGAATAAGAAATGCTCTTATCAACAACGGTGTTCCTGAATATGTGGGTC AC ACTGCAGGATTGGTTATTGGTGCTGGTGGAACATCAAGAGCTGCATTATACGCTCTTCAT AG TTTGGGTTGTAAGAAAATCTTTATCATCAACAGGACAACCTCTAAGTTAAAACCACTTAT CG AGTCACTTCCTAGTGAATTTAACATCATCGGAATAGAGTCCACTAAGTCTATTGAGGAAA TC AAAGAACACGTTGGTGTGGCAGTTTCCTGCGTTCCAGCTGATAAACCTTTGGATGACGAG TT GCTTTCA.AAACTTG AAGATTTTTGGTTAAGGGTGCTCATGCTGCATTCGTGCCAACAC '3?TT TGGAAGCTGCATATAAGCCATCCGTGACCCCTGTTATGACTATCTCTCAGGATAAGTACC AG TGGCACGTGGTTCCTGGATCTCAAATGTTGGTTCATC GGGTGTGGCTCAGTTTGAGAAGTG GACAGGATTCAAAGGACCATTTAAGGCTATTTTCGACGCAGTTACCAAGGAG

SEQ ID NO:4 - ScArol amino acid sequence (Saccharomyces cerevisiae Pentaf nctional arorn protein; CAA88208)

MVQLAKV ILGNDI IHVGY IHDHLVETI IKHCPSS YVICNDTNLS VPYYQQLVLEFKAS LPEGSRLLTYVVKPGETSKSRETKAQLEDYLLVEGCTRDTVMVAIGGGVIGDMIGFVAST FM RGVRVVQVPTSLLAMVDSSIGGKT IDTPLGKNFIGAF QPKFVLVDIKWLETLAKREFING MAEVIKTACIWNADEFTRLESNASLFLI VNGAK VKVTNQLTNEIDEISNTDIEA LDHTY KLVLESIKV AEVVSSDEP.ESSLRNLLNFGHSIGHAYEAILTPQALHGECVSIGMVKEAELS RYFGILSPTQVARLSKILVAYGLPVSPDEK FKELTLHKKTPLDILLKKMSIDKK EGSKKK VVILESIGKCYGDSAQFVS DEDLRFILTDETLVYPFKDIPADQQKVVIPPGSKSISNRALIL AALGEGQCKIKNLLHSDDTKHMLTAVHELKGATISWEDNGETWVEGHGGSTLSACADPLY L

• GNAG ASRFLTSLAALV STSSQKYI LTGNARMQQRPI PLVDSLRANGTKIEYLNNEGSL PIKVYTDSVFKGGRIELAATVSSQWSSILMCAPYAEEPVTLALVGGKPISKLYVDMTIKM M EKFGINVETSTTEPYTYYI PKGHYINPSEYVIESD SSATYPLAFAAMTGT VTVPNIG FES

L0GDARFARDVLKPMGCKITQTATSTTVS GPPVGTLKPLKHVDMEPMTDAFLTACVVAAI SH DSDP SANTT IEGIA QRV ECNRILAMATELAKFGVKTTELPDGIQVHGL SIKDLKVPS DSSGPVGVCTYDDHRVAMSFSLLAGMVNSQNERDEVANPVRILERHCTGKTWPG DVLHSE LGAKLDGAEPLECTSKKNSKKSWI IGMRAAGKTTISKWCASALGYKLVDLDELFEQQHNNQ SVKQFVVENGWEKFREEETRI FKE IQNYGDDGYVFSTGGGIVE 3AESRKALKDFASS GGYV LHLHRDIEETIVFLQSDPSRPAYVEEIREVWNRREGWYKECSNFSFFAPHCSftEAEFQA LRR S FSKYIATITGVREIEI PSGRSAFVCLTFDDLTEQTENLTPICYGCEAVEVRVDHLANYS D FVSKQLSILRKATDSI PI I FTVRTMKQGGNFPDEEFKTLRELYDIALKNGVEFLDLELTL T DIQYEVINKRGNTKIIGSHHDFQGLYS DDAE ENRFNQALTLDVDVVKFVGTAVNFED LR LEFIFRDTHKNKPLIAVNM SKGSISRVLNNVLTPVTSDLLPNSAAPGQLTVAQINKMYTSMG GIEPKELFVVGKPIGHSRSPILHNTGYEILGLPHKFDKFETESAQLVKEKLLDGNKNFGG AA VTIPLKLDIMQYMDELTDAAKVIGAVNTVIPLGNKKFKGDNTDWLGIRNALINNGVPEYV GH TAGLVI GAGGTSP.A ALYALHSLGCKKI F11NR'TTSKLKPLIESLPSEFN11GIESTKSIEEI KEHVGVAVSCVPADKPLDDELLSKLERFLVKGAHAAFVPTLLEAAYKP3VTPVMTISQDK YQ WHWPGSQMLVHQGVAQFEKWTGFKGPFKAIFDAVTKE

SEQ ID N0:5 - CgQsuB pol nucieoiide sequence

ATGAGAACAAGTATTGCAACCGTTTGTTTATCCGGAACTCTTGCTGAiiAAATTGAGAGC AGC TGCAGACGCAGGATTCGATGGTGTTGAGA.TTTTTGAACAAGATTTGGTTGTGTCTCCAC ATT CAGCTGAACAAATCAGACAGAGGGCACAAGATTTAGGTCTTACATTGGACTTATTTCAGC CT T CAGAGATTTTGAAGGAGTTGAAGAGGAACAATTCTTAAAGAi^TCTTCACAGGTTGGAGG A AAAATΤΤΆΆGTTAA GAAC G C TGG ATCGAAATGATCTΪGCTT TGT TCTAACGT GG A CAGCTACCATCAACGATGACGATCTTTTTGTGGAACAATTGCATAGAGCTGCAGATTTGG CT GAGAAGTACAACGTTAAGATCGCTTATGAAGCTCTTGCTTGGGGTAAi^TTCGTTAA GATTT TGAGCATGCTCACGCATTGGTTGAAAAAGTGAACCATAAGGCTTTGGGTACTTGCTTAGA TA CATTCCACATATTAAGTAGAGGATGGGAGACTGATGAGGTTGAAAACATCCCAGCTGAAA AA ATATTTTTCGTGCAATTGGCTGATGCACCTAAGTTATCTATGGATATCCTTTCTTGGTCA AG GCATCACAGAGTTTTTCCAGGAGAGGGTGACTTCGATTTGGTTAAGTTCATGGTGCATCT TG C AAG CAGGATACGATGG CCTAT CTTΪGGAG TTTTC CG G C TT AGGAAAGC GAAGTTGGAAGAACTGCAATTGATGGTTTAAGGTCTCTTAGATGGTTGGAGGACCAAACA TG GCATGCACTTAACGCTGAAGATAGGCCATCAGCACTTGAGTTGAGAGCTTTGCCAGAAGT TG CAGAGCCTGAGGGTGTGGATTTCATTGAGATCGCTACAGGAAGGTTAGGTGAAACCATCA GA GTTTTACACCAGCTTGGTTTTAGACTTGGTGGACATCACTGTTCTAAGCAGGATTATCAA GT TTGGACTCAAGGAGATGTGAGGATCGTTGTGTGCGACAGAGGAGCAACAGGTGCTCCTAC CA CTATATCAGCTATGGGTTTCGACACCCCAGATCCTGAGGCTGCACATGCTAGGGCAGAAC TT TTGAGAGCACAAACAATTGATAGACCACACATCGAGGGAGAAGTTGATCTTAAGGGTGTG TA CGCTCCTGACGGAGTTGAATTGTTTTTCGCAGGACCATCTCCTGATGGTATGCCAGAGTG GT TACCTGAATTTGGTGTTGAGAAGCAAGAAGCTGGACTTATCGAAGCAATCGATCATGTTA AC TTTGCTCAGCCTTGGCAACACTTCGATGAGGCAGTTTTGTTTTATACCGCATTGATGGCT TT AGAAACTGTGAGAGAGGATGAATTTCCATCACCTATTGGTTTAGTTAGGAATCAGGTGAT GA GATCACCAAACGATGCTGTTAGATTACTTTTGTCAGTGGCACCTGAGGACGGAGAACAGG GT GATTTCTTAAATGCTGCATACCCAGAACATATAGCTCTTGCAACTGCTGATATTGTTGCA GT GGCTGAAAGAGCTAGGAAAAGAGGTTTGGATTTCTTGCCAGTTCCTGAAAACTATTACGA CG ATGTGCAGGCTAGATTCGATTTGCCTCAAGAGTTTTTAGACACACTTAAGGAAAACCATC TT CTT ATGACTGCGATGAGAACGGTGAATTTTTGCACTTC TAGACT GAACATTGGGAA ^' CATT ATTTTTCGAGGTTGTGGAAAGAAGGGGTGGATTTGCTGGATGGGGTGAAACCAATGCACC TG TTAGGCTTGCTGCTCAAT AGAGAAGTTAGAGATTTAGAGAGAGGTATCCCAAAC SEQ ID NO:6 - CgQsuB amino acid sequence (Coryne bacterium glulamicum

dehydroshikimate dehydratase; BAF53460)

MRTS IATVCLSGTLAEKLRAAADAGFDGVEI FEQDLWSPHSAEQIRQRAQDLGLTLDLFQP FRDFEGVEEEQFLKNLHRLEEKFKLM RLGIEMILLCSNVGTATINDDDLFVEQLHRAADLA EKYNVKI YEALAWGKFVNDFEHAHALVEKVNHKALGTCLDTFHILSRGWETDEVE IPAEK I FFVQLADAPKLSMDI LSWSRHHRVFPGEGDFDLVKFMVHLAKTGYDGPISLEIFND5FRKA EVGRTAI DGLRSLRWLEDQTWHALNAEDRPSALELRALPEVAEPEGVDFIEIATGRLGETIR VLHQLGFRLGGHHCSKQDYQVWTQGDVRIWCDRGATGAPT ISA GFD'TPDPEAAHARAEL LRAQ IDRPHI EGEVDLKGVYAPDGVELFFAGPSPDGMPEWLPEFGVEKQEAGLIEAIDHVN FAQP QHFDEAVLFYTALMALETVREDEFPS PI GLVRNQVMRS PNDAVRLLLSVAPEDGEQG

DFLNAAY PEHIALATADIVAVAERARKRGLDFL PVPENYYDDVQARFDL PQEFLDTLKENHL L YDCDE]NGE FLHFY RTLG LFFEVVE RRGGFAG GET APVRLAAQYREVRDLERGI PN SEQ ID NO:7 - PaDsDH polynucleotide sequence

ATGCCTTCAAAACTTGCTATCACCTCAATGTCTCTTGGTAGATGCTATGCTGGTCACTCC TT CACTACTAAATTGGATATGGCTAGGAAiiTATGGTTACCAAGGACTTGAATTGTTCCATG AGG ATTTGGCTGACGTTGCATATAGACTTAGTGGTGAAACACCATCCCCTTGTGGACCATCTC CT GCTGCACAGTTGAGTGCTGCAAGACAAATACTTAGGATGTGTCAGGTTAGAAACATAGAA AT TGTGTGCTTACAGCCATTTTCTCAATACGATGGTTTGTTAGACAGAGAAGAGCATGA2 AGAA G GC T T G AC AAT T GGAGTTCTG G AT AG AAT TAGCTCAC G G C T T GAT AC AG C T TAT C C G ATTCCAGCAAATTTTCTTCCTGCTGAAGAGGTTACCGAAGATATTTCTTTGATCGTTTCA GA TTTGCAAGAGGTGGCTGACATGGGTTTGCAGGCAAACCCACCTATTAGATTCGTTTATGA AG CTCTTTGTTGGTCAACTAGAGTGGATACATGGGAAAGGAGTTGGGAGGTTGTGCAAAGAG TT AATAGGCCTAACTTTGGTGTGTGCCTTGATACATTCAATATCGCAGGAAGAGTTTACGCT GA CCCAACCGTGGCATCAGGTAGAACTCCTAACGCTGAAGAGGCAATTAGGAAGTCAATCGC TA GATTGGTTGAAAGGGTTGATGTTAGTAAAGTTTTCTATGTGCAAGTTGTGGACGCAGAGA AG TTGAAAAAGCCATTAGTTCCTGGACACAGATTCTACGATCCAGAACAACCTGCTAGGATG TC TTGGTCAAGAAACTGCAGGTTGTTTTATGGTGAAAAAGATAGAGGAGCTTACTTGCCAGT TA AGGAGATTGCTTGGGCATTTTTCAATGGTTTGGGATTTGAAGGTTGGGTTTCCTTAGAGC TT TTCAACAGAAGGATGTCTGATACTGGTTTTGGAGTGCCTGAAGAGTTAGCTAGAAGGGGA GC AGTTTCCTGGGCTAAACTTGTGAGAGATATGAAGATCACCGTTGACTCACCAACTCAACA GC AAGCTACACAGCAACCTATAAGAATGTTGAGTTTATCCGCTGCATTA SEQ ID NO: 8 - PaDsDH amino acid sequence {Podospora anserina dehydroshikimate dehydratase; CAD60599)

MPSKLAI TSMSLGRCYAGHS FTTKLD ARKYGYQGLELFHEDLADVAYRLSGET PS PCGPS P AAQLSAARQI LRMCQVRNI E IVCLQPFSQYDGLLDREEHERRLEQLE FWIELAHELDTD I I Q I PANFLPAEEVTEDI SLI VSDLQE VADMGLQAN PP I RFVYEALCWSTRV DTWERSWE WQRV NRPNFGVCLDTFNIAGRVYADPTVASGRTPNAEEAIRKS I ARLVERVDVSKVFYVQVVDAEK LKKPLVPGHRFYDPEQPARMSWSRNCRLFYGEKDRGAYLPVKE IAWAFFSGLGFEGWVS LEL FNRRMSDTGFGVPEELARRGAVSWAKLVRDMKI TVDS PTQQQATQQPIRMLSLSAAL

SEQ ID NO:9 - PhPAAS polynucleotide sequence

AT G G G AC TAT C AAG AT C AAC C C AG AG T C G AC GG AC AG T T C T G C AAG AC TAG AT CA T T T AGACCCAGAGGAGTTCAGGAGGAATGGACATATGATGGTTGATTTTCTTGCTGACTACTT CC ACAACATCGAAAAGTACCCAGTTAGATCCCAAGTGGAACCTGGTTATTTGGAGAGGTTGT TA CCAGATTCAGCTCCTATACAGCCAGAACCTATCGAGAAAATTTTGAAGGATGTTAGATCA GA CAT AT T T C C G G T T AAC AC AT T GG CAAAGTC CAAA T T C T T T GC T AG T T C CCT G C T C T T CAAGTACCGCAGGAATTTTAGGTGAAATGCTTTCAGCTGGATTGAACGTTGTGGGTTTTT CA TGGATCGCTAGTCCAGCTGCAACTGAATTAGAGAGTATTGTTATGGATTGGCTTGGAAAA TT GATTAATTTGCCTAAGACATATCTTTTCTCTGGTGGAGGTGGAGGTGTGATGCAGGGTAC TA CATGCGAAGTTATGCTTTGTACTATCGTGGCTGCAAGAGATAAAATGTTGGAAAAGTTTG GA AGGGAGAACATTGATAAGTTAGTTGTGTACGCATCAGACCAAACCCACTTTAGTTTCCAG AA AGCTGTTAAGATCTCAGGTATAAAACCAGAAAACTTCAGAGCTATACCTACCACTAAGGC AA CAGAATTCTCCCTTAACCCAGAGTCTTTGAGAAGGGCTATCCAAGAGGATAAAAAGGCAG GA CTTATCCCTTTGTTTTTATGCACATCAATAGGTACAACCAGTACTACAGCAGTTGACCCA CT TAAACCTTTGTGTGAAATAGCTGAAGAGTATGGAATTTGGGTTCATGTGGATGCTGCATA CG CTGGTTCTGCATGCATTTGTCCTGAATTTCAGCATTTCTTGGACGGTGTTGAGCACGCTA AT TCCTTTTCTTTCAACGCACACAAGTGGTTGTTTACTACTCTTGATTGTTGCTGTCTTTGG TT GAAAGACCCATCCTCTTTGACTAAGGCACTTTCAACAAACCCTGAAGTTTTGAGAAACGA TG CTACCGACAGTGAGCAAGTTGTGGATTATAAAGACTGGCAGATTACTTTATCCAGAAGGT TT AGGTCTCTTAAGCTTTGGTTGGTTCTTAAGTCCTACGGAGTGGCTAATCTTAGAAACTTC AT AAGGTCTCATATCGAAATGGCTAAGCACTTTGAAGAGTTGGTTGCAATGGATGAAAGATT CG AGATCA GGCACCAAGGAATTTTTCCTTAGTTTGTTTCAGAGTGTCTCTTTTGGCTCTTGAA AAGAAGTTTAATTTCG TGATGAAACTCAAGTGAACGAGTTTAACGCTAAGCTTCTTGAATC TATCATCTCAAGTGGTAACGTTTACATGACACATACCGTTGTGGAGGGAGTTTACATGAT TA GATTCGCTGTGGGTGCACCTTTGACAGATTATCCTCACATTGATATGGCTTGGAATGTTG TT AGGAAC CACGC'TAC T G AT G T T G AAC G C A

SEQ ID NO: 10 - PhPAAS amino acid sequence {Petunia hyhrida Phenylacetaldehyde synthase; ABB72475)

MDTIKINPEFDGQFCKTTSLLDPEEFRRNGHidMVDFLADYFHNIEKYPVRSQVEPG YLERLL PDSAPIQPEPIEKILKDVRSDIFPGLTHWQSPNFFAYFPCSSSTAGILGEMLSAGLNWGF S

W I S PA A E L E S I VM D W L G KLINLPKTYL F S G G G G G VM Q G T O E VM L C T I VTA A R D KM L E K F G R E I D K L V Y S D Q T H F S FQ K AV K I S G I K P E F R I P T K A T E F S L N P E S L RR A I QE D K R AG LIPLFLCTSIGTTSTTAVDPLKPLCEIAEEYGIWVHVDAAYAGSACICPEFQHFLDGVEH AN SFSFNAHK LFTTLDCCCLWLKDPSSLTKALSTNPEVLRNDATDSEQWDYKDWQITLSRRF RSLKLWLVLKSYGVANLRNFIRSHIEMAKHFEELVAMDERFEIMAPRNFSLVCFRVSLLA LE KKFNFVDETQVNEFNA LLESIISSGNVYMTHTVVEGVYMIRFAVGAPLTDYPHIDMA NVV R HAT LNA SEQ ID NO: 11 - ObCCMTl polynucleotide sequence

ATGGCGAGAAAAGAGAACTATGTTGTTTCTAACATGAATGTTGAAAGTGTGTTGTGCATG AA

AGGTGGAAAAGGAGAAGATAGCTATGATAACAACTCTAAGATGCAGGAGCAACATGC TCGAT C AG T G C T C C AC CTTCTGATG G AAG C T C T C G A C G G C G T G G G G C T GAG C T C G G T G G C G G C C G G C GCTTTCGTGGTGGCGGATCTCGGCTGCTCCAGCGGAAGAAACGCCATAAACACGATGGAA TT TATGATCAATCACCTGACTGAGCACTACACGGTGGCGGCGGAAGAGCCGCCGGAATTCTC AG C C T T C T T C T G C G AC C T C C C C T C C AA C G AC T T C AAC A C C C T C T T T C AG C T C C T T C. C G C C G T C T GACGGCAGCAGCGGTTCTTACTTCACTGCCGGCGTGGCCG6TTCGTTTTACCGGAGGCTT TT CCCGGCGAAGTCTGTTGATTTCTTTTACTCGGCATTTAGTTTGCACTGGCTATCTCAGAT AC CAAAGGAGGTGATGGAGAAGGGATCGGCGGCTTACAACGAGGGGAGAGTGACCATCAACG GT GCAAAAGAGAGCACCGTAAATGCATACAAGAAACAATTTCAAAGTGATTTGGGTGTCTTC TT GAGATCCAGATCCAAAGAATTGAAACCGGGAGGATCCATGTTCCTCATGCTCTTGGGTCG GA CCAGCCCCGACCCGGCAGATCAGGGCGCATGGATTCTCACTTTCAGCACACGTTATCAAG AT GCTTGGAATGATCTTGTGC IAGAGGGCTTAATTTCGAGCGAAAAACGGGATACGTTCAACAT CCCGATATATACGCCCAGCC AGAGGAGTTCAAAGAGGTGGTAGAAAGAGATGGTGCATTCA TAATCAACAAGCTCCAACTTTTCCACGGTGGCAGCGCTCTCATCATCGATGATCCCAACG AT GCGGTTGAGATTAGCCGTGCCTATGTCAGCCTCTGTCGCAGCCTCACCGGAGGCTTAGTT GA TGCCCACATAGGCGATCAGCTCGGCCATGAGCTCTTCTCGCGCTTATTAAGCCAAGCCGT GG ATCAGGCTAAGGAGCTAATGGACCAGTTTCAGCTCGTCCATATAGTTGCATCCCTTACTT TA GCT

SEQ ID NO: 12 - ObCCMTl amino acid sequence (Ocimum basi!icimi cinnamate/p- eoumarate carboxyl methyltransferases; ABV91100)

ARKENYWSNM VESVLCMKGGKGEDSYDNNSK QEQHARSVLHLL EALDGVGLSSVAAG AE'VVADLGC S S GR AI NTME FMINHLTEHY VAAEE PPE FS AFFC DLPSNDFNTL FQLL PPS DGSSGSYFTAGVAGSFYRRLFPAKSVDFFYSAFSLH LSQIPKEVMEKGSAAYNEGRVTING AKESTVNAY KQFQSDLGVFLRSRSKELKPGGSMFLMLLGRTS?E)PADQGAWILTFSTRYQD AWNDLVQEGLI SSEKRDTFNI PI YTPSLEEFKEVVERDGAFI I NKLQLFHGGSALI I DDP D AVEISRAYVSLCRSLTGGLVDAHIGDQLGHELFSRLLSQAVDQAKELMDQFQLVHIVASL TL A

SEQ ID NO: ! 3 - RgC2'H polynucleotide sequence ATGGCACCAACCAAAGATTCAGT A TCACATGGGAGCAGAGTCCTGGGATGAGATTTCCGA GTTCGTTAG A AAAGGGACACGGTG TAAGGGTCTTTC G ACTTGGIATT AAACTCT C CAAAGCAATTCCATCAGCCTCT GAAGAGAGGTTCAGTGAGAAAAAGATTTTGGAAAGAGC TCAATCCCACTTATCGATATGAGTAAGTGGGACTCCCCTGAGGTTGTGAAGTCTATCTG GA TGCTGCAGAACATTGGGGTTTCTTTCAAATAGTTAATCACGGA.GTGCCATTGGAGACTT TAC AGAGAGTTAAAGAAGCTACACATAGGTTTT CGCTTTGCCTGCAGAAGAGAAAAATAAGTAC TCTAAGGAAAACTCACCAATTAATAACGTTAGATTCGGTTCTTCATTCGTTCCTCATGTT GA GAAAGCACTTGAATGGAAGGATTTTCTTAGTATGTTCTATGTTTCCGAAGAGGAAACrAA CA C ACTGGCCACCTATTTGTAGAGACGAGATGTTAGAATACATGAGGAGTTCCGAGGTTCTT ATCAAAAGATTGATGGAAGTGTTAGTTGTGAAGGGTC TAJAGTTAAGCAAATCGA ^r GAGA'T AAGAG.A¾CCAATGTTGGTGGGATCAAGAAGAAT AATTTGAACTACTACCCTAAATGCCCAA ATCCTGAACTTACATTGGGTGTTGGAAGGCATAGTGATATTTCCACCTTTACTATCTTGT TA CAAGACGAAATCGGTGGACTTCATGTTAGAAAGTTGGATGACACTGGTAACACCTGGGTT CA TGTTACCCCAATATCTGGTTCACTTATTATCAATATCGGAGATGCTTTGCAGATAATGTC TA ACGGAAGGTACAAG CAATAGAACACATGG TGTGGCAAATGGAACACAAGACAGAATCTCT GTTCCTTTATTTGTGAACCCAAAGCCTCAGGCTATACTTTGTCCATTCCCTGAGGTTTTG GC AAATGGAGAAAAACC GTTTATAAGCCTGTGTTGTGCTCTGATTACTCAAGGCATTTCTACA CAAAACCTC CGATGGT AAAAG CAG GGA TCGCAT GATG AC SEQ ID NO: 14 - RgC2'H amino acid sequence (Ruta graveolens 2-oxoglutarate-dependent dioxygenase; Vialart et al plant J 2012, 70:460-470)

MAPTXDSVIH GAESWDEISEFVTKKGHGVKGLSELGI K LPKQFHQPLEERFSEKKILERA S I ?LI DMSK DSPEVVKS ICDAAEHWGFFQIVNHGVPLETLQRVKEATHRFFALPAEEKN Y SKENSPI NVRFGSSFVPHVEKALEWKDFLS FYVSEEETN YWPPICRDEMLEYMRSSEVL IKRLMEVLVVKGLKVKQIDEIREPMLVGSRRINLMYYPKCPNPELTLGVGRHSDISTFT ILL QDEIGGLHVRKLDDTGNT VHVTPISGSLIINIGDALQTMSNGRYKSIEHMVVANGTQDRIS VPLFVNPKPQAILCPFPEVLA GE PVYKFVLCSDYSRHFYTKPHDGKKTVDFALMN

SEQ ID NO: 15 - P!astid targeting signal polynucleotide sequence

ATGGCTTCGA.TCTCCTCCTCAGTCGCGACCGTTAGCCGGACCGCCCCTGCTCAGGC CAACAT GGTGGCTCCGTTCACCGGCCTTAAGTCCAACGCCGCCTTCCCCACCACCAAGAAGGCTAA CG ACTTCTCCACCCTTCCCAGCAACGGTGGAAGAGTTCAATGCATGCAGGTGTGGCCGGCCT AC GGCAACAAGAAGTTCGAGACGCTGTCGTACCTGCCGCCGCTGTCGACGATGGCGCCCACC GT GATGATGGCCTCGTCGGCCACCGCCGTCGCTCCGTTCCAGGGGCTCAAGTCCACCGCCAG CC TCCCCGTCGCCCGCCGCTCCTCCAGAAGCCTCGGCAACGTCAGCAACGGCGGAAGGATCC GG TGCA GCAG

SEQ ID NO: 16 -- Plastid targeting signal amino acid sequence

MASISSSVATVSRTAPAQANMVAPFTGLKSNAAFPTT KANDFSTLPSNGGRVQCMQVWPAY GNKKFETLSYLPPLSTMAPTVMMASSATAVAPFQGLKSTASLPVARRSSRSLG VSNGGRIR CMQ

SEQ ID NO: 17 - IRX5 promoter polynucleotide sequence

ATGAAGCCATCCTCTACCTCGGAAAAACTTGTTGCGAGAAGAAGACATGCGATGGCATGG AT GCTTGGATCTTTGACATTGATGACACTCTTCTCTCAACCATTCCTTACCACAAGAGCAAC GG TTGTTTCGGGTAAATAAACTAAACTTAACCATATACATTAGCCTTGATTCGGTTTTTGGT TT GA TTATGGATATTAAAGATCCGAATTATATTTGAAC.AAAAAAAAATGATTATGTCACA AA AA-AAAAATTGGCTTGAATTTTGGTTTAGATGGGTTTAAATGTCTACCTCTAATCATTTC TT TGTTTTCTGGTTAGCTTTAATTCGGTTTAGAATGAAACCGGGATTGACATGTTACATTGA TT TGAAACAGTGGTGAGCAACTGAACACGACCAAGTTCGAGGAATGGCAAAATTCGGGCAAG GC ACCAGCGGTTCCACACATGGTGAAGTTGTACCATGAGATCAGAGAGAGAGGTTTCAAGAT CT TTTTGATCTCTTCTCGTAAAGAGTATCTCAGATCTGCCACCGTCGAAAATCTTATTGAAG CC GGTTACCACAGCTGGTCTAACCTCCTTCTGAGGTTCGAATCATATTTAATAACCGCATTA AA CCGAAATTTAAATTCTAATTTCACCAAATCAAAAAGTAAAACTAGAACACTTCAGATAAA TT TGTCGTTCTGTTGACTTCATTTATTCTCTAAACACAAAGAACTATAGACCATAATCGAAA T AAAAACCCTAAAAACCAAATTTATCTATTTAAAACAAACATTAGCTATTTGAGTTTCTTT TA GGTAAGTTATTTAAGGTTTTGGAGACTTTAAGATGTTTTCAGCATTTATGGTTGTGTCAT TA AT TGTTTAGTTTAGTAAAGAAAGAAAAGATAGTAATTAAAGAGTTGGTTGTGAAATCATAT TTAAAA.CA.TTAATAGGTATTTATGTCTAATTTGGGGACAAAATAGTGGAATTCTTTAT CATA TCTAGCTAGTTCTTATCGAGTTTGAACTCGGGTTATGATTATGTTACATGCATTGGTCCA TA TAAATCTATGAGCAA CAATATAATTCGAGCATTTTGGTATAACATAATGAGCCAAGTATAA C AGTATCAAACCTATGCAGGGGAGAAGA GATGAAAAGA GAGTGTGAGCCAATACAAA. GCAGATTTGAGGACATGGCTTACAAGTCTTGGGTACAGAGTTTGGGGAGTGATGGGTGCA CA ATGGAACAGCTTCTCTGGTTGTCCAGTTCCCAAGAGAACCTTCAAGCTCCCTAACTCCAT CT ACTATGTCGCCTGATTAAATCTTATTTACTAACAAAACAATAAGATCAGAGTTTCATTCT GA TTCTTGAGTCTTTTTTTTC CTCTCCCTC TTCA TTCTGGTTTATATAACCA TC AAT GCTT TGATCCATGCATG ACCATGA C CTT G GTTTTΐTTTTCCTTC G ACCAT TTTGGGCCTTTGTG TTG TTT GGGCT TTGTTA A ATCTCCTCT C C TTC CT ACCTGATTGGATTCAAGAACATAGCCAGATTTGGTAA-AGTTTATAAGATACAAAATATT AG TAAGACTAiAGTAGA?\ATACA.TAATAACTTGAAAGCTACTCTAAGTTA C AATTCTAAAG AACTC/^iiy^GAATAACAA/^CAGTAGAAGTTGGAAGCTCAAGCAATTAAATTATA AAAAAC CTAACTACACTGAGCTGTCTCCTTCTTCCACCAAATCTTGTTGCTGTCTCTTGAAGCTTT CT TATGACACAAACCTTAGACCCAATTTCACTCACAGTTTGGTACAACCTCAGTTTTCTTCA CA ACAAATTCAAACATCTTACCCTTATATTACCTCTTTATCTCTTCAATCATCAiVAACACA TAG TCACATACATTTCTCTACCCCACCTTCTGCTCTGCTTCCGAGAGCTCAGTGTACCTCGCC SEQ ID NO: 18 - AtC4H promoter polynucleotide sequence

CGGAATGAGAGACG?IGAGCAATGTGCTAAGAGAAGAGATTGGGAAGAGAGAAGAGAAGA TAA AGGAAACGGAAAAGCATATGGAGGAGCTTCACATGGAGCAAGTGAGGCTGAGAAGACGGT CA AGTGAGCTTACGGAAGAAGTGGAAAGGACGAGAGTGTCTGCATCGGAAATGGCTGAGCAG AA AAGAGAAGCTATAAGACAGCTTTGTATGTCTCTTGACCATTACAGAGATGGGTACGACAG AC TTTGGAGAGTTGTTGCAGGACATAAGAGTAAGAGAGTAGTGGTCTTATCAACTTGAAGTG TA AGAACAATGAGTCAATGACTACGTGCAGGACATTGGACATACCGTGTGTTCTTTTGGATT GA AATGTTGTTTCGAAGGGCTGTTAGTTGATGTTGAAAATAGGTTGAAGTTGAATAATGCAT GT TGATAT GTAAATATCAATGGTAA ATTTTCTCATTTCCCAAAACTCAAATGATATCATTT AC TAAACTAACGTAAACTGTTGAC A ACACTTATGGTTAAAAATTTGGAGTCTTGTTTT AGTATACG CACCACCGCACGGTTTCAAAACCACATAATTGTAAATGTTATTGGAAAA A GAACTCGCAATACGTA ^! ]:TGTATTTTGGTAAACATAGCTCTAAGCCTCTAATATATAAGCTCT CAACAATTCTGGCTAATGGTCCCAAGTAAGAAAAGCCCATGTATTGTAAGGTCATGATCT CA AAAACGAGGGTGAGGTGGAATACTAACATGAGGAGAAAGTAAGGTGACAAATTTTTGGGG CA ATAGTGGTGGATATGGTGGGGAGGTAGGTAGCATCATTTCTCCAAGTCGCTGTCTTTCGT GG TAATGGTAGGTGTGTCTCTCTTTATATTATTTATTACTACTCATTGTAAATTTCTTTTTT CT ACAATTTGTTTCTGACTCCAAAATACGTCACAAATATAATACTAGGCAAATAATTATTTT AT TATAAGTCAATAGAGTGGTTGTTGTAAAATTGATTTTTTGATATTGAAAGAGTTCATGGA CG GATGTGTATGCGCCAAATGGTAAGCCCTTGTACTGTGCCGCGCGTATATTTTAACCACCA CT AGTTGTTTCTCTTTTTCAAAAAACACAAAAFTAAAAATAATTTGTTTTCTTAACGGCGTC AAA TCTGACGGCGTCTCAATACGTTCAATTTTTTTCTTTCTTTCACATGGTTTCTCATAGCTT TG CATTGACCATAGGTAAAGGGATAAGGATAATGGTTTTTTCTCTTGTTTGTTTTATCCTTA TT ATTCAAAAAGGATAAAAAAACAGTGATATTTAGATTTCTTTGATTAAAAAAGTCATTGAA AT TCATATTTGATTTTTTGCTAAATGTC AC C GAGACACAAACGTAATGCACTGTCGCCAAT ATTCATGGATCATGACAATAAATATCACTAGAATAATTAAAAATCAGTAGAATGCAIACA AA GCATTTTCTAAGTAAAACAGTCTTTTATATTCACGTAATTGGAATTTCCTTTTTTTTTTT TT GTCGTAATTGGAATTTCCTTTATCAAACCCAAAGTCCAAAACAATCGGCAATGTTTTGCA AA ATGTTCAAAACTATTGGCGGGTTGGTCTATCCGAATTGAAGATCTTTTCTCCATATGATA GA CCAACGAAATTCGGCATACGTGTTTTTTTTTTTGTTTTGAAAACCCTTTAAACAACCTTA AT T C AAA AT A C ΑΆΤ G T AAC T T T AT T G AAC G T G CAT C T AA AAT T T T G AAC T T T G C T T T T GAGA AA AATCAATGTACCAATAAAGAAGATGTAGTACATACATTATAATTAAATACAAAAAAGGA ATCACCATATAGTACATGGTAGACAATGAAAAACTTTAAAACATATACAATCAATAATAC TC TTTGTGCATAACTTTTTTTGTCGTCTCGAGTTTATAT GAGTACTTATAC AAC T AGA TTACAAACTGTGCTCAGATACATTAAGTTAATCTTATATACAAGAGCACTCGAGTGTTGT CC TTAAGTTAATCTTAAGATATCT GAGGTAAATAGAAiiTAGTTGACTCGTTTTTATCTTCTTC TTTTTTTACCATGAGCAJy AAAGATGAAATMGTTCAAAACGTGACGAATCTATATGTTACT ACTTAGTATGTGTCAATCATTAAATCGGGAAAACTTCATCATTTCAGGAGTATTACAAAA CT CCTAAGAGTGAGAACGACTACAT GTACATATTTTGATAAAAGACTTGAAAACTTGCTAAAA CGAATTTGCGAAAA ATAATCA AC AGTGCCAGTGATTTTGATCGAATTATTCATAGCTTT GTAGGATGAACTTAATTAAATAATATCTCACAAAAGTATTGACAGTAACCTAGTACT TACT ATCTATGTTAGAATATGATTATGA A AATTTATCCCCTCACTTATTCATATGATTTTTGAA GCAACTACTTTCGTTTTTTTAACATTTTCTTTTGTTGGTTATTGTTAATGAGC ATTTAGT CGTTTCTTAATTCCACTGAAATAGAAAATACAAAGAGAACTTTAGTTAATAGATATGAAC AT AATCTCACATCCTCCTCCTACCTTCACCAAACACTTTTACATACACTTTGTGGTCTTTCT TT ACCTACCACCATCAACAACAACACCAAGCCCCACTCACACACACGCAATCACGTTAAATT TA ACGCCGTTTATTATCTCATCATTCACCAACTCCCACGTACCTAACGCCGTTTACCTTTTG CC GTTGGTCC C AT T T C T C A AC C A AC C A AAC C T C T C C C T C AT AAAA CC CTCTCCCTTC TTATTTCTTCCT CAGCAG C T C ϊ C G C T CAA T G T C T C GC C

SEQ ID NO: 19 - AtC3H promoter polynucleotide sequence

ATCGTAAGT TTTTTTGTGTGTGTGTTAACAATGTACTCACTACTCACTGT CCATATTTTTG ATGTACGTATATCGAAAACATTCTGCCAACAAATGCAAACATAACAAAAGTCAAAAACAA TA ACA ^AACCGGGAATTAAACCAAAATGTAATTGCTTTTTATTAGTGTCAGGCCT'rCTGCTTAA AAATATTCTCGGCCCAGAGCCC TTAACACCTATCTCAATTCATATTGAAGAAAATGACTAT AT T AC T T GA _* C AAAAAC T T T AG T C AG AAAAAT AT GG A AT C T C T T T C GGT AC T GC T AAGT GC T A ACCTTAAAvTAGTATAGAATTCTTAGTTCATTCTCAAAAACATAGCTATATGTAGATTA AAA AGTTCGATATTATTTCCTGCAAAAGATGTTATAATGTTACAACTTAC AGAA .ATGATG T ATGTAGATTT A AAACTGGTACCGTAATTCA AAAAGATGGTGGTGGGTATGTATCAGTAA CGGAACTTACATATGCGTGTGTATTACTATGTCTATATGGTGTATTCCTTTGTGTGGAAC AA TGCACGTCAGAGTTGTTTATTTTCTTATAGAATTTAAGGAATCAATTATTGGATTTCTCA AG GTGAAAGTGGACTTCTTTGCACGCAAGGTCTAGTTGCCGACTTGCCGTTGCATGTAACAT GA TTGTTGAAATAAAGTGAATTGAGAGAAGTTTGGCC GACATTTTAAATTTAACCCAAAAAAA GTAGGGCCTAACACAAAATATAACCTCTCTTTGTTCAAAGGAAATAACACCTACGTCTTA TA ATTGAACCAAACATTGAATCATTGAACTCACC AT ATAATTATAATAACACGAATTCACAA GACACCTAAAAGAAAAAGTTCACAAAAACAAATAAAAATTTACCTCTCACCAAACACACT CA C C G C C G T C T G G T C C C AC T G AC C C C AAC AT AC AA C AC C G AC T C T C T C C C AC AC CAAT T T T T TTTTTTGGCGTTTTAAAACAAATAAACTATCTATTTTTTTTTCTTACCAACTGATTAATT CG TGAATAATCTATTATCTTCTTCTTTTTTTTGTGACGGATGATTAGTGCGTGGGGAAATCA AA ATTTACAAAATTTGGGATGATTCCGATTTTTGCCATTCGATTAATTTTGGTTAAAAGATA TA CTATTC TTCACCAAGTTTTCAG TGAGTCTAAAAGATAATATCATTTCACTAGTCACTTAA AAAAAGGGTTAAAAGAACATCAATAATATCACTGGTTTCCTTAGGTGACCCAAAAAAAGA AG AAAAAGTCACTAGTTTCTTTTTGGAAATTTTACTGGGCATATAGACGAAGTTGTAATGAG TG AGTTTAAATTTATCTATGGCACGCAGCTACGTCTGGTCGGAC TACCAAGT CCAACTCT C T C T A C T T CAT GT G AT T G C C AAT AAAAGG T G AC GT C T C T C T C T C T C T C AC C AAC C C C AAAC C

A.CTTTCCCCACTCGCTCTCAAAACGCTTGCCACCCAAATCTATGGCTTACGGGGAC ATGTAT TAACATATATCACTGAGTGAAAAGAAGGGTTTATTACCGTTGGACCAGTGATCAAACGTG TT TTATAAAAATTTGGAATTGAAAACATGATTTGACATTTTTAATGATGGCAGCAGACGAAA CC AACAACACTAAGTTTAACGTTCGTGGAGTATACTTTTCTATTTTCGAAGAAGACATATAA CT AAGCTGATTGTTATTCTTCATAGATTTCTTTTCACTGCGAATAAAAGTTTGTGAACATGT CA CCGTTTGAACACTCAACAATCATAAGCGTTTTACCTTTGTGGGGTGGAGAAGATGACAAT GA GAAAGTCGTCGTACATATAATTTAAGAAAATAC TTCTGACTCTGGAACGTGTAAATAATT ATCTAAACAGATTGCGAATGTTCTCTACTTTTTTTTTGTTTACATTAAAAATGCAAATTT TA TAACATTTTACATCGCGTAAATAT C TGTTT A CTAT- ATTAATGAAAGCTACTGAAAAA AAACATCCAGGTCAGGTACATG ATTTCA.CCTCAACTTAGTAAATAACCAGTAAAATCCAAA GTA?iTTACCTTTTCTCTGGAAA.TTTTCGTCAGTAGTTTATACCAGTCAAATTAAAACC TCAA AT C T GAAT G Ϊ T GAAAA GAT T C C A G AA TTTCTCATTG GAAT AAAGT T CAAT C T G AAATAGATA T C T C T G C C T GT T T T T T T T T T C T C C AC C AAC T T T C C C C T AC T T C C ATCAATAATCGACATTATCCATCTTTTTT TTGTCTTGAACTTTGCAATTTAATTGCAT CT AGTTTCTTGTTTTACATAAAAGAAGTTTGGTGGTAGCAAA ATATATGTCTGAAATTGATTA TTTAAAAACAAAAAAAGATAAATCGGT CACCAACCCCCTCCCTAATATAJ^ATCAAAGTCTC CACCACATATATCTAGAAGAATTCTACAAGTGAATTCGATTTACACTTTTTTTTGTCCTT TT TTATTAATAAATCACTGACCCGAAAATAAAAATAGAAGCAAAACTTC

SEQ ID NO;20 - AtHCT promoter polynucleotide sequence

TTCTCTAGGTTTTGAAGCTTTCCTAGTTCTTTTGGAAGCGTGCCGGACAAGTCATTGTCG TA TAGAAACAGATTGATAAGTTCAGAGCAGTTTCCAAGCTCTTTAGGGATCTCACCTGAGAG CA TTGTAGAATAGACAGATAAAGACTGGAGCTTGCTTAGTTGACCCAACGAAACAGGTAAAG AA CCGGATATTTTCGTTGCTGCTAACCCTAAGACCTTGAGATTCCT CAGTTTCCGATCTCCTC C GG GAT C T C C C T G A AGC C GAG TTCCTCCGGCTCT ATGC CT C AG AG T C GAG AT C T TTCCGAGCTCCAACGGGAGATTCTCGGATAAGTAGTTATCGAAAATCTCAAGATTCTTGA GG CTAACGCAGTCGCCGAGT CCGGTGGGATCTTTCCTGTGAGGCCATTGGAGTTTAAACAAAG TTCTTGAAGATTCTTGAGC TCCCTAGACTCGAAGGTATTTCACCAACAAGACTATTTGAGC TTAAATCGATAACTATAAGCTCCGAAC'AATCTCCGATCTCAGAAGA A GCTCCGGTGAGA T TAGTGTTGGAGATAACGAGTTTCTGAAGTGAAGTAAACGAAGAAATGTTAGGAGGGAAAG G TAAAGCTAACTGAACAGAGACGACATTGATCTCTGTAACGAGTTTGTTGTCTGAGGAGGA AC AAGTAATGTAAGGCCATTGACATGGGTCAGAATCAGAAGGATTCCAGCCGGAGAAGACTG AC GGTGGCGGCGAGTTCGAGCTGTGAAGCCAAGAAATCAAAGCTGAGACTTCATTGGTTGAT GC AGAGGTCGAGGAGATGAAGAAAGCTAAAAACAGAGACAATGTAATGGAAAAATGAGAAAC AG TTAAGGCTTTTTTTCT GGAATCGGCATTTGCAAAGACATAAGAGTTTTTTTCTTTGCATTT GGCTCTCAAATCCAAAACAAGCCTTCTTGGTTCTGCATCGATCTGAGTCCTTTGGCTTAG GG TTTAGGGAAGTTTTTGCTTTAGAGATAAGCAATAAGAAAGAATGATATATTAAATATAT AA AGTACTAAACTTCATGTGC'TCTGTCTTTTTCTTTTACCTCGGGGTTCTGTTTCTAGCTT CAG ATTAATTAATTACAGTCATTAACTTTTCTTTGSAATATGTTTGCCAAGAGCCCGAGACAC TA T CCATAGAT GACAAAAGT CAATAGTTATATATACATAAAATAT C ACAAAAC AAAAGG CAT T G GTTATATATATACAGAATCATTTCACTTAGTAGTGTTTTTTCTTATAAGATTATGATAGA AA TATGGAAGCATGCATGTGGTTTTGCATTGTTTTCCTCAATTAAGTCAGGATTGTGAGTTG GT TTGTTTTCGAGACCTGAACCGAGCGTTTAAGATTCTTCCTCGTTTGAAGTAAACTCCATA AT TGTCCACACCTAAGCTAAAAGAAAGTAATAACAAGTTTAAATATTCATGACAAGGAAAAT AT TGCATTCAGAAAATTGTTAACAACGAAGTAAACATTTTTTTCAATCCGATGCCAATAGTC TC TAGCGGCATCAAAAGTCCACAAACTCGATACCTCTGGGTAAATGAGCGAATGGGCCGGTC CG T T G GC C C G AAG GAAAT T G C C C T AAA T C C T AC T T C C GAAT TTTCTCT GT AT AT C C T C G T T T GAT G T A GG T T T T G ΐ T C C G C C T AAT CAT G AC C AAC C C A G G T G AAT TGTCATTTAAGCTTTGATTGGTATTTGGTAGCATGGGTTACCATTGACCAACCCACGGTA CT AGTTGCTTTTCTTTTAGTTTTGCTTTTGCTTTATTTTCTTAGAGAGTGGGAGGACAAAAG GT T T G GAT C AT TAAGC CAAT GAAT G C T CAAAGAAAT T GAAT T T T TAT T G AT CC T CAAAC CAA GTTGGATCATCAAATAATGGCTAAGAAATAATTTTAGAACAGAAAGCAAAGAAAAGCTAT CC GCAAC AAC AAC CAT T AGTT AAT AAAT T AAAAT G AAT GT GAAAT T T T GAC T AAT TGAGGT A TGTTTTCATATAATATAGTATAGTTCGGATATAAATTCAACATAATTTATTTGTGGTGTA CT GAAAAAAAGACTTTCTTGGATTCTGACGTAATTCTCTTAACACGTGAGTTTACGCCGTTA GA TGTTATTGGTGGTTGTTGTTATGCTCTGCTACGTGGTAATGAGTTAAGTTAAGCCAAACT TT GGCATTCGATTGACTAACTTGTACGGTAGCTATAACAATCAACTTGTCAATTTTTTTTCC TT CTTCTTCATTCGAACTTTATACTATTTAAGCCCATTAGTATTATTTGGGCCTTAGGACAG AG GGAACGGGTTTACCAACCCCGGATAGAAAAGTAGGACCGAGTGATGAGATGGACCAATGA TA AACCTTCTGAGAGAGTTGGTCGACAGATGGAGTAGGCGGGGTCGTGGGGCGGTAGGTGAA GG ATTACGACCTTTCCTTTTTTGTTCACACCCACTTATATCTACCCCTCCTCGCTTCTCACA CA ATTTCTCAGATCAAACTCAAAACAAAATTTGTTTGTTCGTTTGATCTTTCTTAAAAAT

SEQ ID O:21 - AtCCRl promoter polynucleotide sequence

TTGCTTTCTCTGTCCATGATATGAGGCATTGACTTCTCACCTGTATTCATATGGTAT AGATTCCTCTT T CAGGAG CCAATACA ACGAGCTTGGTGAAGAACTCGTTGGTAAGAGAGTTAATGTCTGGTGGCCA C CGACAAGAAGTAAGTT TTGTTAAACTTACTAACTTCATTTTTGATACTATATGATGAATGATAG CAATGTTACGATTl'GTATTTGCACAGGTTTTATGAAGGTGTCATAAAATC TTATTGTAGAGTTAAGAA GATGCA CA^STGAG TAACTTCTCTATTTGGTATTTTAAAATTCTCTATTTATTGCATAACTGG TT ATATAGAATTTTCCCACTGATGGTCTCGCAGGTAACATATTCTGATGGCGATGTTGAAGA GCTTAATC TGAAAAAAGA iCGTTTTAAGATAATCGAGGATAAATCTTCAGCCAGTGAGGTGAA/VATTTCTTACATT CTATCATTCACCATTCTTTATATTTACCAAAATTTCAATG A CTGGTTTCCCTAATAAAATCTAAGC AGGATAAGGAAGATGATCTGCTTGAGTCTACTCCTTTATCTGGC GTAAGTGAAACTTCCATAGTTC TATGATAACCCACAATTTATAATTTTAATTTAGCTTTAGTCTTGAGTTTTTTGCTGTTAT GTGCAGTA

:I¾CAAAGGGAGAAATCCAAGAAGAGGAAAATTGTGTCTAAGAATGTGGA \CCGAGTAGTTCTCCAGAA GTCAGGTATGAAAGTATATAAGAATTCTAGTTTTAGTTG TTGi AAGTTTGATCCGTGAGTGAATTAG TTCACAATTATGGATGTAGATCCTCTATGCAAACAATGAAGAAGAAAGACTCTGTAACAG ACTCCATT AAGCAAAC¾AAAAGARCCARAGGTGCACTGAAGGCTGTAAGCAATGAACCAGAAAGCAC TACAGGGAA AAATCTTAAATCCTTGAAAAAGCTGAATGGTGAACCTGATAAAACAAGAGGCAGAACTGG CAAAAAGC AGAAGGTGACTCAAGCTATGCACCGGA AATCGAAAAAGATTGTGATGAGCAGGAAGACCTCGAAACC AAAGATGAAGAAGACAGTCTGAAATTGGGGAAAGAATCAGATGCAGAGCCTGATCGTATG GAAGATCA CCAAGAATTGCCTGAAAATCACAA'rG'rAGAAACCiiAAACTGATGGAGAAGAGCAGGA GGCAGCGAAAG AGCCAACGGCAGAGTCTAAAACTAATGGAGAGGAGCCAAATGCAGAACCCGAAACTGATG GAAAAGAG CAT'AAATCATTGAAGGAGCCAAATGCAGAGCCCAAJ TCTGATGGAGAAGAGC7\GGAGGCJ\GCAAAi\GA GCCAAATGCTGAGCTCAAAACTGATGGAGAAAATCAGGAGGCAGCAAAAGAGCTAACTGC AGAACGCA AAACTGATGAGGAAGAGCACAAGGTAGCTGATGAGGTAGAGCAAAAGTCACAGAAAGAGA CAAATGTA GAACCGGAAGCTGAGGGAGAAGAGCAAAAGTCAGTGGAAGAGCCAAATGCAGAACCCAAG ACCAAGGT AGAAGAGAAAGAGTCAGCAAAAGAGCAAACTGCAGACACAAAATTGATTGAGAAGGAGGA TATGTCTA AGACAAAGGGAGAAGAGATTGATAAAGAAACATATTCA ;CA'rCGCTGAGACTGGTAAAGTAGGAAAC GAAGCTGJ^GAAGATGATCAGAGAGTGATTAAGGAACTGGAAGAAGAGTCTGACAAGGCA GAAGTCAG TACTACGGTGCTTGAGGTTGATCCATGAATGAAGGATTGTTAGGTAAATGTTAATCCAGG AAAAAAAG ATTGGTTCTTGTGGT TAGG AACTTATGTATTAAGTGAAGCTGCTTGTTTAGAGACTAATGGTGTGT TTTATGAGTAGATTCTTCTGACCTATGTCTCGTTATGGAACTAGTTTGATCT ATGTCACCTTGCTAG CAGCAGATATTGATATTTATATATTTAAGAGACATGCGCATGAGAATGAGGGTATGGAAA iVGTCCATA TCAGATGACAi^AACAJVrGATCGTATGTGTAGTCACTTGTGCATTTCGAGTTTTGGACA T/sJii ⁱ ATTCT GATATTGCATAGAAATGTTTTTAAATAACACTAATCCAAACCTAAATAAAATATCTCTAT ACATCATC TAGAAATGTATGGCTTGATCAAGAA TGTAGATAATAATACCCTGAGTTAAATGATTGTAGGTATTAT TTCAGTTTTCAAAATTGTCCAAATTTATGAGC ATATTAAAGATAATATTTTCAATAAGGTGTGTAGT TC AAATGTTTCTTCTTCTTCCACCAACCCCTCTTTCTATATGTA GTTCTTTTTTCTAAAATAATTG TTTGTTCTTTTTTAGATATATCAAATTAAATATAAAAAATATTGACAAAACTTATTTACC ATTGTTAG GTGAACTTGGCAAGTGTGTAAATATAAAGATAACATTCCTTTTCGTTCTTTATATATACG AAACGTAC CACAAATTTCTAACTAAAGCATTCATAGTCTCTCGAAAGCCTCTTTTCAGAACCGAAGCT CT TACTT TCG'TCCACCGGGAAAT

SEQ ID NO:22 - AtCAD4 promoter polynucleotide sequence

CAGAAAGGTCTTCACACTCTGTTTTAGCTAGAGAGTTTTATCCATCTGAGTTTTTAGTCT ATTTTGTT TTATCTAGGAGTTGCTTTGTTTGTTCGAATTCGGTCAT GCTTTTGC GCTTTACTGGAGTCAAATTT GAAGGTAAAATATATGTTAAATATCTGGGTAGGTGGTTGTGGATGATGGAAAATCTGAAC GTATCACT GTTAATGACAATGGAGARCTCGTTTCTACTCAGCATGCTATCACCGAATACCGAGTGATT GAATCTTC ACCACATGGTTAGTGAGACTGACTTCCATTTCTATTCAGTTAAACTTAAAGCAAATGATT TTGCCTTG AGTTTTTAGCACATTGTTGAATTGCAGGATACACATGGCTTGAGCTTCGCCCTTTAACCG GGAGAAAA CATCAGGTCTCTATAGATATTCAGTTTTTGTTTCAACTTTCTCTCTTTTTTATGTTCTCT TAATACTA ATCTGTTTTCAAC GTTCTTCGATTGCCACAGCTTCGTGTACACTGCGCTGAAGTGCTAGGAACACCG ATAGTCGGGGACTACAAATACGGTTGGCAAGCTCATAAAGCCCGGGAACCTTTTG CTCTTC GAAAA CAACCGAACCAAGCAATCATCATCTCCTTTTGGATTGGATCTGGATGG ^r rGGAGA ^r rGTCTCTTCGAAAC AGCCACACCTxCATCTCCATTCAAAGCAAATCGATCTGCCAAACATATCACAGCTCTTGG AGAAAATG CAGGTCTCTTCAGACTCTGATATTTCGGATCTCGATAGGCTTAAATTCGATGCTCCATTG CCTAGTCA TATGCAACTAAGCTTTAATTTGTTGAAATCTAGAGTCGAAACTTGTGACAAAAATTAGAT TTTTTTTC ^r !'TAGCGAGCTTTCTTCTTTGTGTTCATTGAGGCCCAAGTATTTGTGTAl ^{, r} rTGGACCTGAATATTCTCA 1'AG/ AGATAAATAATTATAATTAAATGATTTT CGCATATAATCATTA1 ^: GTGGTATGATTAACACA GTTGGTGTGATGACTGATTGACACAATAATCACCGTTTGGATTCGATTCCTTTAATAGTT GTCACTAG AGTTGTTTGACTAAACAGCTAACTTGTCACTAGAGTTATTGTGTTTGTATTTTGATCTGT TATTAATC TGATTGGGTATAA TACAGATAGAGAGACATCTATATTGT/IATTAAGACAATCTTAAAGTGTAAACTA AAAAGATCTCTCTGACCTCTGGAAAACGAAAGGTGGGTGACACA CACTCTAGCTATGAATATGATGA ATAT CAGTACCTAACCGAACAAAGACTGGTTTGGTATTTTTATTGGAAAAAAGAGATAAATAAT TGT GAATGTGAATTATCCTGTCTGAAAGGTAAGGTGATGACATGGCG TA ATGA TGGACGAGCTTCAGA ACAAAAGAGTAGCGTCGAaTCGAATCTTTACCTACTACACTTTGAAC TTGAAGTACATTACCTACTT CCTCCTTGATCGAACGTCTTTTGTCAAA¾GTATTTTATTTCCCCAATTAAAGTAGTGGT GATAAATTC ACAAAAATACAAACACTTTTATTTTTGAGGTCAAAAACAAATACTTCTTTGAACAGGCTA TTACAATA TTTT AAGAAAAAAGTAAGCAAAATAGTCCACAAi CCAAAATCTGTAACATATTAAACGATTTATGTT TTTTTTTTTTTTTCTTAACTAGAGAACAATTCGGGCTTTTACTAAGGATGATGAGTGTAG TTACCGAA TAGTGTATTCATATAATC T^TGAGCTTAAGATATGATATTATTTCGACTAATCAGATAAGAGTA GTTAGATAATTTCGTAATAGAGCAACTCTTTCGC^^

TTCl' TTTTTTTGGTCACAACCAATTAGTTTGTTTGTTCTAT TTATGAAGTGCGTATTAi AGCTAAC GTGl'?TAC:AG¾AACXX CAGAGAAATAAAAATAAAAATAATTATGTACTTTATGGATTTATA(mAAiiAA CAAGAATAGTCACCAAAAATTGATTGTGTCATATATCTTTTGTCAACTATTTTATCT ATTTTTCTAT GGATATGTATGTCCAAAATGTTAGACAAAAAACCAAAAAATCATGTCCAAAATTTCGT AGGGTGCCG

AT TCTCTGTTTCCCTTTCAACGACTATCTATTTAATTACCGTCGTCCACATTGTTTTTAATA TCTTT ATTCGAGG TGGTTTAGTTTTTTTTACCAAACTCACTTTGCTACGTTTTTGCCTTTTTGGTATGGTTG

TATTTGTACCACCGGGAAAAAAAAGATAAGAGGTTTGGTTGGTCGAGCTTACTGATT AAAAAATATAC CGTCCAGCAAATATTAAAACAATATATCCGATTT TCCTCCTCTCTTT GGTATTACATTAATATTT ATTATTTCCCCATTTGCTCTGTA ATATAAAGATATGTCAATAGAGTGCCTCTACAGTCATGTTTCC ATAGAGATAATCTCTCACCATTGTTTTTCTC GCAA :TAAI GAAACAAAAAAAGAAAAATCGGAGA AACCAAGAAAAAAGAA

SEQ ID NO:23 - AtCADS promoter polynucleotide sequence

CCTCGATAACTCTGATTGTTGTAT GTCCAAGTATTGACTAAACAAC TTGCTAAAAGAGAAGATGCT GCTGGAGCAA TTCAGAAGGTTTTAGCACAACGGGATTACCAGCTGGAATAGCTCCAATGACTGGCTC GACAGACAA ACTAAGGAAAAAAACA2VAGGACCATGAAGACATATAAACTTTAATAGTTTAGAAATTG AGACAAAAT GTCAATAA VrAAAATTGAGCTTACAGAAAGGGAAATTCCAGGCTGAAATAAGGAAAAC AACTCCAAGCGGTTCTGAGACTATTTGTGCAGACGAGGGAAATGTTGTCACAGAAGTTTT GAGCTGAA AGGTCCAAGCATAGAAAAAGCAAGTGGTTTTAGAAAGGACACATATCAATGAAGCAGCAA AGCTTGAA CGGTGTAGTTACCGTTTCTGGAGCCATCCAGTTCTTTAACTCTTTGATTGCAAGCATACA GGATGA T TTGTAVTTCGAAATCTAAAAAACGAGAAJ-AATACCAAAGAGATTCAACAGTGGATAAGT GGAATGCAGT GAAGAAACGGGACATTGAAATTATATAAAAAACCTCAGCTAGAAAACSC TCAAGCTCAGGCTTAGAAA GATCTTGATACAAAGCTTGGGTGATGCATTTCTCGTTCTCATCAATGATCCTAGCAATGT TTTGAAGC TGAGAAATTCTCCAGTCGTAGCTCTTCGTTCTGGGAGAGTTGAAGTTGC TCTGAGCTCATCTACAAG CAAAGCTGCTTCTT TCCACTAAAGTCTGATGCTTGCTCGTTTACCACAGCAGATAGTGTTGCATAAC

AAGTACTGATTCAAGACACGAAAACCGCA TGTGAGAGACTTTAAGACTAAAAATCATGGATAAGACT AAAAAAACATGGATAAGTATCAACTGTTCTCACGATTATTTATTCATACCACTGTACTTA AACT AAA ACCCACTATACTAAA AGAAAGG AATCATCAAAAAATCAGTATGTAAAAACCACTTTTGTGAATAAA ATATGTAAAATGGGTGAA ^R [AAAC;AAATGTGCT ACAATTTCAACCGATAAGGGATACAAGCATTGGTG GAAIATGCACCAGCACCACGACGAGATATCCGAAAAGGTGAAGTTGGAACATT AATCTGGAACAAAA GAI^GCGATTCATTAAIIATGGTACTAATTAGATCTAATCATATCAT TTGAATGAGCAAA'RGAL'TGACA GI^GCA GCAI'TGGTCCAATTAACATTCTAGACCAAATTCAACTTAAAGGTAAGTG^'I'TTATAC AGGA A^ GCGAGAIII CCGAAAACGCAATTCACATAAAAAGGAAGGCT GTTTGGAGAAGCAGAATGGAAGAAG 'RCAATC CAAACCCTGATGAGCAGGTTTTTCAAGTTACCTGGCAGGAGAAAAACGCTTGGGAAAACA A AGGGTTTGAATATGATTAATCTCTAGAAGCTTCGTCATGACTTGGGTTGAGTTAAAAATC TCAAAT G GAGACATTATTGGTGTTTA ATATTTGAGAGAGAGAGCCAGAGAGGAGACGTTGAATTGAATGAAGGG TGTGGTCGGAAGAGAAGACGTGTAGAAGAGAGGAGAGAAGTAAATT AAGCATTGGCCCCATTTACAG CCACAAGTCCGCTACAACAAATTA TTCCAAGAAAGTCTGAGATAACGTCGTGATGAAACGGCTGATG CTGCTGTTGTGATTGGTGAATTAGAGGTTTATGL'TTTGGGTTTTTGAATGTTACTTAAT TGGACGGTC GATTTTTGAAACTGGGTGTGAAATGTGAATGGGTCATTCATAATGGGCTTTTGTTT AATGTGAAGCC TTCACACACTCTTTGTCCTTCTTTTCTATTA TCATAACTGTCAC CTTTGTTCTTCG A TAGTAA AGAGCAAATCGATTCTTTG TGATCTGGGCCGTAAAATTTCCATGGTTGTGGGAAGT TTCTCGCAGC TGATCTGGC3GCG CAATGGTACAGTTTCATGTCAGAGAGAGGTCAAGAATCAACAGGTGGCCI ACCAT GATTTTAAAGCAAAGCAAAGACACGATTAGACCCCACATTGTTTGTTCACCAACGGGCGT GGACCCTC CTTTAGGGGACGTGTCCACGTCAATAGTGGTTTTTC TCCTTTCAAAGTAGRCAAATTCCATTCTTTC TCATTTTACTTTTTGGATTACGTTGTTGTTATAAACTGGTAAAATGAATTATGAATGCAA ATAAATTT CATTTAAGTTTTGTTGGCTTCTAATATTTTTTTCACC AAAA TCTAATAAACTAGACAGCCATGAGC CATCGTATGAAAAGAAGAAGAAAAAAAATGTCTTTTTCTAGAAGGATC

TT AAGCTTTTGACTAATTTTGTCAATAATATACACAAATTTAGACTCA? TTATAGCCATCAAATGTG TGCTATGCAGAAACACCAATTATTTCATCACACATACGCATACGTTACGT CCAi CTTTCTCTATAT ATATATATAGTAATACACACACATAAACAGCAAAAGCGTGRAAGCAGCAGATCAAGATAA GftAAGAAG AAAGAA T CAT CAAAAA

SEQ ID NO:24 - AtF5H promoter polynucleotide sequence

TGTGTGTCTTTTTGCGAGTAGTTGTTGGCTTCAGACAGTTCATAGCGGAGTTACTCTATA CGCGAAGT ACTTGTCTCATACTGATAJVTTTTGATGGCAATTAAGGCTTTAAAAGCTTATGTATTTTC TTATAACCA TTTTATTCTGTATATAGGGGGACAGAAACATAATAAGTAACAAATAGTGGTTT ^.TTTTTTTAAATAT ACAAAAACTGTTTAACCATTTTATTTCTTGGTTAGCAAAATTTTGATATATTCTTAAGAA ACTAATAT TTTAGGTTGATATATTGCAGTCACTAAATAGTTTTAAAAGACACGAAGTTGGTAAGAACA GGCATATA TTATTCGATTTAATTAGGAATGCTTATGTTAATCTGATTGGACTAATTAGAAACGACGAT ACTATGAG CTCATAGATGGTCCCACGACCCACTCTCCCATTTGATCAATATTCAACTGAGCAATGAAA CTAATTAA AAACGTGGTTAGATTAAAAAAATAAATTGTGCAGGTAGCGGATATATAATAC'iAG AGGGGTTAAAAA TAAAATAAAACACCACAGTATTAAATTTTTGT TCAAAAGTATTATCA VIAGTTTTTTTGCTTCAAAA ATATCACAAATTTTTGTATGAAATATTTCTTTAACGAAAATAAATTAAATAAAATTTAAA ATTTATAT TTGGAGTTCTATTTTTAATTTAGAGTT TTATTGTTACCACATTTTTTGAATTATTCTAATATTAATT TGTGATATTATTACAAAAAGTAAAAATATGATATTTTAGAATACTATTATCGATATTTGA TATTATTG ACCTTAGCTTTGTTTGGGTGGAGACATGTGAT ATCTTATTACCTTTTTATTCCATGAAACTACAGAG TTCGCCAGGTACCATACATGCACACACCCTCG GAAACGAGCGTGACTTAATATGATCTAGAACTTAA ATAGTACTACTAATTGTGTCATTTGAACTTTCTCCTATGTCGGTTTCACTTCATGTATCG CAGAACAG GTGGAi^TACAGTGTCCTTGAGTTTCACCCAAATCGGTCCAATTTTGTGATATATATTGC GA'tACAGAC ATACAGCCTACAGAGTTTTGTCTTAGCCCACTGGTTGGCAAACGAAATTGTCTTTATTTT TTTATGTT TTGTTGTCAATGTGTCTTTGTTTTTAACTAGATTGAGGT TAATTTTAATACATTTGTTAGTTTACAG ATTATGCAGTGTAATCTGATAATG AAGTTGAACTGCGTTGGTCAAAGTCTTGTGTAACGCACTGTAT CTAAATTGTGAGTAACGACAAAATAATTAAAATTAAAGGGACCTTCAAGTATTATTAGTA TCTCTGTC TAAGATGCACAGGTATTCAGTAATAGTAATAAATAATTACTTGTATAATTAATATCTAAT TAGTAAAC CTTGTGTCTAAACCTAAATGAGCATAAAIOCAAAAGCA-AAAATCTAAACCTAACTGAAA AAGTCATTA CGAAAAAAAGAAAAAAAAAAGAGAAAAAACTACCTGAAAAGTCATGCACAACGTTCATCT TGGC AAA TTTATTTAGTTTATTAAATACAAA/ JVrGGCGAGTTTCTGGAGT TGTTGAAAATATATTTGTTTAGCC ACTTTAGAATTTCTTGTTTTAATTTGTTATTAAGATATATCGAGATAATGCGTTTATATC ACCAATAT T TTGCCA/mCTAGTCCTATACAGTCATTTTTCAACAGCTATGTTCACTAATTTAAAACCCA CTGAAA GTCAATCATGATTCGTCATATTTATATGCTCGAATTCAGTAAAATCCGTTTGGTATACTA TTTATTTC GTATAAGTATGTAATTCCACTAGATTTCCTTAAACTAAATTATATATTTACATAATTGTT TTCTTTAA AAGTCTACAACAGTTATTAAGTTATAGGAAATTATTTCTTTTATTTTTTTTTTTTTTTAG GAAATTAT TTCTTTTGCAACACATTTGTCGTTTGCAAACTTTTAAAAGAAAATAAATGATTGTTATAA TTGATTAC ATTTCAGTTTATGACAGATTTTTTTTATCTAACCTTTAATG TTGTTTCCTGTTTTTAGGAAAATCAT ACCAAAATATATTTGTGATCACAGTAAATCACGGAATAGTTATGACCAAGATTTTCAAAG TAATACTT AG A CCTATTAAATAAACGAAATTT ^r rAGGAAGAAATAATCAAGATTTTAGGAAACGATTTGAGCAAG GATTTAGAAGATTTGAATCTTTAATTAAATATTTTCATTCCTAAATAATTAATGCTAGTG GCATAATA TTGTAAATAAGTTCAAGTACATGATTAATTTGTTAAAATGGTTGAAAAATATATATATGT AGATTTTT TCAAAAGGTATACTAATTATTTTCATATTTTCAAGAAAATATAAGAAATGGTGTGTACAT ATATGGAT GAAGAAATTTAAGTAGATAATACAAAAATGTCAAAAAAAGGGACCACACAATTTGATTAT AAAACCTA CCTCTCTAATCACATCCCAAAATGGAGAACTTTGCCTCCTGACAACATTTCAGAAAATAA TCGAATCC AAAAAAAACACTCAAT

SEQ ID NO:25 - AtPALl promoter polynucleotide sequence

CAAATAGTACGATGTATTTAGTGATTTTATT ATGTACTTTGTTCATTAAATTAGTCATAATTG TCT GATTTTTAGGGGTTTTGATCGAACCCTTAGATCAAAAGTTACCTTAATTGTTTTTTTAGC TAAGTACT TTA TAAAAATTTAATGTTTAGTTCTGATTGAGTAGTACTATJ^AAGGAGACATGTGTCAATCT TGTCA ATTGGTTTTGAGTTCAACAATATGCAATATTGCACATGCA T/L¾CGACCAAAAGAAGA?GCAATGCAC TTAAATCATTGAAACTGATTTTGTTTTTGTAGTGTATA IAATATCTATTTAATTACCAACGAAAGAAG TGAGCTTTTAAAAACAAAGAGTCAGA¾(;MATATA ^R L7\/ GTACAAAACCTACAGAAGATAAGCTGGAT TCAAAAGAAGAGAAAGAGTAAI :CAATAI ATTGACCAAAGCAAAATCGGATATTTGACATAAGTTTCC ATTCACATTGACCCAAI TCCACCAGGATL'LX^AAATAAAGTTACTTAATATAATTTTTGTGTTTATAA ATATTCCGCGCACTCTTGCCTTCATTTGGACCTTATCCTAAAAGTCAAAACAGGTGAAAA AAATGAGA ATACAATTAACACGAAAAATGCAAAAGAJJTGTTAJIACCGAAATCGAATTCTAGTGTAA TCAATCCT T TCCCAATGATACAACTATAAATCAAAAAGAAAAAATGTACTGATAAACGAAACTAAACGT ATAAATTA AT TATTTGTTGACATAAATAGGAGGCTTTTGCCTGCTAGTCTGCTACGATGGAAGGAAAAAT GCA G CACACATGACACATGGAAAATGTTTCAATGAAGACGCATTGCCCAATTAACCAACACACG AGTTCTTC CATTCC AGCCATATTATTTATTTCTACCATTTTCTTTAATTTATTGTTTTT GTTTGATT(;ATACACT GTTTATGAC^ ^: ATTACATT TCCCTTTCGACTAATATTAACGCGT7TAAACCA7¾I\GAATGGATT ^R ['GATA ATGAAATTT ATTTTATTAGCATATAGATAATGGATGGCTTCATGCTTGGTTTCCATGAGAII-GGAATG ACACAAGATAATTA TTTGAATAAAATCATAAATATGATAATACTAGTTGTAAIVAAAACTTGAGTGTT TCGTG GTTATTTTTCGGTTTCTTGACTTTTTATATTTCTGGTTTTTGTAATTTTAGGATGGATTA TT TAGCTTGCTTTTCTCTTTTATTACTTTCTAAAATTTTATTTATAAACTCATTTTTAA ATATTGACAA TCAATAAATGAGTTATCTTTTAATTAATAAAAAATTTGTAILACTCTTGTAAACAGATCA TAGTCACTA AAAGCTATTATAAGTTATTTGTAGCTATATTTTTTTATTTCATGAACTTAGGATAAGATA CGAAAATG GAGGTTATA TTACATAAATGTCAGCACATTGCCTTTGTGAL'GCAAACGGCGTGTTGCGTCACTCGCC TCCTATTGGGAATCTTATAATCGGGTGAATATTATTAGAGTT'I'GCGATATTTCCACGT AATAG TATC TTTCACAAATTTTATACTCAATTACAAAATGAACGAAAATGTACATTTGTATCTTTAACT ATTTACGT TTTTTTTACGTATCAACTTTCAG TATATGTTTTGGATA¾TATATTTTTTTACTTT GACTTTTCAGT TTTCACCTAATGATTGGGATATAGATATGCATGCATAGTTCCCATTATTTAAATGTAAGC TAAGTGCA TATGAACTGTTAGTCAAAATTACGAAGTTTATTTGTACA^

AATTAAACAGAACATCAAGAAAGTACAAAAGACTGAACACAATAATT ACATGAAAACAAAACACTT AAAAAATCATCCGATAAAATCGAAATGATATCCCAAATGACAAAAATAACAATATAGAAA ATACAAAA

ACAAAAACAAI^ATATGAAAGAGTGTTATGGTGGGGACGTTAATTGACTCAATTACG TTCATACATTAT ACACACCTAC;TCCCATCACAATGAAJ CGCTTTACTCCAAAAAAAAAAAAAAAACCACTCTTCAAAAAA TCTCGTAG CTCACCAACCGCOAAATGCAACTATCGTCAGCCACCAGCCACGACCACTTT ^R ! ^: ACCACCG TGACGTTGACGAAAACCAAAGAAAT CAGCACCGTG TAAAATGAAATTAAAAATAAGTCTCTTTTTG

CGACTTAAACCAAATCCACGAATTA AATCTCCACCACTAAAATCCATCACTCACTCTCCATCTAACG GTCATCATTAATTCTCAACCAACTCCTTCTTTCTCACTAAT TTCATTTT TCTATAATCTTTATATG GAAGAJ^AAAAAGAAACTAGCTATCTCTATACGCTTACCTACCAACAAACACT^^

CACCCT CATTCATCTAATTTTCC CAGGAACAAATACAATTCCTTAACCAACAATATTACAAATAAG CTCCTATCTTCTTTCTTTCTTTTAGAGATCTTGTAATCTCCTCTTAGTTAATCTTCTATT GTAAAACT AG T CAAAAG T CT AA

SEQ ID NO:26 - AtPAL,2 promoter poiynuc!eotide sequence

GATTGATGGTT AATAATCTGCCTCGTGATACATGGTGTTATCTTAAAATGGTCTCTCAATTAGTC TGTATTTGTATAAAATAAGGCX:TAAA; VTATCATCAATGGGGTCCTGTTAAAAACAAAAACAGATACA CCTTTCACTAATAAAAAAAJ ^CTGTTACCGACAAGTCAAACAATATCTGCGGACAAAAAAATGAAGAA TGTTTAGTAAGAAATAGAAGATGTGGTAAAGAGCCATACACACATGCAAGTGTTTT^

TCTTACCAI CCCACTACTTCTTTGAGCCATAATTGTTTGGTTCGGAGACCCTTTACATTTCGGTCTCA GCTTTAT L'GTTTACGCATTGATTTGTCT AAAT ATGTTAGATATTGTTTTTTGGCTATTTATTAGC AGCAA ^: GAAGTTAAAAGAGTGGTTCGATATCACCATCGAACTCTCGTTTAGATAI"[ ^,R ! ^,R RG'RATATAAAA CCAAACAAAAACAAAAAAATTGGTCCGATGATCTAIITA'RAGAAGT AGACGATTTCACGTTATGTTAT TACAACCTACAACAAAATAGAGTATGATCGAAATCAT TTGAATGTTTTACCTTTGAACGTAATACAA ATCTGGCTTTACAAAGCAATAATTCATGTTTGTTTGTCTAII TTAAATTTCCCTGTTTTTTTTCCCCT CTTTCTGTTTCCCA TTGAAAGTAAAAGATGATTTAAGCAGCTAACTCAATTTTATTTTATTTTAAAC ACCTAATGTCATGC CCTTGGCTCCTTG AATTAGTTGATCGTTTCAATTTAGACCAGCAAAACATTT TAGTATGTTCGTAAATATTGCGTACATGCCATTTCGTTTGTCATGCAAACGGTGTGTGTT TCT TACT TAGCTTCTAGTTGGTGTATATTGCGTCGCATTAATATCGGTTTACCTTCCTCCTGTCTAC GTAATGAT ATATTGTCCAGCACAIVATTTAAATTGTTATTGAAATTTCCTAATTTTTTAGGTAGCTCA AGGTCTGAA GTATACTACGTACCCTATTTTTTTGAATATCTATCTATATTATAACAAGAGTT TTCTGAGCTAGTTA ATGAGATGAGAI ^R [ATTCTAGATAAATAAATGACCCTCGAAAGTTTCAAGTACTTTAGGATCTGAGG AA ATCGGGGTAAAI\CATTTTGAAACTAATTACGTTCACATCTACCATCGATGATTGAGAAG GT ^R IAT'RGTC ACCTTTTATGTTAAAGTGACATGGTCTTGACGTTAATTTGCA GTTATTCTACATCTATAGTCCAAAG ATAGCAAACCAAAGAAAAAAATTGTCACAGAGGGTTCAATGTTACTTAGATAGAAATGGT TCTTTACA ATAATAAATTTATGTTCCATTCTTCATGGACCGATGGTATATATATGACTATATATATGT TACAAGAA AAACAAAAACTTATATTTTCTAAATATGTCTTCATCCATGTCACTAGCTCATTGTGTATA CATTTACT TGC TCTTTTTGTTCTATTTCATTTCCTCTAACAAATTATTCCTTATATTTTGTGATGTACTGA ATTA TTATGAAAAAAAACCTTTACACTTGATAGAGAAGCATfATTTGGAfiACGTATATAATTT GTTTAATTGG AGTCACCAAAATTATACAAATCTTGTAATATCATTAACATAATAGCAAACTAATTAAATA TATGTTTT GAGGTCAAATGTTCGGTTTAGTGTTGAAACTGAAAAAAATTATTGGTTAATAAAAT TCAAATAAAAG GACAGGTCTTTCTCACCAAAACAAAiTTTCAAiGTATAGATAAGAAAAATATAATAAGAT AAACAATTCA TGCTGGTTTGGTTCGACTTCAACTAGTTAGTTGTATAAGAATATATTTTTTTAATACATT TTTT AGG AACTTTTGTTTTTGATACATATAAACAAATATTCACAATAAAACCAAACTACAAATAGCA ACTAAAiVr AATTTTTTGAAAACGAAATTAGTGGGGACGACCTTGAATTGACTGAACTACATTCCTACG TTCCACAA CTACTCCCATTTCATTCCCAAACCATAATCAATCACTCGTATAAACATTTTTGTCTCCAA AAAGTCTC ACCAACCGCAAAACGCTTATTAGTTATTACCTTCTCAATTCCTCAGCCACCAGCCACGAC TACCTTTT CGATGCTTGAGGTTGATATTTGACGGAACACACAAATTTAACCAAACCAAACCAAAACCA AACGCGTT TTAAATCTAAAAACTAATTGACAAACTCTTTTTGCGACTCAAACCAAATTCACGTTTTCC ATTATCCA CCATTAGATCACCAATCTTCATCCAACGGTCATCATTAAACTCTCACCCACCCCTCATAC TTCACTTT TTTTCTCCAAAAAATCAAAACTTGTGTTCTCTCTTCTCTCTTCTCTTGTCCTTACCTAAC AACAACAC TAACATTGTCCTTCTTATTTAAACGTCTCTTCTCTCTTCTTCCTCCTCAGAAAACCAAAA ACCACCAA CAATTCAAACTCTCTCTTTCTCCTTTCACCAAACAATACAAGAGATCTGATCTCATTCAC CTAAACAC AC T T C T T G AA C C A

SEQ ID MO:27 - At4Cl l promoter polynucleotide sequence

ACATAAGATTTGGATTATGAGAGGAGTTGAGAAGTTATATGATGGAAACTGAAAAGTAAA TCTTTTTG CAGAGCTGTAGAATCAA'TCAACATTTGATGACTTGGACTTCTTCACCATGTGTGTTGGT GTGGACCAT TGAATTGACGGTTTTGCCATTCACCAACAACAGCATGAGTTTTTGAGTCTTCATGTTTGG TAAAGGTT AGGCTTATTAGGAGACACGGGTAAGAGACTAGAGAGAGACATTCTCCAAACCTTTCTTTT GCATGTTT TGTAAGAAACATTTCCGAAAATGAAAGAAATCTTACACAACATTCATATAATTTGTTTGA AATATAAC AAAATGATAATTTATACTCTCAAGTAAAATGCCTAAACTTTTATCAATTGGAAAAGACAT CACACACA AGCGTGAAGCGTATCTTATTACCAAACCCAACTAAGCATGGGTC'TCGATACT'TGCCAT AAT'TACTTTA ATCCATTCTCTTTTTGAGAAATGTATAAAACATGACTTTGCATAAA AGTCTTTTACTAA-TTACTATG TAAATAATTCCTAAGACTGGTTTCATGGTACATATTATCGTTTTATCCTTGTTTTAAGAA TATTCAGA TGTTTGGTCTATGGAATATAGTCTATTCTTCATGTTTAAAACTATTATTTGATAAGAAAA TATGTACT AATATGTTTTTGCATACAAATGTTGATCAGTTCGTAGCATTTGAATTAATACATTCTCAA TCACTTTC AAGCATTATTATGTAATAAATGATTCATGTCGAAAAGTAATAGTATCACTGTCCATTACA TTTGGCAT ATATATTTTTTTGTCAAAGCCTTACATTTGGCATATTGACGAAGCAGTTTTGTATTCACT TATATTTT GACATCGCTTTCACAAAAATAAATAGCTATATATGATTATTATCCATTAATTGTCTCTTT TCTTTTGC TGACACAATTGGTTGTAAATGCAATGCCAATATCCATAGCATTTGTGTGGTGAATCTTTT TCTAAGCC TAATAGTAAATAAATCTCAATACAAGAACCCATTTACGAACAAATCAAACCAAGTTGTGA TGGGTTAG TACTTAGTAGCCCGTTTGAAATGTAGAATTTTTGATGAGATTTTACGTTTTATATAGATT TTTCTCAG AAAACAAAAAATTCTTGCATCTTGCATTTTGGTCATTTGTAAATATTTTTTTAGTCTTAA AAAAGACC CAAATTCTTATTAATTTCAAAATTTTCGGTCTCTAATACCTCCGGTTTTAAAAAAAAACA TATCAGTT GAAGGATGAGTTTGGTGAAGGCTATATTGTCCATTGATTTTGGAGATATATGTATTATGG TCATGATT ATTACGA TTTTATATAAAAGAATATTAAAAATGGTGGGGTTGGTGAAGAAATGAAGATTTATCGTCA AATATTTCAATTTTTACTTGGACTATTGCTTCGGTTATATCGTCAACATGGGCCCACTCT TCCACCAA AGCCCAATCAATATATCTCTCGCTATCTTCACCAACCCACTCTTCTTCTCTTACCAAACC CATTTCCT TTATTTCCAACCC ACCCC TTATTTCTCAAGCTTTACACTTTTAGCCCATAACTTTCTTTTTATCCA AATGGATTTGACTGGTCTCCAAAGTTGAATTAAATGGTTGTAGAAATAAAATAAAATTAT ACGGGTTC AAT GTTCAATTGTTCATATACCGTTGACGTTCAATTGTTCATATACGGGTTCCGTGGTCGTTG GTAA TATATATGTCTTT ATGGAACCAAAATAGACCAAATCAACAACAAATGAAGAAATTGTTAGAGTATGA TACACTCATATATACCCAAATATAGCATATATTTATAATATAACTTTTGGCTATGTCATT TTACATGA TTTTTTTGGCTTATCTATTAAAAGTATCATACAAACTGTTTTTACTTCTTTTTTTTCTTA GAA ATAT ATGCCCAAAATGGAAAAGAACATATGCCAAGGTTGATTTTATCGCTTATATGGTAAAAAT TGGAAAAA CATACAAATCATTACTTTATTTAATTAAATCATGTGAAGAAACATATTCAATTACGGTAA TACGTTAT CAAAACATTTTTTTTTACATTAA'T GTTACATTTTTTTTT TTGCAAATATTCTTAAATAACCATTCT TTTTTTATTTACTATAATTAACATAAAAATAAATAAAATATAACATTTCAACAAAGAAAT TTGCTTAT GAAAAATACAAAATCCAGTTAATTTTTCAGAAAAATACAAATTTGCTTATAAAT T TTACCACTAGT TTATGTGATTTTAAAAGAAAGAAATGCAGCTTACCAAACGCAACGTGAAAATTTGAGAAA CCCATACT Ci\AAAAAGATTAAATGACAAAA^

ACCCCACCGTC ACTCCGGTGAATTGTCTATATGAACTCCTCCGATACAACTCCTGTTTCCTTCAGGC CAAAGCCTAAAATTCACACiiACCAALAAAACCAACCTTTTTTTTCCACCTAAATCTTTG AATATCACA AT TTTACT TTTACA

SEQ ID NO:28 - AiCcoAOMT promoter polynucleotide sequence

ACACATTAAAACAAAAACCATTTCCACATAAAILIAAA/\CGATCCAGTAAATGAAATAG ATTCAAGACC GATCGTCGAGCGGTAGAGAAAGTAAACAAAACAAAGACAGAGAA TGAAGAAACTGTGTACCTGCAAA AATACCAATCAGATGGGTCTCCGCCAIUGTAATCTGCTTAGAAGTTTTGTAAGAAAAAAC AATTAAAG GCGTTTCATTTATTGAATTTTCCGGTTGTTTGATTCTCAGGATGAGATTGCCTATTTCCT TCAAAAAA GAACTCTTTAATXTACACAGAAAAGCTCTGAAAATTTCCACAGAAAAT^^

AAGGGGAAAGAGATGAAATC;GG TATTAAAAAJUGAAGCAGTGGATGAGGGAAGAGAGGATTAAGAGG CGTAGAGATTACATGTGATGAATGATACTATCTTTTCTTACAAACACATTTTCGTGTAAT TAAAATTT AATTTGGTTCCAAAGATTTTAATCA/UIAGAAGTTTGGTAAATTGAAACAGGCAGACATA ATTTA TGT AAAGAGTTTTTATTTA TTATTCATGACGTTGCTTGATGGTGCTTTACCAATTTTCTTCTCCTACGTT AGATTTTTTTCACL ^,R [' ^R !' TTTTTTGGTGTTTGTAATAAATGTGAAAAATGGACCGTTTAAAAACTTAAA GACGTTTGAT ACTATATAAAGTAATTGTTTATAATAGAAAGTTAATTGAGACGTGAAATGGTA AAT ATTATTGTG A¾CAGT ^: RGTGTACACGTAGCTCTCATGCAGTTTTAGTGGACCCATATGGCTTGACTTG TATRC GTL ^: L'TTGGGCTATTAAAGTCCAAAACAGAGACCCCTCTCAAGCCCTTCCTATTAAT CCATCT AGCTAATAGAAACTATAAACGTGTCCTCTCTCTCAATTAAATAAGCTAGAAACATACTCA ACCATTCG CATTACX5CACTTCATAGCGGTAGGTTTAGATTTGTCTAAAATACTTAAAAAAATTTTTG TCTAAGTTG TTGTCCGTTACAAAG TTTTTTCTTTGTGACAACTTGACAACATTGACAAVRAGAAA IVRAAATTTCG ATGAAACCTATGAAATGGGCTATGGCCCAACTAAAAAGAGTGGGAAAT AA GATGGGATGGTTCAAG TGTATACTTCGAACTTCCGACATTAGGGTCAAAGGATTTTTAAAAGGCAACCATT GTTCCACTTTCT CGAACAAAAACGAGCCATTTATTAATA ATAGTACX3GCTGA/TTGGTTTTGTTCGTCATTGTGTAAAC ACAAAGTCATTCGAATTATGTTAGGGTCCGT ^R I ^; GATAATATAGACGGCCCATCCCACGCACATATTAAG TGTTCAACTCCATAGAATATCATATGGGACACTGTTTTTAIVRTLATAATCACCATTTAA AATGTTTAA ATGTTTATGCAAATTGGATGGCTTCTTCACACAACATTTATTTATTGGCCTT CATTCCA CAAAG A AA AGC TTTCAAVTACATTATACTCTATACTCCTATACATGTAAATAACCATATGCATATATAT T TTTTCAAATATAGGTCAACGCCATTΐΑΑΐ ΤΑΑΪTTTΑΆΑΑΑΑΑΤTTGTTCGGAAAATATCACATΪTC TTTCACTAGACAAGCCTTGTTACCACACAATGTATCAATATGATCTAAAGGGCAAACGAA AGATCCTG ACATGAJ^ACGTTTAATTCTCATTTTCTCCAAATTTTATTTTTTATGTGAAGTAGATAAA TTAGTATAT ATATATATATACCAAACTAGTGTGTTATGTTATGGCAAATGTTATATCAATTCGAAGGTT CCGCTATT GCAATATTCATTAATTTTTTCATACCAATACTATTTTTCTT1 TCTTTTATTTTGTTTTTTAATAAAT AAAAGAAATTAAGGATGATTAGTAAGGAAGTCGCCTACCAAGAGATTCACCTACCACGGT ACACTTCA ACACCGAAGCAGAGTTGTTGAATCCACTTTTTAT ^! TCCCTTCTCTAATCTCTACTCACCAAGTCTCCAC TTTTTTTTCTCTTTATTATATACATTTAAATTATTTAATATACX>CAJCTACATACA TATCCAGTGTA ATTTCTCGTTAC:GTCACACCCCTTTCGTAATCGTCTAATTTCAGAAAAATATCCAGAGG TTTAAATAC ATATTCCCATCAT[AAATCTAGACATAAACACATCATACTCACAAAATTTGGCAGCAAAC AGTTACTA CAGACCCATAAATGAAAAIIACGTATTCACTTGTTTTCAATTTTCACATAACCACTTCCC TGAGTTTGG TCTCAATTTGATTGCCCCGCCGAGGCATTACTACGCCAAGTGCGATTAAGGTCCCATACA GTGTAACG GGACCCACTATAAGACAGCGACCGACCAATTGCGTGTTAGGAGAGTTTCACCAACCCCGG ACCGGTTT TTACCGGATATAACAGAACCGGTACGAACCGGTCTCATTATCTTCCATCTTCTTTATATA GACCTCAT

GCCATGTGTGTGACTCACCAAGALAAAACACAATCGTTTAATCTCACCCAAGAAGAC AAAAACACAGAG AGAGAAAGAGAGAGAA

SEQ ID NO:29 - TcPAM amino acid sequence (Taxus chinensis phenylalanine

aminomutase; AAT471 86)

MG F VESRSHV DILGLI TFNEVKKI VDGTTPITVAHVAJiLARRHDVKVALEAEQCRA V ETCSSWVQRKAEDGADIYGVTTGFGACSSRRTNQLSELQESLIRCLLAGvFTKGCASSVD EL PATATRSAMLLRLNSFTYGCSGIR EVMEALEKLLNSNVSPKVPLRGSVSASGDLIPLAYIA GLLIGKPSWARIGDDVEVPAPEALSRVGLRPFKLQAKEGLALVNGTSPATALASTVMYDA N VLLLLV ETLCGMFCEVI FGREEFAHPLIHKVKPHPGQIESAELLEWLLRSSPFQDLSREYYS IDKLKKPKQDRYALRSSPQWLAPLVQTIRDATTTVETEYNSANDNPIIDHANDRALHGAN FQ GSA GFYMDYVRIAVAGLGKLLFAQFTELMIEYYSNGLPGNLSLGPDLSVDYGLKGLDIAMA

ftYSSELQYLA PVTTHVHS EQHNQDI SLALISARKTEEALDILKLMIASHLTA CQAVDL RQLEEALVKVVENVVSTLADECGLP DTKARLLYVAKAVPVYTYLESPCDPTLPLLLGLEQS CFGSI LALRKKDGIETDTLVDRL AE FEKRLS DRLENEMTAVRVLYEKKGHKTADNNDALVRI QGSRFLPFYRFVREELDTGVMSARREQTPQEDVQKVFDAIADGRITVPLLHCLQGFLGQP NG CA GVES FQSVWNKSA

SEQ ID NO:30 - PDC amino acid sequence (Pediococcus pentosaceus Phenylacryiic decarboxylase; CAC 16794)

MEKTFKT LD DFLGTHFI YTY DNG E YEWYAKNDHTVDYRIHGGMVAGRWVKDQEAHI MLTE G I YKVA TE PTGT DVAL DFVPNEKKLNGT I FFPK VEEHPE I TVT FQNEH I DLMEES REKYE TYPKLWPEFATITYMGDAGQDNDEVIAEAPYEGMTDDIRAGKYFDENYKRIN

SEQ ID NO:31 - CHS amino acid sequence (Physcomitreila patens chalcone synthase; ABB84527)

MASAGDVTRAALPRAQPRAEGPACVLGIGTAVPPAEFLQSEYPDFFFNITNCGE EALKAKF RICDKSGIRKRHMFLTEEVLKANPGICTY EPSLNVRHDIVVVQVPKLAAEAAQKAIKE G GRKSDITHI VFATTSGV MPGADHALAKLLGLKPTVKRVMMYQTGCFGGASVLRVA DLAEN NKGARVLAvASEVTAVTYRAPSENHLDGLYGSALFGDGAGVYVVGSDPKPEVE PLFEVHWA GET I L PE S DGAI DGH LTE GLI FHL KDV PGLISKNIEKFLNEARKPVGSPA NEMF AVHP G G P 1 L D Q V E A K L K L T D K M Q G S R D I L S E FG S S A S V L F VL D Q I R H R S V KMG A S T L G E G S E FGFFIGFGPGLTLEVLVLRAAPNSA

SEQ ID NO:32 - CHS amino acid sequence {Arabidopsis thaliana chalcone synthase: AAA3277!)

VMAGASSLDEIRQAQRADGPAGILAIGTANPE HVLQAEYPDYYFRI NSEHMTDLKEKFK RMCDKST I RKRHMHLTEEFLKEN PHMCAYMAPS LDTRQD I WVEVPKLGKEAAVKAI KE GQ PKSKI HVVFCTTSGVDMPGADYQLTKLLGLRPSV RLMMYQQGCFAGGTVLRIAKDLAENN RGARVLVVCSEITAV FRGPSDTHLDSLVGQALFSDGAAALI GSDPDTSVGEKPIFE VSA AQT ILPDS DGAI DGHLREVGLT FHLLKD PGLI S KN I V KSLDEAFKPLGIS DWNSLFWI HP GGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSAKDGVATTGEG LE WGVLFGFGPGLTVETVVLHSVPL

SEQ ID NO;33 - SPS amino acid sequence (Viiis vinifera stilbene synthase; ABE68894)

MASVEEFR AQRAKGPATILAIGTATPDHCvYQSDYADFYFRVTKSE?i TALK KFNRICDK SMIKKRY3HLTEEMLEEHPNIGAY APSLNIRQEIITAEVP LGKEAAL ALKEWGQPKSKI THLVFCTTSGVEMPGADYKLANLLGLEPSVRRVMLYHQGCYAGGTVLRTAKDLAENNAGA RV LWCSE JrvVT FRCPS E DAL DSL VGQALFGDGS AA ⁷ ! VGSDPDI SI ERPLFQLVSAAQTFI P NSAGAIAGNLREVGLTFHL PNVPTLISENIEKCLTQAFDPLGISD NSLF IAHPGGPAIL DAVEAKL L DKKKLE A'TRH VL S E YGNMS S AC VL FI L DEMRKKS LKGERATTGE GL DWGVL FG FGPGLTI ETVVLHS I PMVTN

SEQ ID NO:34 - COS amino acid sequence (Oryza saliva curcuminoid synthase short version; 30IT A)

MRRSQRADGLAAVLAIGTANPPNCVTQSEIPDFYFRVTNSDHLTALKDKFKRICQEMGVQ RR YLHHTEEMLSAHPEFVDRDAPSLDARLDIAADAVPELAAEAAKKAIAE GRPAADITHLvVT TNSGAHVPGVDFRLVPLLGLRPSVRRT LHL GCFAGCAALRLAKDLAENSRGARVLVVAAE LTLMYFTGPDEGCFRTLLVQGLFGDGAAAVI VGADADDVERPLFEIVSAAQ I IPESDHALN MRFTERRLDGVLGRQVPGL IGDNVERCLL DM FGPLLGGDGGGGW DLFWAVHPGSS IMDQV DAALGLEPG LAASRRVLSDYGNMSGAT I FALDELRRQRKEAAAAGEWPELGVMMAFGPGM TVDAMLLHATSHVN

SEQ ID NO:35 - CUS amino acid sequence (Oryza sativa curcuminoid synthase !ong version; 3ALJE_A) APTTTMGSALYPLGEMRRSQRADGLAAVLAIGTANPPNCVTQEEI PDFYFRVTNSDHLΪAL KDKFKRICQEMGVQRRYLHHTEE LSAHPEFVDRDAPSLDARLDIAADAVPELAAEAAKKAI AEWGRPAADITHLWTTNSGftHVPGVDFRLVPLLGLRPSVRRTMLHLNGCFAGCAALRLA KD LAENSRGARVLVVAAELTLMYFTGPDEGCFRTLLVQGLFGDGAAA.VIVGADADDVERPL FEI VSAAQTI IPESDHALNMRFTERRLDGVLGRQVPGLIGDNVERCLLDMFGPLLGGDGGGGWND LFWAVHPGSSTIMDQVDAALGLEPGKLAASRRVLSDYGNMSGATVIFALDELRRQRKEAA AA GEWPELGVM AFGPGMTVDAMLLHATSHVN

SEQ ID NO:36 - BAS amino acid sequence {Rheum pal ^' malum benzalacetone synthase; AAK82824)

MATEEMKKLATVMAIGTANPPNCYYQADFPDFYFRVTNSDHLINLKQKFKRLCENSRIE RY LHVTEEILKENPNIAAYEATSLNVRHKMQvKGVAELGKEAALKAIKEWGQP SKITHLIVCC LAGVDMPGA.DYQLTKLLDLDPSVKRFMFYHLGCYAGGTVLRLAKDIAENNKGARVLIVC SEM TΪTCFRGPSETHLDSMIGQAΪLGDGAAAVIVG DPDLTVERPI FELVS QTIVPESHGAIE GHLLESGLS FHLYKTVPTLISN IKTCLSD F PLNISDWNSLFWIAHPGGPAILDQVTAKv GLEKEKLKVTRQVLKDYGNMSSATVFFIMDEMRKKSLENGQATTGEGLEWGVLFGFGPGI ETVVLRSVPVIS

SEQ ID O:37 - AtPAPl amino acid sequence (Arabidopsis thaliana R2R3 Myb transcription factor, AtMyb75 ; AAG42001 )

EGSSKGLR GAWTTEEDSLLRQCINKYGEGK HQVPVRAGLNRCRKSCRLRWLNYLKPSIK RGKLSSDEVDLLLRLHRLLGNR SLIAGRLPGRTANDVKNY NTHLSKKHEPCCKIKMKKRD I PI TTPALKNNVYKPRPRSFTVNNDCNHLNAPPKVDVNPPCLGL I NVCDNS I IYNKDK KKDQLV NLI DGD MWLEKFLEESQEVDILVPEATTTEKGDTLAFDVDQLWSLFDGETVKFD

SEQ ID NO:38 - A1PAP2 amino acid sequence {Arabidopsis thaliana R2R3 Myb transcription factor, AtMyb90; AAG42002)

MEGSSKGLRKGAWTAEEDSLLRLCIDKYGEGKWHQVPLRAGLNRCRKSCRLRWLNYLKPS IK RGRLSNDEVDLLLRLHKLLGNRWSLIAGRLPGRTANE)VKNY NTHLSKKHESSCCKSKMKKK NIISPPTTPVQKIGVFKPRPRSFSVNNGCSHLNGLPEVDLI PSCLGLKK NVCENSI CNKD DEKDDFV NLMNGD MWLENLLGENQE D IVPEATTAEHGATLAFDVEQL SLFDGETVEL

D

SEQ ID NO:39 - AtTT2 amino acid sequence {Arabidopsis thaliana R2R3 Myb transcription factor, AtMybl 23; AED93980)

GKRATTSVRREELNRGAWTDHEDKILRDYITTHGEGK STLPNQAGLKRCGKSCRLRWKNY LRPGIKRGNISSDEEEL 11 LHNLLGNRWSLIAGRLPGRTDNEIKNHWNSNLRKRLPKTQTK QPKRIKHSTNNE VCVIRTK IRCSK LLFSDLSLQKKSSTSPLPLKEQEMDQGGSSLMGD LEFDFDRIHSEFHFPDLMDFDGLDCGNVTSLVSS EILGELVPAQGNLDLNRPFTSCHHRGD DED LRDFTC

SEQ ID NO:40 ~ NtAn2 amino acid sequence (Nicotiana tabacum R2R3 Myb transcription factor; ACO52470)

M ICTNKSSSGVKKGAWTEEEDVLLKKCIE YGEGKWHQVPLRAGLNRCRKSCRLRWLNYLR PHIKRGDFSFDEVDLILRLHKLLGNRWSLIAGRLPGRTANDV NYWNSHLR KLIAPHDQKE SKQKAKKITIFRPRPRTFSKT CVKS T TVDKDIEGSSE 1 1RFNDNLKPTTEEL DDGIQ WWADLLANNYNNNGIEEADNSSPTLLHEEMPLLS

SEQ ID NO:41 - MtLAPI amino acid sequence (Medicago iruncalida R2R3 Myb transcription factor; ACN79541 )

MENTGGVRKGAWT KEDELLKACIN YGEGK NLVPQRSGLNRCRKSCRLR LNYLSPNINR GRFSEDEEDLILRLHKLLGNRWSLI GRLPGRTANDVKNY HTNLAKKVVSEKEEEKENDKP KETMKAHEVIKPRPITLSSHSN LKGKNSIPRDLDYSENMASNQIGRECASTSKPDLG API PCEMWCDSL LGEHVDSEKIGSCSSLQEENLMEFPNVDDDSF DFNLCDLNSLWDLP

SEQ ID NO:42 - ZmMYB-C amino acid sequence [Zea mays R2R.3 Myb

transcription factor; AAK09326)

MGRRACCA EGVKRGAWTS EDDALAAYVKAHGEGK REVPQKAGLRRCGKSCRLRWLNYLR PNIRRGNIS YDEEDLI IRLHRLLGNR SLIAGRLPGRTD EIKNYWNSTLGRRAGAGAGAGG SWVVVY^PDTGSHATPAATSGACETGQ SAAHRADPDSAGTTTTSAAAV APKAVRCTGGLFF FHRDTTPAHAGE AT PMAGGGGGGGGEAGSSDDCSSAASVSLRVGSHDEPCFSGDGDGDWMD DVRALAS FL E S DE D L RCQT GQ LA

SEQ ID NO:43 - ZmMYC-Lc amino acid sequence (Zea mays BHLH transcription factor; ABD72707)

MALSASRVQQAEELLQRPAERQLMRSQLA _^ AAARSINWSYALFWSISDTQPGVLTWTDGFYNG EVKTRKI SNSVELTS DQLVMQRS DQLREL YEALLSGEGDRRAAPARPAGSLS PEDLGDTEWY Y SMTYAFRPGQGLPGRSFASDEHVWLCNAKLAGSKAFPRALLAKSASIQSILCIPVMGGV LELGTT DTVPEAPDL VS RA AAFWE PQCPSS S PSGRA ETGEAAADDGT FAFEELDHNNG D D I E M Ϊ AAG G H G Q E E E L R L R E A E L S D DA S L E H I T K E IEEFYSLCDEMDLQ LPLPLEDG W VDASNFE VPCS S PQPAPPPVDRATANVAADAS RAP VYGS RATS FMAWTRS S QQS S CS DDAAP AAWPAIEEPQRLL KWAGGGAWESCGGATGAAQEMSGTGTKNHVMSERKRREKLNEMFLV LKSLLPSIHRVNKASILAETIAYLRBLQRRVQELESSREPASRPSETTTRLITRPSRGNN ES VRKEVCAGSKRKSPELGRDDVERPPVLTMDAGTSNVTVTVSDKDVLLEVQCRWEELLMTR VF DAI KS LHLD VL S VQAS AP DG FMG LK I P, AQ FAGS G AVV P MI S E ALRKAI GKR

SEQ ID NO:44 - AtTT8 amino acid sequence (Arabidopsis thaliana BHLH transcription factor; AEES2802)

MDESS I I PAEKVAGAEKKELQGLLKTAVQSVDWTYS FWQFCPQQRVLVWGNGYYNGAl KTR KTTQPAEVTAEEAALERSQQLRELYETLLAGESTSEARACTALSPEDLTETEWFYL CVSFS FPPPS G PGKAYARRKHVWLSGANE VDSKT FSRAI LAKSAKI QTVVC I PMLDGVVELGTTKK VREDVEFVELTKSFFYDHCKT PKPALSEHS YEVHEEAEDEEEVEEEMTMSEEMRLGSPDD EDVSNQNLHSDLHIESTHTLDTHMDMMNLMEEGGNYSQTVTTLLMSHPTSLLSDSVSTSS YI QSS FATWRVENGKEHQQVKTAPS SQWVLKQ I FRVPFLHDNTKDKRL PREDLSHWAERRRR EKLNEKFI LRSMVPFVTK DKVSILGD IAYVMHLRKRVHELENTHHEQQHKR RTCKRKT SEEVEVS I IENDVLLEMRCEYRDGLLLDI LQVLHELGIETTAVHTSVNDHDFEAEIRAKVRG KKAS I EVKRAI HQVI I HDTNL

SEQ ID NO:45 - VvMycl amino acid sequence i itis vinifera BHLH transcription factor; ACC68685)

AAPPN8RLQSMLQSAVQSVR TYSLFWQICPQQGILVWGDGYYNGAIKTRKTVQPMEVSAE EASLQRSQQLRELYESLSAGETNQPARRPCAALSPEDLTESE FYLMCVSFSFPPGVGLPGK AYAKRHHIWLAGANEVDSKVFSRAILAKSARVQTVVCI PL DGVVEFGTTEKVQEDLGFVQH VKSFFTDHHLHNHPPKPALSEHSTSNPATSSDHSRFHSPPIQAAYAAADPPAS NQEEEEEE EEEEEEEEEEEEEEEEEEAESDSEAETGRNNRRVRTQNTGTEGVAGSHTAAEPSELIQLE MS EGIRLGSPDDGSN LDSDFHML VSQPGSSVDHQRRADSYRAESARRWPMLQDPLCSSGLQQ PPPQPPTGPPPLDELSQEDTHYSQTVSTILQHQPNR SESSSSGCIAPYSSQSAFAKWTTRC DHHHHPMAVEGTSQWLLKYI LFS VP FLHTK YRDEN S PKS RDG DS AGRFRKGT PQDEL S ANHV LAERRRREKLNERFI ILRSL PFVTKMDKASILGDTIEYVKQLRKKIQDLEARTRQMEVEQR SRGSDSVRSKEHRIGSGSVDRNRAWAGSD RKLRIVEGSTGAKPKWDSPPAAVEGGTTTV EVSIIESDALLEMQCPYREGLLLDViyiQMLRELRLETTTVQSSLTNGVFVAELRAKVKE NASG KKAS IMEVKRAI QI I PQC [0184] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in Sight thereof will be suggested to persons skilled n the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.