This application claims the benefit of priority from Australian Patent Application No. 20099051 19 filed October 21 , 2009 and Australian Patent Application No. 2009905122 filed October 21 , 2009, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a method of producing plants with desirable properties by modulating the expression and/or activity of Auxin Response Factors (ARFs), and plants produced by the method. It also relates to transgenic plants having modulated expression and/or activity of Auxin Response Factors (ARFs), and seeds, plant cells, plant parts and other types of propagating materials, and uses thereof.

BACKGROUND OF THE INVENTION

An object of plant genetic engineering or conventional breeding is to produce novel plants with agronomically, horticulturally, or economically important traits including increased tolerance to a variety of environmental stresses, e.g., water availability, altered growth characteristics, improved plant yield, and seed quality. Plant yield and altered growth characteristics further impacts economically valuable properties of marketable plant products.

For example, modified wood properties are considered desirable by the forest and pulp industries. The expensive, energy-intensive, and environmentally hazardous process of turning wood into paper is attributed in part to the need to separate cellulose from lignin in wood and, as a consequence, plants having modified xylem structure or composition e.g., fast-growing, low-lignin trees, are highly desirable. In this respect, wood comprises layers of secondary xylem impregnated with lignin and comprises both living and dead cells. Functional xylem, in layers adjacent to cambium, is primarily a water and mineral-conducting tissue, whereas functional phloem, also in layers adjacent to the cambium, primarily conducts photoassimilates and signalling molecules.

Drought and flooding rain are defining features of the Australian landscape and are becoming increasingly prevalent globally due to climate change. It is desirable to minimize the impact that this fluctuating supply and the associated reduction in soil moisture content has on our primary production. Accordingly, it is desirable to identify genes that are implicated in a plant's ability to cope with a drying soil so that the knowledge can be used to the advantage of breeders and growers alike. Since xylem vessels are the main arteries for the transport of water and phloem is the main means for distribution of photoassimilates by plants, modification of xylem structure or function may provide desirable benefits to plants in times of environmental stresses such as water deficiency and/or mineral nutrient deficiency.

A major disadvantage with traditional tree breeding, especially for forest tree species, is the slow progress due to their long generation periods. However, by taking advantage of recent developments in gene technology the time required to produce a new variety could be reduced significantly. In addition, a biotechnological approach would allow closer targeting of traits considered desirable by the forest and pulp industries, in specific tree species.

Auxins are known to regulate many of the physiological events in a plant's life cycle. The Auxin Response Factor (ARF) and Aux/IAA families are two families of transcription factor proteins important for transducing the perception of auxin through to changes in gene expression (Guilfoyle et al. (1998) Plant Physiol. 1 18:341 -347). ARFs bind to auxin response cis-acting elements through an N-terminal DNA binding domain, while the carboxy-terminus contains two protein-protein interaction domains that are also found in the Aux/IAA family of early auxin-response genes. In vitro homodimerization and heterodimerization within each family, as well as interactions between the families, suggest that combinatorial action of these proteins confers cell or tissue specificity in auxin responses (Kim et al. ( 1997) Proc. Natl. Acad. Sci. USA 94: 1 1786-1 1791 ; Ulmasov et al. (1997a) Science 276: 1865-1868; Ulmasov, et al. ( 1997b) Plant Cell 9: 1963- 1971 ; Ulmasov et al. ( 1999) Plant J. 19:309-319).

In Arabidopsis thaliana, the ARFs are encoded by a large gene family comprising at least 23 members, of which many have unknown function. A typical ARF protein comprises an N-terminal B3-like DNA-binding domain, C-terminal domains III and IV similar to those found in the C terminus of Aux/IAAs, and an intervening region that determines whether the ARF functions as a repressor or enhancer of gene expression. ARFs bind to auxin-responsive cis-acting elements (AuxREs) found in the promoter regions of auxin-responsive genes. Certain ARFs are known to regulate plant growth responses to blue light, and plant morphologies such as gynoecium patterning, vascular strand formation in the early embryo, and hypocotyl elongation and bending during germination. Recently, Okushima et al. (2005) The Plant Cell 17, 444-463 attempted to characterize the functions of most A. thaliana ARFs by producing T-DNA insertion mutants for 18 of the 23 ARF gene family members and, notwithstanding the authors failed to demonstrate an obvious growth phenotype for most ARFs, they disclose severely-impaired lateral root formation and abnormal gravitropism in both hypocotyl and root of double ARF7/ARF19 mutants, unusual gynoecium and floral patterning defects in ARF3-deficient mutants, abnormal root meristem and cotyledon development in ARF5-deifcient mutants, impaired phototropic responses toward blue light for ARF7-deficient mutants, and pleiotropic phenotypes for ARF2-deficient mutants, including abnormal inflorescence stem, leaf and flower morphology, and flowering time.

There remains a need to identify genetic factors in auxin signalling relevant to xylem and secondary xylem development and/or structure, including effects on water use efficiency and/or stomatal conductance and/or transpiration rate and/or wood quality and/or pulpability and/or paper quality and/or coarseness.

General

Conventional techniques of biochemistry, molecular biology, recombinant DNA technology, are described, for example, in the following texts that are incorporated by reference:

• Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;

• DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;

• Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp l -22; Atkinson et ai, pp35-81 ; Sproat et a/., pp 83-1 15; and Wu ef a/., pp 135-151 ;

• Perbal, B., A Practical Guide to Molecular Cloning (1984);

• Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; • Smith et at., (2002) Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th Edition (Illustrated), John Wiley & Sons Inc., ISBN 0471250929.

• Sambrook and Russell (2001 ) Molecular Cloning, Cold Spring Harbor Laboratory, New York, ISBN 0879695773.

SUMMARY OF THE INVENTION

In work leading up to the present invention the inventors sought to identify genes useful for manipulating plant properties, for example, properties of economic importance. For example, the inventors sought to identify factors that affect secondary xylem development and/or structure, including effects on water use efficiency and/or stomatal conductance and/or transpiration rate and/or wood quality and/or pulpability and/or paper quality and/or coarseness. As exemplified herein, the inventors have shown that modified expression of the Auxin Response Factor 2 (ARF2) gene in Arabidopsis thaliana produces modified or altered secondary xylem. For example, ARF2-deficient plants were found to have significantly enhanced stem and/or hypocotyl thickness, such as determined by measuring stem and/or hypocotyl diameter and/or the diameter of secondary xylem (wood) tissue, compared to wild-type (WT) plants. Alternatively, or in addition, ARF2-deficient plants exhibited enhanced growth level or growth rate compared to WT plants grown under identical environmental conditions. For example, the growth rate in terms of above and/or below ground biomass accumulation of ARF2-deficient plants is about 2- fold greater or about 3-fold greater or about 4-fold greater or about 5-fold greater or about 10-fold greater than WT plants. Alternatively, or in addition, the number of small vessels e.g., vessels having a cross-sectional area of about 0-20μιη ² or less, is enhanced in ARF2-deficient plants relative to WT counterparts. Alternatively, or in • addition, the number of large vessels e.g., vessels having a cross-sectional area of more than about 0-20μπι ² is reduced compared to the level of large vessels in WT plants. These data indicate that ARF2 expression and/or activity affects e.g.,- growth level and/or growth rate and/or xylem structure and/or xylem development.

Also exemplified herein, the inventors have shown that modified expression of the Auxin Response Factor 18 (ARF18) gene in Arabidopsis thaliana produces modified xylem structure, and water use efficiency, in addition to altered leaf size, modified primary root growth and lateral root production, modified hypocotyl length and growth rate, modified flowering time, and modified seed yield. More particularly, the inventors have shown that hypocotyls of the ARF18-deficient plants have larger fibres compared to wild-type (WT) plants, indicating that ARF18 is important for determining xylem structure. Alternatively, or in addition, water usage rate is reduced in ARF18- deficient plants relative to WT plants expressing ARF18. Alternatively, or in addition, stomatal conductance is reduced in ARF18-deficient plants relative to WT plants expressing ARF18. Alternatively, or in addition, transpiration rate is reduced in ARF18-deficient plants relative to WT plants expressing ARF18. Alternatively, or in addition, the rate of photosynthesis is reduced in ARF18-deficient plants relative to WT plants expressing ARF1 8. Alternatively, or in addition, leaf area is enhanced in ARF18-deficient plants relative to WT plants expressing ARF1 8. These data indicate that ARF18 expression or activity also affects xylem structure and/or xylem development and/or water use efficiency in plants.

In summary, the data presented herein suggest that ARF2 and A RF 18 exert opposing actions on one or more auxin-responsive genes affecting cell expansion in cells of the functional xylem and/or secondary xylem, including cells comprising vessels. In ARF2-deficient plants, cell expansion is limited in vessels, whereas ARF I 8-deficient . plants exhibit larger xylem vessels than wild-type plants. Without being bound by any theory or mode of action, these data suggest that ARF2 may serve as an antagonist of ARF18 and block expression of one or more genes regulating cells expansion in xylem, whereas in the absence of ARF2, ARF18 acts to enhance expression of one or more genes regulating cell expansion in xylem thereby leading to xylem cell expansion. Accordingly, ARF2 and ARF18 may exist in dynamic equilibrium in xylem cells, such that a relative level of ARF2 and ARF18 determines vessel and fibre development, including e.g., radial expansion and/or fibre length and/or vessel length and/or vessel diameter. Accordingly, in one example, the present invention provides a method of modifying a phenotype mediated by ARF2 in a plant, said method comprising modulating the expression of one or more Auxin Response Factor- 18 (ARF18) genes in the plant. For example, a phenotype that is enhanced by expressing ARF2 in a plant may be inhibited or reduced by expressing at least one ARF18-encoding polynucleotide in the plant. Alternatively, a phenotype that is repressed or inhibited by expressing ARF2 in a plant may be enhanced or de-repressed by expressing at least one ARF18-encoding polynucleotide in the plant.

In another example, the present invention provides a method of modifying a phenotype of a plant, said method comprising reducing or inhibiting the expression of one or more Auxin Response Factor-18 (ARF18) genes in the plant, wherein the modified phenotype is selected from increased root growth, reduced leaf length, reduced petiole length, delaying flowering, enhanced flower size, increased hypocotyl length, enhanced seed yield, enhanced embryo size, enhanced seed size, enhanced tracheid fibre diameter, enhanced xylem vessel diameter, enhanced water use efficiency, enhanced drought tolerance, and enhanced or improved recovery from water stress or water deficiency. For example, reduced or ablated expression of ARF18 and/or activity of ARF18 enhances cellular expansion, thereby leading to increased cell size in xylem vessels and to a larger lumen size, enhanced biomass, larger seeds, enhanced root growth and/or size, and/or enhanced water usage efficiency or improved productivity during 'and/or following water stress or water deficit. An enhanced water usage efficiency is a desirable property as it leads to a greater ability of the plant to cope with drying soil, when-water supply is reduced, intermittent, or during drought conditions. Enhanced water usage efficiency may also be related to the root growth and/or size and/or to the increased cellular expansion in xylem. Enhanced biomass is desirable for increasing plant and crop yields. Woody plants according to the invention exhibiting enhanced cellular expansion in secondary xylem and/or increased coarseness are useful for production of solid wood products. In another example, increased expression of ARF18 and/or activity of ARF18 according to any embodiment described herein decreases cellular expansion, reduced average xylem cell size, tracheid fibre diameter, xylem vessel diameter, coarseness and lignin content. Decreased cellular expansion lead to decreased cell size in secondary xylem and/or reduced coarseness and/or reduced lignin content. Woody plants according to the invention exhibiting reduced cellular expansion in secondary xylem and/or low coarseness or low lignin content have utility in pulp and paper milling.

In another example, the present invention provides a method of modifying a phenotype of a plant, said method comprising reducing or inhibiting the expression of one or more Auxin Response Factor-2 (ARF2) genes in the plant, wherein the modified phenotype is selected from reduced average xylem cell size, reduced tracheid fibre diameter, reduced xylem vessel diameter, decreased cell size in xylem, reduced coarseness and reduced lignin content.

In another example, increased expression of ARF2 and/or activity of ARF2 according to any embodiment described herein increases cellular expansion, particularly in fibre and vessels, increases root growth, reduces leaf length, reduces petiole length, delays flowering, enhances flower size, increases hypocotyl length, enhances seed size, enhances embryo size, enhances tracheid fibre diameter, enhances xylem vessel diameter, enhances water use efficiency, enhances drought tolerance, or enhances or improves recovery from water stress or water deficiency.

In another example, the present invention provides a method for modifying a xylem structure and/or xylem development in a plant such as a fibre-producing plant or a woody plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant. For example, by ■ increasing a level of expression of ARF2 and/or decreasing a level of expression of ARF18 in a plant, a plant having larger vessels and/or fibres is produced, such as determined by radial expansion and/or fibre length and/or vessel length and/or vessel diameter. In a particularly preferred example, ARF2 expression is increased and ARF18 expression is decreased in the plant to achieve this increase in vessel and/or fibre production.

In another example, the present invention provides a method for improving a paper milling and/or pulp milling property of a woody plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant. For example, decreasing a level of expression of ARF2 and/or increasing a level of expression of ARF18 in a plant produces a plant having smaller vessels and/or fibres such as determined by radial expansion and/or fibre length and/or vessel length and/or vessel diameter, and preferably, reduced lignin content, thereby leading to improved pulp and paper milling properties. ¹

In another example, the present invention provides a method for reducing lignin content of a plant such as a fibre-producing plant or a woody plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant. For example, by decreasing a level of expression of ARF2 and/or increasing a level of expression of ARF18 in a plant, a plant having reduced lignin content of secondary xylem is produced. In a particularly preferred example, ARF2 expression is decreased and ARF18 expression is increased in the plant. In another example, the present invention provides a method for enhancing water use efficiency of a plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant. For example, by increasing a level of expression of ARF2 and/or decreasing a level of expression of ARF18 in a plant, a plant having enhanced water use efficiency e.g., as determined by reduced transpiration and/or reduced stomatal conductance is produced. .In a particularly preferred example, ARF2 expression is increased and ARF18 expression is decreased in the plant.

In another example, the present invention provides a method for enhancing drought tolerance of a plant and/or recovery from water deficit in a plant, wherein said method comprises modulating the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 in the plant. For example, by increasing a level of expression of ARF2 and/or decreasing a level of expression of ARF 18 in a plant, a plant having enhanced drought tolerance and/or ability to recover form water deficit e.g., as determined by reduced wilting during drought and improved survival following water deficit is produced. In a particularly preferred example, ARF2 expression is increased and ARF18 expression is decreased in the plant.

In the foregoing examples, the expression of one or more Auxin Response Factors e.g.; ARF2 and/or ARF18 is increased or enhanced by providing plant cells with a chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell;

ii) a polynucleotide comprising a nucleotide sequence that encodes the Auxin Response Factor; and .

iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell.

Preferred polynucleotides that encode an auxin responsive factor will encode ARF2 or ARF18 and comprise a sequence selected from the group consisting of:

(i) a sequence that encodes a polypeptide having an amino acid sequence that is at least about 50% identical or at least about 60% identical or at least about 70% identical or at least about 80% identical or at least about 90% identical or at least about 95% identical or at least about 99% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32;

(ii) a sequence that is at least about 50% identical or at least about 60% identical or at least about 70% identical or at least about 80% identical or at least about 90% identical or at least about 95% identical or at least about 99% identical to any one of SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, or 31 ;

(in) a sequence that is complementary to a sequence that hybridizes under at least moderate stringency or high stringency conditions to (i) or (ii); and

(iv) a sequence that is amplified e.g., by a polymerase chain reaction or isothermal amplification process using one or more nucleic acid primers each comprising at least about 15 contiguous nucleotides of (i) or (ii).

Alternatively, the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 is decreased or reduced or inhibited by providing an antisense R A, co- suppression molecule, interfering RNA (iRNA) or double-stranded RNA (dsRNA) or RNAi molecule to a plant cell. For example, a chimeric gene construct comprising nucleic acid that is expressed to produce an antisense RNA, co-suppression molecule, interfering RNA (iRNA) or double-stranded RNA (dsRNA) or RNAi molecule may be expressed in the plant cell to thereby reduce expression of the Auxin Response Factor(s).

In one example, the expression of one or more Auxin Response Factors e.g., ARF2 and/or ARF18 is decreased or inhibited or reduced by providing plant cells with a chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell;

ii) a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor as described herein and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the auxin responsive factor; and

iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell. Exemplary dsRNA molecules for inhibiting the expression of an Auxin Response Factor will comprise a sense strand and an antisense strand that are complementary to each other, wherein the antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding an Auxin Response Factor wherein a region of complementarity with said mRNA is less than 30 nucleotides in length, and more generally 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 nucleotides in length. The dsRNA, upon contacting with a cell expressing the Auxin Response Factor may inhibit expression of a gene encoding the Auxin Response Factor by at least about 30% or 40% or 50% or 60% or 70% or 80%. Exemplary sense strand sequences thus comprise at least about 19 contiguous nucleotides from a polynucleotide that encodes an auxin responsive factor as described herein. Exemplary antisense strand sequences comprise at least about 19 contiguous nucleotides complementary to at least about 19 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor as described herein.

Exemplary antisense RNAs will comprise a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding an Auxin Response Factor wherein a region of complementarity with said mRNA is more than 30 nucleotides in length, and more generally 50 or 100 or 150 or 200 or 250 or 300 or 400 or 500 nucleotides in length and preferably complementary to the 3'-end of said mRNA. In a particularly preferred form, antisense mRNA is substantially complementary to the full- length of the coding region of mRNA and the 3'-untranslated region thereof. Exemplary antisense sequences thus comprise at least about 30 contiguous nucleotides complementary to at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor as described herein.

In one example, the promoter is a constitutive promoter e.g., CaMV 35S promoter or ubiquitin gene promoter. In another example, the promoter is a fiber-specific promoter. In another example, the promoter is operable in the cambium and/or xylem.

The transcription terminator and polyadenylation signal may be any 3-untranslated region of a plant gene.

The chimeric gene may be contained within an expression vector, which is transferred and stably incorporated into a plant genome by standard procedures e.g., Agrobacterium-mediated transformation as described for example by Fraley et al. ( 1983) Proc. Natl. Acad. Sci. USA. 80: 4803-4807, or by employing microparticle bombardment of plant cells by the biolistics method described by Klein et al ( 1987) Nature. 327:70-73. Cells are regenerated into whole plants according to standard procedures for regenerating plants. Transformed plants carrying the chimeric gene or an expression vector comprising same are then selected and screened to demonstrate that they exhibit the desired phenotype(s).

In the foregoing examples, the plant according to. any example hereof may be a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.

Alternatively, the plant according to any example hereof may be a fiber-producing plant e.g., flax or cotton.

Alternatively, the plant according to any example hereof, especially those examples producing a modified pulp or paper milling property may be a wood-producing plant e.g., oak, aspen, eucalyptus, maple, pine, spruce, poplar, or larch. For example, the plant may be a species of Eucalyptus ( E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi E. camaldulensis, E. ceracea, E. cloeziana, E. coccifera, E. cordata, E. cornuta, E. corticosa, E. crebra, E. croajingoleisis, E. curtisii, E. dalrympleana, E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. diversifolia, E. dives, E. dolichocarpa, E. dundasii, E. dunnii, E. elata, E. erythrocoiys, E. erythrophloia, E. eudesmoides, E. falcata, E. gamophylla, E. glaucina, E. globulus, E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis *urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora, E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua, E. obtusiflora, E. occidentalis, E. optima, E. ovata, E. pachyphylla, E. pauciflora, E. pellita, E. perriniana, E. petiolaris, E. pilularis, E. piperita, E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, E. risdoni, E. robertsonii E. rodwayi, E. rubida, E. rubiginosa, E. saligna, E. salmonophloia, E. scoparia, E. sieberi, E. spathulata, E. staeri E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragona, E. tetrodonta, E. tindaliae, E. torquata, E. umbra, E. urophylla, E. vernicosa, E. viminalis, E. wandoo, E. wetare sis, E. willisii, E. willisii subsp. falciformis, E. willisii subsp. willisii, E. ^' woodwardii ); or a species of poplar ( e.g.,

Populus alba, P. alba *P. grandidehtata, P. alba *P. tremula, P. alba *P. tremula var. glandulosa, P. alba * P. tremuloides, P. balsamifera, P. balsamifera subsp. trichocarpa,

P. balsamifera subsp. trichocarpa *P. deltoides, P. ciliata, P. deltoides, P. euphratica,

P. euramericana, P. kitakamiensis, P. lasiocarpa, P. laurifolia, P. maximowiczii, P. maximowiczii *P. balsam/era subsp. trichocarpa, P. nigra, P. sieboldii *P. grandideiztata, P. suaveolens, P. szechuanica, P. tomentosa, P. tremula, P. tremula tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis ), or a conifer as, for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine

(Pinus ponderosa), lodgepole pine (Pinus contorta), or Monterey pine (Pinus radiata); or Douglas-fir (Pseudotsuga menziesii); or Western hemlock (Tsuga canadensis); or

Sitka spruce (Picea glauca); or redwood (Sequoia sempervireris); or a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea); or a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis). ,

A further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF2 in a plant such as by increasing expression of ARF18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments: .

i) a promoter that is operable in the plant cell, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor- 18 ( ARF 18), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

Preferred polynucleotides that encode ARF 18 comprise a sequence selected from the group consisting of:

(i) a sequence that encodes a polypeptide having an amino acid sequence that is at least about 50% identical or at least about 60% identical or at least about 70% identical or at least about 80% identical or at least about 90% identical or at least about 95% identical or at least about 99% identical to any one of SEQ ID NOs: 26, 28, 30, or 32; (ii) a sequence that is at least about 50% identical or at least about 60% identical or at least about 70% identical or at least about 80% identical or at least about 90% identical or at least about 95% identical or at least about 99% identical to any one of SEQ ID NOs: 25, 27, 29, or 31 ;

(iii) a sequence that is complementary to a sequence that hybridizes under at least moderate stringency or high stringency conditions to (i) or (ii); and

(iv) a sequence that is amplified e.g., by a polymerase chain reaction or isothermal amplification process using one or more nucleic acid primers each comprising at least about 15 contiguous nucleotides of (i) or (ii).

A further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF2 in a plant such as by down-regulating expression of ARF 18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell, as described according to any example hereof, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor-18 (ARF 18) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF18; and

iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

It is to be understood that this chimeric gene is capable of expressing antisense RNA, a co-suppression molecule, interfering RNA (iRNA), a double-stranded RNA (dsRNA) or RNAi molecule as described herein above in a plant cell, and that the sequence of the polynucleotide at (ii) supra is readily derived from a sequence encoding ARF18 as described according to any example hereof. Structures of exemplary dsRNA molecules and antisense molecules are as described herein above and said structural features apply mutatis mutandis to this example of the invention. A further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF18 in a plant such as by increasing expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell, as described according to any example hereof; ii) a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor-2 (ARF2), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

Preferred polynucleotides that encode ARF2 comprise a sequence selected from the group consisting of:

(i) a sequence that encodes a polypeptide having an amino acid sequence that is at least about 50% identical or at least about 60% identical or at least about 70% identical or at least about 80% identical or at least about 90% identical or at least about 95% identical or at least about 99% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24;

(ii) a sequence that is at least about 50% identical or at least about 60% identical or at least about 70% identical or at least about 80% identical or at least about 90% identical or at least about 95% identical or at least about 99% identical to any one of SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , or 23;

(iii) a sequence that is complementary to a sequence that hybridizes under at least moderate stringency or high stringency conditions to (i) or (ii); and

(iv) a sequence that is amplified e.g., by a polymerase chain reaction or isothermal amplification process using one or more nucleic acid primers each comprising at least about 15 contiguous nucleotides of (i) or (ii).

A further example of the present invention provides a chimeric gene for modifying a phenotype mediated by ARF18 in a plant such as by down-regulating expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an auxin responsive factor-2 (ARF2) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF2; and

iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof. It is to be understood that this chimeric gene is capable of expressing antisense RNA, a co-suppression molecule, interfering RNA (iRNA), a double-stranded RNA (dsRNA) or RNAi molecule as described herein above in a plant cell, and that the sequence of the polynucleotide at (ii) supra is readily derived from a sequence encoding ARF2 as described according to any example hereof. Structures of exemplary dsRNA molecules and antisense molecules are as described herein above and said structural features apply mutatis mutandis to this example of the invention.

A further example of the present invention provides the use of a chimeric gene as described according to any example hereof to modify a phenotype mediated by ARF2 in a plant by increasing expression of ARF18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor- 18 (ARF18), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

A further example of the present invention provides the use of a chimeric gene as described according to any example hereof to modify a phenotype mediated by ARF2 in a plant by down-regulating expression of ARF18 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA ₍ fragments:

i) a promoter that is operable in the plant cell, as described according to any example hereof, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an Auxin Responsive Factor-18 (ARF18) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF18; and

iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

' A further example of the present invention provides the use of a chimeric gene as described according , to any example hereof to modify a phenotype mediated by ARF18 in a plant by increasing expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments: i) a promoter that is operable in the plant cell, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that encodes an Auxin Response Factor-2 (ARF2), as described according to any example hereof; and iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

A further example of the present invention provides the use of a chimeric gene as described according to any example hereof to modify a phenotype mediated by ARF18 in a plant such as by down-regulating expression of ARF2 in the plant or a cell, tissue or organ of the plant, said chimeric gene comprising the following operably linked DNA fragments:

i) a promoter that is operable in the plant cell, as described according to any example hereof;

ii) a polynucleotide comprising a nucleotide sequence that is complementary to at least about 19 contiguous nucleotides or at least about 30 contiguous nucleotides of a polynucleotide that encodes an Auxin Responsive Factor-2 (ARF2) and optionally, further comprising a nucleotide sequence comprising at least about 19 contiguous nucleotides of a polynucleotide that encodes the ARF 18; and

iii) a 3' region that is capable of transcription termination and/or polyadenylation of mRNA in a plant cell, as described according to any example hereof.

It is to be understood that this chimeric gene is capable of expressing antisense RNA, a co-suppression molecule, interfering RNA (iRNA), a double-stranded RNA (dsRNA) or RNAi molecule as described herein above in a plant cell, and that the sequence of the polynucleotide at (ii) supra is readily derived from a sequence encoding ARF2 as described according to any example hereof. Structures of exemplary dsRNA molecules and antisense molecules are as described herein above and said structural features apply mutatis mutandis to this example of the invention.

A further example of the present invention provides an expression vector comprising a chimeric gene according to any example hereof and for the use of the expression vector to modify a phenotype mediated by ARF2 and/or ARF18 in a plant. A further example of the present invention provides a plant cell comprising one or more chimeric genes according to any example of the invention described herein. In another example, the present invention provides a plant, seed, plant cell, plant tissue, or propagating material thereof having modulated expression of ARF2 and/or ARFl 8 produced according to any example hereof.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure I provides copies of photomicrographs of transverse sections of hypocotyl of an A. thaliana ARF2-deficient mutant (panel A) and wild-type A. thaliana ecotype C24 (panel B). Data indicate small xylem cell size for the ARF2-deficient mutant plant.

Figure 2 provides copies of photomicrographs of transverse sections of hypocotyl of an A. thaliana ARF2-deficient mutant (panel A), an A, thaliana ARFl 1 -deficient mutant (panel B), an A. thaliana ARF13-deficient mutant (panel C), an A. thaliana ARF l 4- deficient mutant (panel D), an A. thaliana ARF15-deficient mutant (panel E), an A. thaliana ARF3-deficient mutant (panel F), an A. thaliana ARF19xARF7-double mutant deficient in both ARF19 and ARF7 (panel G), an A. thaliana ARF22-deficient mutant (panel H), an A. thaliana ARF4-deficient mutant (panel I), an A. thaliana ARF5- deficient mutant (panel J), and an A. thaliana ARFl O-deficient mutant (panel ). Data indicate small xylem cell size for the ARF2-deficient mutant plant relative to other ARF-deficient mutants. Figure 3 provides a graphical representation showing the smaller xylem cell cross- sectional area for A. thaliana ARF2-deficient mutant (ARF2; right columns in each pair) relative to wild-type A, thaliana ecotype C24 (WT-Col; left columns in each pair). Four size classes of xylem cell area are indicated on the x-axis. Numbers of cells in each size class are indicated on the abscissa. Sample size was 335 for each of ARF2- deficient mutant and WT-Col. Data indicate a predominance of smaller cells in the ARF2-deficient mutant. Figure 4 provides copies of photomicrographs of transverse sections of hypocotyl of an

A. thaliana ARF18-deficient mutant (panel A, left) and wild-type A. thaliana ecotype C24 (panel A, right), and for transverse sections of flower stems of the A. thaliana ARF18-deficient mutant (panel B, left) and wild-type A. thaliana ecotype C24 (panel

B, right). Data indicate enlarged xylem cells for the ARF18-deficient mutant plant.

Figure 5 provides copies of photographic representations showing A. thaliana ARF18- deficient mutant plants (N9299, left hand side) and wild-type A. thaliana ecotype C24 (C24, right hand side), after 12 days of water deficit (above) and 6-days after recommencement of watering (below). Data indicate reduced wilting of ARF18- deficient plants during the period of water deficit and more rapid recovery and enhanced survival rate of A F18-deficient plants following recommencement of watering, compared to wild-type C24.

Figure 6 provides graphical representations showing the rates of photosynthesis (top left panel), stomatal conductances (tope right panel), transpiration rates (lower left panel), and water use efficiencies (lower right panel) of A. thaliana ARF1 8-deficient mutant plants (N9299) and wild-type A. thaliana ecotype C24 (C24) at day 3 and day 7 after cessation of watering of plants. Data indicate that ARF 18-deficient plants maintained a photosynthetic rate, stomatal conductance, transpiration rate and overall water use efficiency (WUE) during water deficit stress, whereas wild-type plants exhibit reduced rates of photosynthesis, transpiration, stomatal conductance and water use efficiency under the same conditions.

Figure 7 provides graphical representations showing water content of leaves at day 1 through day 12 of water deficit (top panel) and average leaf areas at day 3 of water deficit (lower panel) for A. thaliana ARF18-deficient mutant plants (diamonds in top panel; 299 in lower panel) and wild-type A. thaliana ecotype C24 (squares in top panel; C24 in lower panel). Differential water content between the lines is also indicated in the top panel (triangles). Data indicate that the A RF18-deficient plants lose less water during a period of water stress than wild-type C24 plants, as indicated by the negative value for differential water content between the lines, and marginally higher leaf area than wild-type C24 plants. Figure 8 provides copies of photographic representations showing additional phenotypes of ARF18-deficient plants relative to wild-type C24 plants, including a rounder and wavier leaves (panels a and b), a delayed flowering (panel c), larger seeds (panel d), larger flowers (panel e), and longer primary roots (panel ί)·

Figure 9 provides a graphical representation showing an elevated root elongation rate for ARF18-deficient plants relative to wild-type C24 plants during the first 12 days of growth. DETAILED DESCRIPTION OF THE PREFERRED EXAMPLES

The present invention provides methods and chimeric genes for modulating one or more phenotypes of a plant or a plant cell, plant organ or plant tissue.

The methods and chimeric genes employ polynucleotides comprising one or more sense strand sequences each of which is derived from a gene encoding an ARF2 or ARF 18 polypeptide. It will be understood by the skilled artisan that a sense strand sequence corresponds to sequence in mRNA encoding an ARF2 or ARF 18 polypeptide, however if the sense strand sequence is in DNA as opposed to mRNA it will generally comprise thymidine residues in place of uracil. Wherein the sense strand sequence encodes an ARF2 or ARF 18 polypeptide, or both and ARF2 polypeptide and an ARF 18 polypeptide, the chimeric gene and method may provide for ectopic expression of ARF2 and/or ARF18 polypeptides in a plant cell, tissue, organ or throughout the plant. A sense strand sequence may also be employed to reduce or inhibit ARF2 and/or ARD18 expression in a plant cell, tissue, organ or whole plant, for example wherein it comprises an incomplete or partial open reading frame. Polynucleotides comprising only a partial open frame of an ARF2 or ARF 18 gene may be employed as dominant negative mutants or for co-suppression of an endogenous level of expression of ARF2 and/or ARF18. Alternatively, a sense strand sequence, generally comprising only about 19-30 contiguous nucleotides of a gene encoding ARF2 or ARF18, or a combination of such sense strand sequences, may be employed in the construction of RNAi or dsRNA molecules capable of reducing or inhibiting or preventing an endogenous level of expression of ARF2 and/or ARF 18 in a plant cell, tissue, organ or throughout the plant. Alternatively, or in addition, the methods and chimeric genes of the present invention employ polynucleotides comprising one or more antisense strand sequences i.e., complementary to at least a part of mRNA encoding an ARF2 or ARF18 polypeptide and, if the antisense strand sequence is in DNA as opposed to mRNA it will generally comprise thymidine residues in place of uracil. Antisense strand sequences are generally employed to reduce or inhibit or prevent expression of mRNA to which it is complementary i.e., mRNA encoding an ARF2 or ARF18 polypeptide. Wherein the antisense strand sequences are complementary to a plurality of mRNAs such as because they are presented in a chimeric gene in a tandem array or linked contiguously or non- contiguously, they are employed to reduce or inhibit or prevent expression of the plurality of mRNAs. Preferred methods for reducing expression of one or more mRNAs in a cell by employing antisense strand sequences include antisense technology, ribozyme technology, RNAi and dsRNA. For example, an antisense RNA may employ at least about 50 contiguous nucleotides complementary to ARF2- encoding mRNA and/or at least about 50 contiguous nucleotides complementary to ARF18-encoding mRNA, such as an antisense sequence comprising or consisting of at least 50 contiguous nucleotides of a 3'-untranslated sequence of mRNA and/or protein- encoding sequence. For ribozyme, RNAi and dsRNA approaches, shorter regions of contiguous antisense strand sequence may be employed e.g., at least about 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 and preferably no more than 30 nucleotides in length. Accordingly, the ARF2-derived and/or ARF18-derived sense and antisense strand sequences are employed in the manufacture of chimeric genes for modifying one or more properties and/or traits. For example, the present invention provides a method for modifying a desirable property and/or trait in a plant, which method comprises modulating the expression of an ARF2 gene and/or an ARF18 gene whose expression or transcription product is capable of directly or indirectly modifying a desirable property and/or trait in the plant or plant cell, plant part, tissue, seed or plant propagating material thereof. An ARF2 gene and/or transcription product or an ARF18 gene and/or transcription product may comprise the nucleotide or amino acid sequence from Arabidopsis thaliana or an ortholog thereof from another plant species e.g., poplar or Eucalyptus spp. The method of the invention may comprise the step of transforming a plant or plant propagating material with a nucleic acid molecule comprising a regulatory sequence, typically a- promoter sequence, capable of modulating expression within the plant of a nucleic acid molecule corresponding to an ARF2 gene or an ARF18 gene whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby, on expression of that sequence, the desirable property and/or trait is modified. For example, an expression cassette comprising the chimeric gene may be used to either enhance, reduce or inhibit the expression of ARF2 and/or ARF 18, or to enhance, reduce or inhibit the activity of ARF2 and/or ARF18 in the plant, seed, plant cell, tissue or plant propagating material thereof, thereby modulating the desirable property and/or desirable trait in the plant. For example, a plant-operable promoter, including a plant promoter, may be operably linked to a coding region of the ARF2 and/or ARF 18 gene in the sense orientation. Alternatively, expression of ARF2 and/or ARF18 gene(s) may be modulated by operably linking a plant promoter to a nucleic acid fragment from the gene(s) to thereby form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed. For example, the expression of ARF2 and/or ARF 18 may be modulated by introducing one or more nucleic acid fragments of the gene(s) into an appropriate vector such that double-stranded RNA is transcribed where directed by i.e., under the control of an operably-linked plant promoter, thereby producing decreased levels of mRNA and/or protein encoded by endogenous copies of the gene(s). Levels of mRNA and protein encoded by orthologs or homologs of the gene(s) may also be reduced. Alternatively, expression of ARF2 and/or ARF 18 gene(s) may be modulated by operably linking a plant promoter to a dominant-negative allele of the gene, which interferes with the function of the gene product.

IThe modulated expression of ARF2 and/or ARF 18, or the modulated activity of ARF2 and/or ARF18 in the plant, seed, plant cell, tissue or plant propagating material thereof may comprise an enhanced, reduced, or inhibited expression and/or activity relative to the expression of an otherwise isogenic plant not comprising the chimeric gene of the invention.

Without being bound by any theory or mode of action, the modulated expression of ARF2 and/or ARF 18 and/or the modulated activity of ARF2 and/or ARF 18 results in an altered response of an auxin gene responsive element which drives the transcription of auxin controlled genes to thereby modulate the desirable property and/or desirable trait in the plant. ARF2 and/or ARF18 may each act on a factor in the auxin signalling pathway, e.g., another Auxin Response Factor, to modulate expression of auxin- controlled genes thereby modulating the desirable property and/or desirable trait in the plant. For example, ARF2 may modulate the expression/activity of ARF18 whose expression or transcription product directly or indirectly modifies a desirable property and/or trait in the plant or plant cell, plant part, tissue, seed or plant propagating material thereof. Alternatively, or in addition, ARF18 may modulate the expression of the ARF2 gene whose expression or transcription product is capable of directly or indirectly modifying a desirable property and/or trait in the plant or plant cell, plant part, tissue, seed or plant propagating material thereof. In these examples, ARF2 and ARF18 may be in dynamic equilibrium to control auxin-regulated gene expression.

In these examples of the present invention, the desirable trait according to any example described herein is selected from, but not limited to altered leaf and/or vascular structure, altered secondary xylem structure including lumen size and cell wall thickness, and altered growth characteristics including cellular proliferation and/or cellular expansion. By modifying these traits, a desirable, property is obtained such as increased plant size, root growth and/or root size, seed quality and/or quantity, xylem (wood) quality and water usage efficiency. For example, reduced or ablated expression of ARF2 and/or activity of ARF2 according to any example described herein may enhance cellular expansion, leading to increased cell size in secondary xylem and to a larger lumen size, enhanced biomass, larger seeds, enhanced root growth and/or size, and/or enhanced water usage efficiency. Woody plants according to the invention exhibiting enhanced cellular expansion in secondary xylem and/or reduced wall thickness and/or low density are useful for production of pulp and paper products.

In another example, reduced or ablated expression of ARF18 and/or activity of ARF18 according to any example described herein may also enhance cellular expansion leading to increased cell size in secondary xylem and to a larger lumen size, enhanced biomass, larger seeds, enhanced root growth and/or size, and/or enhanced water usage efficiency, and a greater ability of the plant to cope with drying soil when water supply is. reduced, e.g., during intermittent drought conditions. Enhanced water usage efficiency may also be related to the higher root growth rate and/or increased size and/or increased cellular expansion in xylem of ARF 1 8-deficient plants. Enhanced biomass is desirable for increasing plant and crop yields. Woody plants according to the invention exhibiting enhanced cellular expansion in secondary xylem and/or increased coarseness are useful for production of solid wood products. Increased expression of ARF18 and/or activity of ARF18 according to any example hereof may decrease cellular expansion, leading to a decreased cell size in secondary xylem and to reduced or low coarseness of xylem, and a small seed size when desirable e.g., for the production of parthenocarpic fruits. Woody plants according to the invention exhibiting reduced cellular expansion in secondary xylem and/or low coarseness are useful for production of pulp and paper products. Because performance of the inventive method yields desirable plant products, the present invention extends to a plant, seed, plant cell, plant tissue, or propagating material thereof produced by the method according to any example described herein, such as a plant, seed, plant cell, plant tissue, or propagating material having modulated expression of the ARF2 and/or ARF 18 gene(s).

Chimeric genes and ARF2/ARF sequences

In one example, a chimeric gene of the present invention comprises a plant-operable promoter operably linked to a DNA region coding for an ARF2 and/or ARF 18 protein comprising the amino acid sequence of SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32 or a variant thereof having similar activity as the mentioned proteins, and a 3'-untranslated region involved in transcription termination and polyadenylation.

As used herein, "chimeric gene" or "chimeric nucleic acid" refers to any gene or any nucleic acid, which is not normally found in a particular eukaryotic species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.

As used herein, the term "promoter" denotes any DNA which is recognized and bound (directly or indirectly) by a DNA-dependent RNA-polymerase during initiation of transcription. A promoter includes the transcription initiation site, and binding sites for transcription initiation factors and RNA polymerase, and can comprise various other sites (e.g., enhancers), at which gene expression regulatory proteins may bind.

The term "regulatory region", as used herein, means any DNA, that is involved in driving transcription and controlling (i.e., regulating) the timing and level of transcription of a given DNA sequence, such as a DNA coding for a protein or polypeptide. For example, a 5' regulatory region (or "promoter region") is a DNA sequence located upstream (i.e., 5') of a coding sequence and which comprises the promoter and the 5'-untranslated leader sequence. A 3' regulatory region is a DNA sequence located downstream (i.e., 3') of the coding sequence and which comprises suitable transcription termination (and/or regulation) signals, including one or more polyadenylation signals.

In one example, the promoter is a constitutive promoter. In another embodiment of the invention, the promoter activity is enhanced by external or internal stimuli (inducible promoter), such as but not limited to hormones, chemical compounds, mechanical impulses, abiotic or biotic stress conditions. The activity of the promoter may also be regulated in a temporal or spatial manner (tissue-specific promoters; developmentally regulated promoters).

In a particular preferred example of the invention, the promoter is a plant-operable promoter. As used herein, the term "plant-operable promoter" means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell e.g., certain promoters of viral or bacterial origin such as the CaMV 35S promoter (Hapster et al., Mol. Gen. Genet. 212, 182-190, 1988), the subterranean clover virus promoter No 4. or No. 7 (WO 1996/006932), or a T-DNA gene promoter. Tissue-specific or organ-specific promoters, including but not limited to seed-specific promoters (e.g., WO 1989/003887), organ-primordia specific promoters (An et al., The Plant Cell 8, 15-30, 1996), stem-specific promoters (Keller et al., EMBO J. 7, 3625-3633, 1988), leaf specific promoters (Hudspeth et al., Plant Mol Biol 12, 579-589, 1989), mesophyll- specific promoters such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al., Genes Dev. 3, 1639-1646, 1989), tuber-specific promoters (Keil et al., EMBO J. 8, 1323- 1330, 1989), vascular tissue specific promoters (Peleman et al., Gene 84, 359-369, 1989), stamen-selective promoters (WO 1989/010396, WO 1992/013956), dehiscence zone specific promoters (WO 1997/013865) and the like, may also be employed.

Preferred plant-operable promoters are cambium-operable or xylem-operable promoters. For example, the promoter may be an expansin gene promoter, a sucrose synthase (SuSy) gene promoter, an alpha-tubulin (TUB) gene promoter, an arabinogalactan protein (ARAB) gene promoter, a caffeic acid 3-O-methyltransferase (COMT) gene promoter, a cinnamyl alcohol dehydrogenase (CAD) gene promoter, a cinnamate 4-hydroxylase (C4H) gene promoter, a cinnamoyl CoA reductase (CCR) gene promoter, a ferulate-5-hydroxylase (F5H) gene promoter, a sinapyl alcohol dehydrogenase (SAD) gene promoter, a UDP-D-glucuronate carboxy lyase (UDP) gene promoter, a lipid transfer protein (LTP) gene promoter, or an ag-13 (AG 13) gene promoter, see e.g., U.S. Patent Application 20090229016 or International Application No. PCT/AU2003/001660.

The transcription terminator and polyadenylation signal may be any 3-untranslated region of a plant gene, preferably a gene that is expressed at a high level in a plant e.g., a CaMV 19S or CaMV 35S transcription termination and polyadenylation signal sequence, or a ubiquitin gene terminator, NOS terminator, etc.

The DNA region coding for ARF2 or ARF18 may comprise an open-reading frame or protein-encoding region such as the nucleotide sequence of SEQ ID No. 1 from nucleotide 502 to nucleotide 3081 , or the nucleotide sequence of SEQ ID No. 3 from nucleotide 376 to nucleotide 2955, or the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 2580, or the nucleotide sequence of SEQ ID No. 7 from nucleotide 376 to nucleotide 2955, or the nucleotide sequence of SEQ ID No. 9 from nucleotide 341 to nucleotide 2377, or the nucleotide sequence of SEQ ID No. 1 1 from nucleotide 502 to nucleotide 3081 , or the nucleotide sequence of SEQ ID No. 13 from nucleotide 310 to nucleotide 2889, or the nucleotide sequence of SEQ ID No. 15 from nucleotide 289 to nucleotide 2868, or the nucleotide sequence of SEQ ID No. 17 from nucleotide 291 to nucleotide 2870, or the nucleotide sequence of SEQ ID No. 19 from nucleotide 339 to nucleotide 2918, or the nucleotide sequence of SEQ ID No. 21 from nucleotide 339 to nucleotide 2918, or the nucleotide sequence of SEQ ID No. 23 from nucleotide 339 to nucleotide 2918, or the nucleotide sequence of SEQ ID No. 25 from nucleotide 283 to nucleotide 2091 , or the nucleotide sequence of SEQ ID No. 27 from nucleotide 1 to nucleotide 1809, or the nucleotide sequence of SEQ ID No. 29 from nucleotide 221 to nucleotide 2029, or the nucleotide sequence of SEQ ID No. 31 from nucleotide 1 to nucleotide 1809.

In another example, the DNA region codes for a variant of the proteins comprising the amino acid sequence of SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID o. 28, SEQ ID No. 30 or SEQ ID No. 32. For the purposes of nomenclature, reference is made to the following nucleotide and amino acid sequences:

SEQ ID No. l : Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number N _180913.2).

SEQ ID No. 2: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number NM l 80913.2)

SEQ ID No. 3: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number NM_203251.2).

SEQ ID No. 4: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number NM_203251.2)

SEQ ID No. 5: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AY669787.1 ).

SEQ ID No. 6: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AY669787.1 )

SEQ ID No. 7: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number NM_125593.3).

SEQ ID No. 8: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number NM_125593.3).

SEQ ID No. 9: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AF378862.1 ).

SEQ ID No. 10: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AF378862.1 ).

SEQ ID No. 1 1 : Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number BT000784).

SEQ ID No. 12: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number BT000784).

SEQ ID No. 13: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number A 221305.1 ).

SEQ ID No. 14: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221305.1 ).

SEQ ID No. 15: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK221282.1 ).

SEQ ID No. 16: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number A 221282.1 ). SEQ ID No. 17: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (Gen Bank; Accession number AK.221277.1 ).

SEQ ID No. 18: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK.221277.1 ).

SEQ ID No. 19: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK.221274.1 ).

SEQ ID No. 20: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221274.1 ).

SEQ ID No. 21 : Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK221254.1 ).

SEQ ID No. 22: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221254.1 ).

SEQ ID No. 23: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF2 (GenBank; Accession number AK221252.1 ).

SEQ ID No. 24: Predicted Arabidopsis thaliana amino acid sequence ARF2 (GenBank; Accession number AK221252.1 ).

SEQ ID No. 25: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF 1 8 (GenBank; Accession number NM_1 16048.2);

SEQ ID No. 26: Predicted Arabidopsis thaliana amino acid sequence ARF 18 (GenBank; Accession number NM_1 16048.2).

SEQ ID No. 27: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF18 (GenBank; Accession number AF334717.1 ).

SEQ ID No. 28: Predicted Arabidopsis thaliana amino acid sequence ARF18 (GenBank; Accession number AF334717.1 ).

SEQ ID No. 29: Complete Arabidopsis thaliana mRNA nucleotide sequence ARF 18 (GenBank; Accession number AY059746.1 ).

SEQ ID No. 30: Predicted Arabidopsis thaliana amino acid sequence ARF18 (GenBank; Accession number AY059746.1).

SEQ ID No. 31 : Complete Arabidopsis thaliana mRNA nucleotide sequence ARF 18 (GenBank; Accession number AY091392.1 ).

SEQ ID No. 32: Predicted Arabidopsis thaliana amino acid sequence ARF18 (GenBank; Accession number AY091392.1 ).

As used herein, "variant" proteins refer to proteins wherein one or more amino acids are different from the corresponding position in the proteins having the amino acid sequence of SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32, by substitution, deletion, insertion; and which have at least one of the functions of the proteins encoded by SEQ ID No 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32 such as e.g. the same enzymatic or catalytic activity. Methods to derive variants such a site-specific mutagenesis methods are well known in the art, as well as assays to identify the enzymatic activity encoded by the variant sequences. Preferred substitutions are so called conservative substitutions in which one amino acid residue in a polypeptide is replaced with another naturally occurring amino acid of similar chemical character, for example GlyoAla, Val =>Ile<=>Leu, Asp«->Glu, LysoArg, Asn<»Gln or PheoTrpoTyr. Allelic forms of the nucleotide sequences which may encode variant proteins, including any orthologs or homologs of the exemplified sequences hereof, may be identified by hybridization of libraries, under moderate or high stringency hybridization conditions, such as to cDNA or genomic libraries of a different plant species or plant lines. This includes nucleotide sequences which hybridize under moderate or high stringency conditions to nucleotide sequences encoding the amino acid sequence of SEQ ID Nos 2, 4, 6, 8, 10; 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 or to the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29 or 31 or a sufficiently large part thereof (preferably at least about 19 contiguous nucleotides, or at least about 20 contiguous nucleotides or at least about 25 contiguous nucleotides or at least about 30 contiguous nucleotides or at least about 50 contiguous nucleotides, or at least about 100 or 200 or 300 or 400 or 500 or 1000 or 2000 contiguous nucleotides of said SEQ ID NO(s).

"Moderate stringency hybridization conditions" as used herein mean that hybridization will generally occur if there is at least about 50% and preferably at least 70% sequence identity between the probe and the target sequence. Examples of moderate stringency hybridization conditions includes an overnight incubation in a solution comprising 50% formamide, 2 x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 2 x SSC at approximately 55°C. "High stringency hybridization conditions" as used herein mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5 x SSC ( 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 x SSC at approximately 65°C.

Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), particularly chapter 1 1 . Variants, homologs and orthologs of the exemplified sequences herein are readily obtained by hybridization or amplification according to standard procedures known to the skilled artisan. For example, an isothermal or polymerase chain reaction may be employed using one or more oligonucleotide primers, each comprising at least about 12 or 13 or 14 or 15 or 16 or 17or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 contiguous nucleotides, preferably at least about 30 or 35 or 40 or 45 or 50 or 60 or 70 or 80 or 90 or 100 contiguous nucleotides of a nucleotide selected from SEQ ID No. 1 , SEQ ID No. 3, SEQ ID No. 5 , SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 1 1 , SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 , SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29 and SEQ ID No. 31 , or a complementary sequence to any one of said SEQ ID NOs.

Preferred variants of the exemplified sequences are homologs or orthologs from plants other than A. thaliana and which encode a functional protein that can complement at least one function, but preferably all of the affected functions, in an ARF2-deficient or ARF18-deficient A. thaliana plant. Such complementation is accepted in the art as evidence of homologous or orthologous functionality.

For example, variants of the exemplified sequences may be obtained from a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato. Alternatively, variants of the exemplified sequences may be obtained from a fiber-producing plant e.g., flax or cotton. Alternatively, variants of the exemplified sequences may be obtained from a wood-producing plant e.g., oak, aspen, eucalyptus, maple, pine, spruce, poplar, or larch. For example, the plant may be a species of Eucalyptus ( E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi E. camaldulensis, E. ceracea, E. cloeziana, E. cocci/era, E. cordata, E. cornuta, E. corticosa, E. crebra, E. croajingoleisis, E. curtisii, E. dalrympleana, E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. diversifolia, E. dives, E. dolichocarpa, E. dundasii, E. dunnii, E. elata, E. erythrocoiys, E. erythrophloia, E. eudesmoides, E. falcata, E. gamophylla, E. glaucina, E. globulus, E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis x-urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora, E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua, E. obtusiflora, E. occidentalis, E. optima, E. ovata, E. pachyphylla, E. pauciflora, E. pellita, E. perriniana, E. petiolaris, Έ. pilularis, E. piperita, E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, E. risdoni, E. robertsonii E. rodwayi, E. rubida, E. rubiginosa, E. saligna, E. salmonophloia, E. scoparia, E. sieberi, E. spathulata, E. staeri E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragona, E. tetrodonta, E. tindaliae, E. torquata, E. umbra, E. urophylla, E. vernicosa, E. viminalis, E. wandoo, E. wetarensis, E. willisii, E. willisii subsp. falciformis, E. willisii subsp. willisii, E. woodwardii ); or a species of poplar ( e.g., Populus alba, P. alba *P. grandidentata, P. alba *P. tremula, P. alba *P. tremula var. glandulosa, P. alba *P. tremuloides, P. balsamifera, P. balsamifera subsp. trichocarpa, P. balsamifera subsp. trichocarpa * P. deltoides, P. ciliata, P. deltoides, P. euphratica, P. euramericana, P. kitakamiensis, P. lasiocarpa, P. laurifolia, P. maximowiczii, P. maximowiczii xP, balsamfera subsp. trichocarpa, P. nigra, P. sieboldii ^P. grandideiztata, P. suaveolens, P. szechuanica, P. tomentosa, P. tremula, P. tremula ^χ . tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis ), or a conifer as, for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), or Monterey pine (Pinus radiata); or Douglas-fir (Pseudotsuga menziesii); or Western hemlock (Tsuga canadensis); or Sitka spruce (Picea glauca); or redwood (Sequoia sempervirens); or a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea); or a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis).

In one example of the invention, the expression or activity of an ARF2 and/or ARF18 gene is decreased in a plant, plant cell, plant tissue or plant organ. Thus, in another example of the invention, a method is provided to decrease expression or activity of an endogenous ARF2 and/or ARF18 gene in a plant, said method comprising the step of providing plant cells with a chimeric gene capable of reducing the expression of an endogenous ARF2 and/or ARF18 gene to the plant, wherein said endogenous gene codes for a protein comprising the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32 or a variant thereof, said variant having the same functional or enzymatic activity.

In one example of this method of the invention, a chimeric gene is provided to cells of the plant, wherein the chimeric gene comprises a nucleotide sequence of at least 19 contiguous nucleotides from a gene encoding an amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10. or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32, such as at least 19 contiguous nucleotides of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 7 or SEQ ID No. 9 or SEQ ID No. 1 1 or SEQ ID No. 13 or SEQ ID No. 15 or SEQ ID No. 17 or SEQ ID No. 19 or SEQ ID No. 21 or SEQ ID No. 23 or SEQ ID No. 25 or SEQ ID No. 27 or SEQ ID No. 29 or SEQ ID No. 31. The nucleotide sequence is operably linked to a plant-operable promoter and a 3' region involved in transcription termination and polyadenylation (so-called "sense" RNA mediated gene silencing). In another example, a chimeric gene is provided to cells of the plant, wherein the chimeric gene comprises a nucleotide sequence of at least 19 contiguous nucleotides selected from the antisense strand sequence of a gene encoding a protein comprising the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32, such as a nucleotide sequence of at least about 19 contiguous nucleotides selected from the complement of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 7 or SEQ ID No. 9 or SEQ ID No. 1 1 or SEQ ID No. 13 or SEQ ID No. 15 or SEQ ID No. 17 or SEQ ID No. 1 or SEQ ID No. 21 or SEQ ID No. 23 or SEQ ID No. 25 or SEQ ID No. 27 or SEQ ID No. 29 or SEQ ID No. 31 operably linked to a plant operable promoter and a 3' region involved in transcription termination and polyadenylation.

The length of the antisense or sense nucleotide sequence may vary from about 19 nucleotides in length to a length equal to a length of the target nucleic acid e.g., mRNA. For example, a length of the antisense or sense nucleotide sequence may be at least about 50 or 100 or 150 or 100 or 500 nucleotides. There is really no upper limit to the total length of the antisense nucleotide or sense nucleotide sequence, however there is no advantage a priori in gene manipulations comprising antisense strand sequnces longer than a length of the target nucleic acid, and smaller fragments are easier to handle and may work more effectively for certain operations e.g., dsRNA. For practical reasons e.g., stability of a chimeric gene, a length of the antisense strand sequence should not exceed about 1 or 2 or 3 kb, and the total length of a chimeric gene for most practical applications should not exceed about 12 kb or 15 kb. Preferably, the total antisense nucleotide sequence should have a sequence identity of at least about 75% with the corresponding target sequence, particularly at least about 80 %, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100% identity. It will be appreciated that longer antisense strand nucleotide sequences may be more effective for distantly-related species, to achieve more effective hybridization to the target mRNA where the percentage identity between the antisense strand and the target is low e.g., less than about 50%. However, it is preferred that the antisense or sense nucleotide sequence always includes a sequence of at least about 19-30 nucleotides in length having at least about 90% or 95% or 99% or 100% sequence identity to a part of the target nucleic acid e.g., mRNA. Preferably, for calculating the sequence identity and designing the corresponding antisense or sense sequence, the number of gaps should be minimized, particularly for the shorter antisense or sense sequences.

For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x l OO) divided by the number of positions compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970) Computer-assisted sequence alignment, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wisconsin, USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3. Another example of the invention, relates to a method for reducing the expression of endogenous genes of a plant, wherein said endogenous gene codes for a protein comprising an amino acid sequence having at least about 50% identity to SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32.

The use of a chimeric gene which when transcribed results in so-called double stranded RNA (dsRNA) molecules comprising both sense and antisense sequences, is particularly preferred. Such technology is described e.g., in WO 1999/053050 incorporated herein in its entirety by reference. According to this example of the invention, a chimeric gene may be provided to a plant cell comprising a plant-operable promoter operably linked to a nucleic acid comprising a sense strand sequence comprising at least 19 consecutive nucleotides from a coding region of a nucleic acid encoding a protein having an amino acid sequence of SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32 and an antisense strand sequence comprising at least about 19 consecutive nucleotides having a region of complementarity or wholly complementary to the sense strand sequence. The chimeric gene may comprise additional regions, such as a transcription termination and polyadenylation region > functional in plants. When transcribed an RNA can be produced which may form a double stranded RNA stem between the complementary parts of the sense and antisense region. A spacer region may be present between the sense and antisense nucleotide sequence that forms a loop region of a hairpin loop in the expressed dsRNA. The chimeric gene may further comprise an intron sequence, preferably located in the spacer region. In yet another example of the invention, the chimeric gene used to reduce the expression of a gene endogenous to said plant, wherein said endogenous gene codes for a protein comprising the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32 or a variant thereof, said variant having the same functional or enzymatic activity, encodes a ribozyme which recognizes and cleaves RNA having the nucleotide sequence of an RNA coding for a protein comprising the amino acid sequence of SEQ ID SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No, 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32 or a variant thereof. In another embodiment, the ribozyme recognizes and cleaves RNA having the nucleotide sequence of an RNA comprising the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, or 31. Methods for designing and using ribozymes have been described by Haseloff and Gerlach ( 1988) and are contained e.g.,. in WO 1989/005852.

It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference to nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application. In yet another embodiment of the invention, nucleic acids (either DNA or RNA molecules) are provided which can be used to alter a plant phenotype.

Thus, in one example the invention provides chimeric genes (DNA molecule) which comprise the following operably linked DNA fragments

i) a promoter expressible in said cell of said plant;

ii) a DNA region comprising a nucleotide sequence of at least 19 contiguous nucleotides selected from a nucleotide sequence coding for the protein comprising the amino acid sequence of SEQ ID SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No. 8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 or SEQ ID , No. 28 or SEQ ID No. 30 or SEQ ID No. 32 (or a variant of that protein having the same enzymatic activity), such as the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29 or 31 ; and/or

iii) a DNA region and comprising a nucleotide sequence of at least 19 contiguous nucleotides selected from the complement of a nucleotide sequence coding for the protein comprising the amino acid sequence of

SEQ ID SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6 or SEQ ID No.

8 or SEQ ID No. 10 or SEQ ID No. 12 or SEQ ID No. 14 or SEQ ID No.

16 or SEQ ID No. 18 or SEQ ID No. 20 or SEQ ID No. 22 or SEQ ID No.

24 or SEQ ID No. 26 or SEQ ID No. 28 or SEQ ID No. 30 or SEQ ID No. 32 or a variant thereof, said variant having the same enzymatic activity, such as the nucleotide sequence of SEQ ID Nos 1 , 3, 5, 7, 9, I I , 13, 15,

17, 19, 21 , 23, 25, 27, 29 or 31 ; and

iv) a 3'end region involved in transcription termination and polyadenylation. In another example, the present invention also provides RNA molecules that can be obtained from the chimeric genes according any example hereof. Such RNA molecules can be produced by in vivo or in vitro transcription of the chimeric genes. They can also be obtained through in vitro transcription of chimeric genes, wherein the transcribed region is under control of a promoter recognized by single subunit RNA polymerases from bacteriophages such as SP6, T3 or T7. Alternatively, the RNA molecules may be synthesized in vitro using procedures well known in the art. Also chemical modifications in the RNA ribonucleoside backbone to make the chimeric RNA molecules more stable are well known in the art. Different examples for chimeric genes or RNA molecules have been described above in relation to the methods exemplified herein for altering cellulose biosynthesis and can be applied mutatis mutandis to the examples relating to substances.

Production of transformed plant cells

Chimeric genes or RNA may be provided to plant cells in a stable way, or transiently. Conveniently, stable provision of chimeric genes or RNA molecules may be achieved by integration of the chimeric genes into the genome of the cells of a plant. Methods for the introduction of chimeric genes into plants are well known in the art and include Agrobacterium-medizAed transformation, particle gun delivery, microinjection, electroporation of intact cells, polyethylene glycol-mediated protoplast transformation, electroporation of protoplasts, liposome-mediated transformation, silicon-whiskers mediated transformation etc. The transformed cells obtained in this way may then be regenerated into mature fertile plants.

In another example, the chimeric genes or chimeric RNA molecules of the invention are provided on a DNA or RNA molecule capable of autonomously replicating in the cells of the plant, such as e.g. viral vectors. The chimeric gene or the RNA molecules of the invention may be also be provided transiently to the cells of the plant.

In another example, the present invention also provides plant cells and plants containing the chimeric genes or the RNA molecules according to any example hereof. Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants comprising the chimeric genes of the present invention, which are produced by traditional breeding methods are also included within the scope of the present invention.

The methods and means of the invention are particularly suited for use in a food crop plant such as wheat, rice, maize, barley, rye, sorghum, pearl millet, oil seed rape, canola, soybean, peanut, sunflower, kidney bean, white bean, black bean, broad bean, pea, chick pea, lentil, or tomato.

Additionally, the methods and means of the invention are particularly suited for use in a fiber-producing plant e.g., hemp, jute, flax or cotton. Exemplary cottons include Gossypium hirs tum and Gossypium barbadense, such as the varieties Coker 312, Coker310, Coker 5 Acala SJ-5, GSC251 10, FiberMax® 819, FiberMax® 832, FiberMax® 966, FiberMax® 958, FiberMax® 989, FiberMax® 5024, transgenic FiberMax® varieties exhibiting herbicide or insect-resistant traits, Siokra 1 -3, T25, GSA75, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C 1 , Acala B 1644, Acala B 1654-26, Acala BI 654-43, Acala B3991 , Acala GC356, Acala GC510, Acala GAM 1 , Acala C I , Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B 1810, Acala B2724, Acala B4894, Acala B5002, non Acala "picker" Siokra, "stripper" variety FC2017, Coker 31 5, STONEVILLE 506, STONEVILLE 825, DP50, DP61 , DP90, DP77, DES 1 19, McN235, HBX87, HBX191 , HBX107, FC 3027, CHEMBRED A l , CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED Bl , CHEMBRED B2, CHEMBRED B3, CHEMBRED C I , CHEMBRED C2, CHEMBRED C3, CHEMBRED C4, PAYMASTER 145, HS26, HS46, SICALA, PIMA S6 and ORO BLANCO PIMA. FiberMax is the registered Trademark in Australia of Cotton Seed Distributors Pty Ltd.

Additionally, the methods and means of the invention are particularly suited for use in a wood-producing plant e.g., oak, aspen, eucalyptus, maple, pine, spruce, poplar, or larch. For example, the plant may be a species of Eucalyptus ( E. alba, E. albens, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi E. camaldulensis, E. ceracea, E. cloeziana, E. coccifera, E. cordata, E. cornuta, E. corticosa, E. crebra, E. croajingoleisis, E. curtisii, E. dalrympleana, E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. diversifolia, E. dives, E. dolichocarpa, E. dundasii, E. dunnii, E. elata, E. erythrocoiys, E. erythrophloia, E. eudesmoides, E. falcata, E. gamophylla, E. glaucina, E. globulus, E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis *urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora,. E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua, E. obtusiflora, E. occidentalis, E. optima, E. ovata, E. pachyphylla, E. pauciflora, E. pellita, E. perriniana, E. petiolaris, E. pilularis, E. piperita, E. platyphylla, E. polyanihemos, E. populnea, E. preissiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, E. risdoni, E. robertsonii E. rodwayi, E. rubida, E. rubiginosa, E. saligna, E. salmonophloia, E. scoparia, E. sieberi, E. spathulata, E. staeri E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragona, E. tetrodonta, E. tindaliae, E. ' torquata, E. umbra, E. urophylla, E. vernicosa, E. viminalis, E. wandoo, E. wetarensis,

E. willisii, E. willisii subsp. falciformis, E. willisii subsp. willisii, E. woodwardii ); or a species of poplar ( e.g., Populus alba, P. alba *P. grandidentata, P. alba *P. tremula,

P. alba *P. tremula var. glandulosa, P. alba *P. tremuloides, P. balsamifera, P. balsamifera subsp. trichocarpa, P. balsamifera subsp. trichocarpa *P. deltoides, P. ciliata, P. deltoides, P. euphratica, P. euramericana, P. kitakamiensis, P. lasiocarpa,

P. laurifolia, P. maximowiczii, P. maximowiczii ^P. balsamfera subsp. trichocarpa, P. nigra, P. sieboldii *P, grandideiztata, P. suaveolens, P. szechuanica, P. tomentosa, P. tremula, P. tremula *P. tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis ), or a conifer as, for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), or

Monterey pine (Pinus radiata); or Douglas-fir (Pseudotsuga menziesii); or Western hemlock (Tsuga canadensis); or Sitka spruce (Picea glauca); or redwood (Sequoia sempervirens); or a true fir such as silver fir (Abies amabilis) or balsam fir (Abies balsamea); or a cedar such as Western red cedar (Thuja plicata) or Alaska yellow-cedar (Chamecyparis nootkatensis).

For a better understanding of the invention and to show how it may be performed, the present invention will now be illustrated by way of the following Examples, which are not intended to be limiting in any way. The teachings of all references cited herein are incorporated herein by reference.

Example 1: Materials and methods

Plant materials

Arabidopsis thaliana was selected as a model plant for xylem development in woody plants such as poplar. All experiments were performed using Arabidopsis thaliana line N906 (wild-type syn. ecotype C24), an ARF2-deficient transgenic RNAi line, and an ARF18rdeficient transgenic mutant line. The ARF18-deficient transgenic line was a GAL4-GFP enhancer trap line produced by Haseloff et al.. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom, and is publicly available from the Nottingham Arabidopsis Stock Centre. The ARF2 transgenic mutant line was obtained from Okushima et al., Plant Gene Expression Center, 800 Buchanan Street, Albany, CA 94710, USA, and is publicaly available from the Arabidopsis Biological Resource Centre. Plant growth conditions

Arabidopsis thaliana wild-type and mutant plants were grown at 21 °C and 16 hr day period, and at 18°C during an 8hr dark period, in a controlled growth room • environment. ' Seeds were germinated on plugger soil comprising 45 g/L Osmocot general mix fertilizer. Germinated seedlings were separated and grown for 35 days until mature.

, Alternatively, seeds were surface-sterilized for 10 min in 70% (v/v) ethanol/0.05% (v/v) Triton X-100, and washed three times in 95% (v/v) ethanol, sown onto the surfaces of ½MS agar plates, vernalized for 3 days at 4°C, and incubated in a growth chamber at 120- 150 μΕ m ^"2 s ^' ' , with a day period of 16 hr at 23°C and a dark period of 8 hr at 21°C. After 1 1 days, seedlings were transplanted to potting media and grown as describe din the preceding paragraph.

Tissue sections

Transverse sections of stem and hypocotyls were harvested, cleaned to remove excessive plant material, including lateral roots, trimmed to a maximum length of 2mm, and immersed in fixative according to standard procedures for 4 hr. The fixative was replaced and plant tissue fixed for a further 4 hr period. Plant material was stored at 5°C in fixative. Fixed plant material was wax-embedded according to standard procedures. A Leica RM2235 rotary microtome was employed to slice tissue sections to an appropriate thickness e.g., 2 μτη. Wax -embedded blocks were pre-cooled on ice, and transverse sections of stem and hypocotyl were obtained, wherein the wrinkle effect was reduced by incubating tissue ribbons in 1% (w/v) horse serum for 2min, and sections were transferred onto glass slides for staining. Multiple sections were produced from each specimen to ensure a complete representation of the specimen structure.

To stain sections for secondary xylem vessels and fibers sections were de-waxed by washing three times in absolute ethanol, and hydrated by dipping into ethanol baths of decreasing strength i.e., 90% ethanol then 80% ethanol then 70% ethanol then 60% ethanol then finally 50% ethanol. The slides were then incubated in the presence of 2% safranin for lOmin. The slides were dipped into absolute ethanol, followed by a 50:50 (v/v) ethanol:xylol solution, and then into ethanolic baths comprising a gradually increasing concentration of xylol to a final wash in 100% xylol. Oil and a ^" cover slip were then applied to the slides, which were air-dried overnight at room temperature.

Alternatively, hypocotyls and flower stems were incubated in a fixative solution (5 mL of 16% paraformaldehyde, 2 mL of 25% gluteraldehyde, 10 mL of 0.2 M phosphate- buffered saline (PBS), 3 mL of distilled water) for 2 hours, washed twice for 5 min per wash in 0.2 M PBS, and washed successively in 30% ethanol for 15 min, , then in 70% ethanol for 15 min, then in 00% ethanol for 15 min then in 99% ethanol for 15 min, and finally in absolute ethanol for 60 minutes. After the final dehydration step, the ethanol was removed and plant material was incubated in 25% LR White Resin for 1 hr followed by 50% LR White Resin for 24 hr, then 75% LR White Resin for 2 hr and undiluted LR White Resin for 2 hr. The plant material was then transferred to fresh 100%) LR White Resin for at least 24 hours, and then dried at 55°C for at least 24 hours. Pant material was then sectioned using a Leica (Reichert) Ultracut S to produce tissue sections of Ο.όμπι thickness.

Microscopy

Digital images of secondary xylem and vascular bundles were obtained using a light microscope with in-built camera comprising a 1 OX or 40X objective and an 8X eye piece.

Plant morphological and physiological determinations

Root length and root elongation rate were determined for seedlings grown on ½MS agar plates. Root tips of seedlings were marked from day 6 post-germination to day 12 post-germination and root lengths determined at day 12 by imaging using ImageJ software. Elongation rates of roots were then calculated. Hypocotyl lengths were determined for plants grown in potting medium, or for etiolated seedlings grown on ½MS agar plates. Leaf areas were determined using EZ-Rhizo software.

To determine responsiveness of plants to water deficit, plants that had been grown for 23 days under normal conditions were subjected to water deficit by withholding water for a period of 12 days, and then re-watered. At day 3 and day 7 after water was withheld, photosynthetic rate, stomatal conductance and transpiration rate were determined on the largest most fully mature leaf of each plant using standard procedures e.g., employing a Li-Cor LI 6400 gas exchange and fluorescence system. Measurements were taken at 10 second intervals. A reference CO _∑ flow of 400 μηιοΐ s ^'1 , block temperature of 25°C, quantum flux of 500 μπιοΐ photosynthetic active radiation m ^'2 s ^{' 1} and an air flow rate of 100 μπιοΐ s ^'1 were employed.

Example 2: Effect of modulating ARF2 expression

Figure 1 provides representative transverse sections of hypocotyl of the ARF2-deficient mutant (Figure l a) and otherwise isogenic wild-type (WT) plants (Figure l b). Data indicate that that the number of small vessels is increased in xylem of the ARF2- deficient A. thaliana mutant. Tears in secondary xylem were apparent in all sections of ARF2-deficient secondary xylem, suggesting possible reduced lignification, because only non-lignified structures of wild-type plants showed tearing. Data presented in Figure 2 show that, in contrast to ARF2-deficient (and ARF18- deficient) mutant plants, the vasculatures of ARF 1 1 -deficient mutant, ARF 13-deficient mutant, ARFI4-deficient mutant, ARF15-deficient mutant, ARF3-deficient mutant, ARF22-deficient mutant, ARF4-deficient mutant, ARF5-deficient mutant, and the ARF19-deficient and ARF7-deficient ARF19xARF7 double-mutant, more closely resembled the vasculature of wild-type plants.

Quantification of the vessel size in the ARF2-deficient plants and wild-type plants is provided in Figure 3. For wild-type A. thaliana, about 44% of vessels are small or very small vessels having cross-sectional area in the range of 0-20 μιτι ² , and about 25% of vessels have a cross-sectional area in the range of 21-49 μm ² and about 56%. of vessels are large vessels having a cross-sectional area greater than 20 μηι ² . This includes about 20% of very small vessels and about 30% of very large vessels. In contrast, there is a markedly enhanced proportion of small vessels in the ARF2-deficient mutant plant, wherein about 65% of vessels are small or very small vessels, and only about 35% of vessels are larger. Thus, about 38% of vessels of the ARF2-deficient mutant have a cross-sectional area in the range 0-7 μπι ² , and about 27% have a cross-sectional area in the range 8-20 μπι ² . The small cell area in secondary xylem of ARF2-deficient plants indicates reduced cell expansion and vessel formation. The ARF2-deficient mutant has enhanced lignification relative to wild-type plants, as evidenced by increased thickening of cell walls (not shown). Hypocotyl diameter of the ARF2-deficient mutant was also larger than in wild-type plants, the increase in size being correlated with increased cell number and density. These data indicate that ARF2 affects cell division, cell expansion and vessel formation and structure in secondary xylem.

Example 3: Effect of modulating ARFl 8 expression

Data presented in Figure 4 indicate that the vasculature of ARFl 8-deficient plants is abnormal. Comparisons of the cross sections of hypocotyls and base flower stems, of ARFl 8-deficient plants and wild-type plants demonstrates larger fibre cells and vessels in ARFl 8-deficient plants relative to wild-type plants (Figure 4a and 4b).

Consistent with the larger vasculature of the ARFl 8-deficient plants, the mutant exhibited enhanced drought tolerance or reduced susceptibility to water deficit stress than wild-type plants, as determined by reducing wilting after water supply was reduced (Figure 5). Following water-deficit, the mutant line also recovered better than wild-type plants, as determined by a higher percentage of plants that survived when watering was resumed after a period of water deficit. As shown in Figure 5, about 60% of ARF 18-deficient plants survive a period of water deficit compared to only about 10% of wild-type plants.

Data in Figure 6 also indicate that ARF 18-deficient plants maintained a photosynthetic rate, stomatal conductance and transpiration rate during and following water stress, whereas wild-type plants exhibit reduced rates of photosynthesis, transpiration, and stomatal conductance under the same conditions. To investigate the morphological basis of the enhanced water use efficiency of A RF18-deficient plants, the weights of potted plants and average total leaf areas of plants were determined for 39-day old plants e.g., after 3 days of water deficit. ARF18-deficient plants had higher average total leaf areas, and reduced water content (p<0.001 ) than wild-type plants. These data suggest that the modified vasculature of the ARF 18-deficient plants contributes to or is associated with enhanced water use efficiency relative to wild-type plants. Other phenotypes were observed for ARF18-deifcient plants, including a clear and packed rosette pattern of rounder and wavier leaves as compared to the wild-type C24 plant (Figure 8a, b), a delayed flowering e.g., by about 10 days (Figure 8c), larger seeds (Figure 8d) and larger flowers (Figure 8e). The roots of 12-day-old ARF 18-deficient plants were also found to be significantly longer (p<0.001 ) than the roots of 12-day-old wild-type C24 seedlings, and this was correlated with a higher rate of root elongation (pO.001 ) i.e., about 5.73 mm/day for the ARF 18-deficient mutant compared to 3.82 mm/day for wild-type plants (Table 1 ; Figure 8f, Figure 9). The hypocotyl length of the ARF18-deficient mutant was also found to be significantly higher than that of the wild- type seedlings (p<0.001 ; Table 1 ). Seeds of ARF 18-deficient plants were also heavier than seeds of wild-type plants (p<0.001 ; Table 1 ).

In summary, the ARF 18 gene affects secondary xylem structure and water use efficiency, and also hypocotyl elongation in the light and in the dark, root elongation, cell size, cotyledon size, leaf size, flowering time and seed yield. TABLE 1

Measurements of develo pmental traits during normal growth

Trait Line Mean Difference S.E. P

H pocotyl length ARF I 8-deficient 8.13 mm 2.72 1.30 <0.001 (8-days old)

Wild type C24 5.41 mm 1.28

Etiolated hypocotyl ARF18-deficient 24.81 mm 6.70 6.06 <0.001 length (12-days old)

Wild type C24 18.1 1 mm 3.86

Root length ARF 18-deficient 36.34 mm 13.71 ' 9.1 1 <0.001

Wild type C24 22.63 mm 5.94

ARF18-deficient 5.73 mm day 1.91 1.47 <0.001

Root elongation rate

Wild type C24 3.82 mm/day 0.94

ARF18-deficient 0.1 18 g 0.037 0.021 <0.001

Seed mass

Wild type C24 0.081 g 0.018