CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No.

62/356,185, filed June 29, 2016, which is herein incorporated by reference for all purposes. STATEMENT AS TO RIGHT TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Contract No. DE-AR0000204 awarded by the U.S. Department of Energy. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Photosynthetic organisms, including bacteria, algae, and plants, have evolved extensive arrays of light-harvesting pigments, comprising chlorophylls, carotenoids, and bilins that absorb sunlight and transfer the excitation energy to photochemical reaction centers. The latter convert the absorbed irradiance to chemical energy via the photochemical charge separation reaction, which is viewed as the beginning step of photosynthesis. The evolution of sizable arrays of light-harvesting antennae in all photosynthetic systems confers a selective advantage for the organism in nature, where sunlight intensity is often the growth-limiting parameter. Successful competition in nature requires capturing more sunlight for self, even if wasted, and preventing light capture by competing neighbors (Melis 2009). Consequently, top layers of plant canopies and upper layers of microalgae in high-density monocultures absorb sunlight far in excess of what is needed to saturate photosynthesis (Melis et al. 1999; Polle et al. 2003; Melis 2009; Ort et al 2011), Excess absorbed irradiance is dissipated in an orderly manner by the photosystems via non-photochemical quenching (NPQ) mechanisms, which evolved to protect the photosynthetic apparatus and prevent photosensitized bleaching (Miiller et al. 2001 ; Ruban 2016).

[0004] In organisms of oxygenic photosynthesis, large arrays of light-harvesting pigment- protein complexes are peripheral components of photosystem-I (PSI) and photosystem-II (PSII). Minimizing, or truncating, the chlorophyll antenna size of the photosystems can improve photosynthetic solar energy conversion efficiency and productivity in high foliage density monocultures (reviewed in Melis 2009). The rationale is that individual chlorop!asts and photosystems with a smaller chlorophyll antenna size in the upper canopy leaves will have a diminished probability of absorbing sunlight, thereby permitting greater penetration and a more uniform distribution and utilization of irradiance throughout the foliage of the monoculture. Such altered optical properties alleviate over-absorption and wasteful dissipation of sunlight by the upper canopy and enhance photosynthetic productivity of the foliage of the monoculture as a whole. The Truncated Light-harvesting Antenna (TLA) concept, referring to a smaller than wild type chlorophyll antenna size of the photosystems, has found application and noteworthy success in the case of high-density cultivation of microalgae (Nakajima and Ueda 1997, 1999; Melis et al. 1999; Nakajima and Itayama 2003 ; Polle et al. 2003 ; Mussgnug et al. 2007) and

cyanobacteria (Kirst et al. 2014), but has not been applied to plant canopies.

BRIEF SUMMARY OF THE INVENTION

[0005] In one aspect, this disclosure relates to high-density plant canopies and is based, in part on the discovery that greater biomass accumulation occurs in TLA plant canopies over that measured in wild-type counterparts grown under the same agronomic and ambient conditions. Distinct plant anatomical appearance differences also occur.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Fig. I Visual appearance and coloration of Nicotiana tabacum wild type and TLA leaves. Wild type (WT) tobacco leaves have dark green coloration whereas the TLA tobacco leaves have light green coloration.

[0007] Fig. 2 Coomassie stai n of total protein extracts from Nicotiana tabacum wild type (WT) and TLA leaves resolved by SDS-PAGE. Lanes were loaded with 2 ¾ chlorophyll (a and b) for the TLA analysis and with 0.5, 1.0, or 2.0 μ chlorophyll (a and b) for the wild type (WT) analysis. On a chlorophyll basis, the TLA sample contained more RBCL, the large subunit of RubisCO, than the wild type. However, the TLA sample contained substantially lower levels of LHC-II, apoproteins of the major light-harvesting complex of PSII, than the wild type.

[0008] Fig. 3 Western blot analysis of total protein extracts from Nicotiana tabacum wild type (WT) and TLA leaves. Total protein extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with specific polyclonal antibodies raised against the PsaA/PsaB photosystem-I reaction center proteins (PS! RC), the PsbD photosystern-11 reaction center protein (D2), or the light-harvesting chlorophyll-proteins LHCB1 and LHCB2 of PSIL Note the approximately equal levels of D2 protein (relative measure of PSII) in the WT and TLA lanes, and the substantially lower PSI RC and LHCB protein levels in the TLA tobacco relative to the wild type.

[0009] Fig. 4 Light-saturation curves of photosynthesis in Nicotiana tabacum wild type (dark green) and TLA leaves (light green) at ambient CO _? , concentration (400 μηιοΐ ιηοΓ ¹ ). Saturation of photosynthesis in the wild type was estimated to occur at 425 μηιοΐ photons m-2 s-1 , whereas in the TLA mutant saturation occurred at 635 μηιοΐ photons m-2 s-1. [0010] Fig. 5 Visual appearance of Nicotiana tabacum wild type and TLA canopies, shown near the end of their respective growth period. The wild type tobacco leaves have a dark green coloration (upper panel) and the TLA tobacco leaves have a light coloration (lower panel). The wiid type plants also have longer internode distances with fewer leaves in their upper canopy of the wild type as compared with that of the TLA plants. The overall foliage density is higher in the TLA monoculture compared to the wild type.

[0011] Fig. 6 A and 6B (6 A) Average values of biomass harvested from three different pairs of wild type and TLA monocultures grown at different periods of time during the growth season in the greenhouse. TLA monocultures produced about 25% more biomass than the corresponding wild-type monocultures. (6B) Biomass accumulation by individual wild type - TLA pairs of monocultures, showing variation among the absolute yiel ds of the three separate pairs, depending on tune of the year and greenhouse location of the monocultures, but in all cases the TLA monoculture produced more biomass than the corresponding wild type.

[0012] Fig. 7. Schematic of traditional plant (tobacco) agronomy shows the typical 1 8-inch distance between adjacent plants, translating into an average of 4.35 plants cultivated per m ² . This planting density prevents individual plants from unduly shading one-another.

[0013] Fig. 8, Schematic of modified tobacco agronomy shows a narrow 9-inch distance between adjacent plants, translating into a monoculture density of 1 4.9 plants per m ² . This substantially greater planting density takes advantage of the TLA property of the photosysteras, and of the ensuing lower chlorophyll content per leaf area, properties that afford a greater transmittance of sunlight through the foliage, thereby enhancing monoculture productivity.

[0014] Fig. 9. Schematic of alternative plant (tobacco) agronomy shows planting in rows of three with an 18-inch distance between the groups of three. A 9- inch distance from each other separates individual plants in a group of three. This configuration translates into 12.7 plants per m ² . This planting density also benefits from the TLA size of the photosystems, and of the ensuing lower chlorophyll content per leaf area, properties that afford a greater transmittance of sunlight through the foliage, thereby enhancing monoculture productivity.

DETAILED DESCRIPTION

Terminology

[0015] In the context of this invention, a "Truncated Light-harvesting Antenna" or "TLA" plant has a genetic modification that results in a smaller antenna size of the photosystems compared to a wild-type counterpart plant of the same strain that does not have the genetic modification. In the present disclosure, TLA antennae are typically less than about 80%, or less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, or less than about 50% of the antenna size of the corresponding wild type plant, and are at least about 20%, or at least about 25% or about 30%, of the antenna size of the corresponding wild type plant.

[0016] A "broad" leaf in the context of this disclosure refers to a leaf having a length-to-width ratio of less than 5: 1.

[0017] A "blade-like leaf in the context of this disclosure refers to a leaving having a length- to-width ratio of 5: 1 or greater.

[0018] The terms "foliage" as used herein refers to the totality of the leaves and other green tissues of a plant, shrub, or tree, including leaves on the stems or branches, which collectively contribute to photosynthesis and plant growth and development.

[0019] The term "canopy" and "upper canopy" as used herein refers to the upper layer of leaves in a monoculture that are directly exposed to the sun and may over-absorb and wastefully dissipate sunlight energy, while shading leaves that are lower or internal to the foliage. A plant canopy changes dynamically, as plants grow and develop.

[0020] As used herein "bright sunlight" refers to incident photosynthetically active radiation (PAR, or visible light) exceeding 500 μηιοΐ photons per m ^' per s ^! . [0021] As used herein, the term "about" means that a value may vary +/- 15%, +/- 10% or +/- 5% and remain within the scope of the invention.

[0022] A "plant" as used herein refers to a whole plant that is cultivated. The class of plants that can be used in the method of the disclosure includes monocots and dicots and any plant that is planted in soil for culture. TLA plants

[0023] The present disclosure is based in part on the discover}' that cultivating TLA plants so that they are closely spaced such that, there is significant overlap of the leaves results in plants that have increased biomass compared to wild-type counterpart plants.

[0024] In the present, disclosure, a TLA plant harbors a mutation that results in a decrease in antenna size relative to the wild-type plant that does not have such a mutation. A TLA plant has an antenna size that are decreased by at least 20%, at least 25%, typically at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 60%, at least 70%, or 80% compared to the counterpart wild type plant. Antenna size of the photosystems can be measured using any method. For example, antenna size of the photosystems in situ can be determined using the spectrophotometric and kinetic method of Melis (see, e.g., Phil. Trans. R. Soc. Lond. B 323: 397 409, 1989. ) Illustrati ve results of antenna size measurements are described in the examples.

[0025] TLA plants that have a mutation results in decreased antenna size are known. For example, mutations that result in small or truncated antenna size can in occur in genes such as TLAl (US Patent 7,745,696), TLA2 (US Patent Application 20140295448), TLA3 (Kirst et al. 2012), CAO (Chlorophyllide a oxygenase; Ghirardi et al. 1986; Polie et al. 2000), Mg-chelatase subunit I, and CFIL-I (Hansson et al. 1999; Fitzmaurice et al. 1999). In some embodiments, a TLA plant, i.e., a plant having reduced chlorophyll antenna size, has a mutation in a gene as indicated in the references cited in the preceding sentence where the mutated gene is unknown. In some embodiments, a TLA plant has a genetic modification resulting in decreased chlorophyll antenna size where the mutation has not been identified. Example of plant lines that having decreased antenna size are provided in Table 3. Methods of identifying or generating such plants are described in the references cited herein, each of winch is herein incorporated by reference for this purpose.

[0026] TLA plants that are planted at high density in accordance with the disclosure, exhibit a chlorophyli a to chlorophyll b ratio that is increased from that of wild type plants, which wildtype ratio is typically about 3+1. Thus, in some embodiments, TLA plants cultured as described herein have a chlorophyll a to chlorophyli b of at least 4±1 , 6±1, 8±1, 12+1, or 20±1. In some embodiments, the chlorophyll to chlorophyll b ratio is greater than 20+1 , e.g., 25+1 , or greater. In some embodiments, TLA plants do not have measureabie amounts of chlorophyll b. The chlorophyll a and chlorophyll b content and compositions of leaves can be determined using any method. Often, chlorophyll a and chlorophyll b content are measured upon pigment extraction from the leaves in organic solvent, typically methanol ethanol, or acetone, and quantified by their specific absorbance in the red region of the spectrum, as per the Arnon method (1949). Example using methanol extraction and quantification of the chlorophylls spectrophotometrically is provided in the Examples section of the present, disclosure.

[0027] A "grouping" of plants in the context of the present disclosure refers to the overall pattern in which plants are planted. Each grouping contains individual plants. Often, the individual plants are spaced such that plants within the grouping are closer to one another than are the grouping to one another. For example, a grouping can be rows, clusters, circles, or other geometrical configurations.

[0028] In typical agronomic practice, e.g., in planting wild type tobacco and grape vine, individual plants are arranged in rows, separated by about 18 inches from each other, with rows also separated by about 18 inches from one another (see, Fig. 7 by way of illustration). This illustrative agronomic configuration, e.g., for wild type tobacco and grape vine plants, results in a field with a plant density of about 4 to 4.5 plants per nr. In order to obtain greater biomass per surface area compared to a wild-type plant, TLA plants, e.g., tobacco or grape vine plants.

Plants may be planted in rows, circles, or other configurations. Typically, TLA plants such as tobacco or grape vine plants, within a grouping, such as a row or cluster, are separated by less than about 9 inches, whereas the groupings of plants are separated by less than about 18 inches and as close as 9 inches from one another, with the rows also separate by less than about 18 inches and as close as about 9 inches from each other (see. Fig. 8 by way of illustration). This agronomic configuration results in a field plant density of about 15 plants per m ^' \ TLA plants are capable of making efficient sunlight utilization under such high planting density, thereby supporting growth and productivity that exceeds the wild type under the same high-density monoculture agronomic conditions. Whereas the schematic of Fig. 8 depicts a better than 300% increase in planting density, alternative groupings may result in planting density increase by 50%, 100%, or by 200% over what could be practiced with their wild type counterpart plants. [0029] The description in the preceding paragraph is described using tobacco and grape vine plants as illustrative embodiments; however, plant density increases by 100%, or 200%, or 300% can be achieved with other TLA crop species having reduced chlorophyll antenna size (see, e.g., Table 3) or can be genetically engineered using such techniques as random mutagenesis coupled with selection for TLA antenna size as described herein or by specifically targeting a gene known to influence TLA antenna size, e.g., TLA1, TLA2, TLA3, CAO, Mg-chelatase subunit I, and CHL-I.

[0030] TLA plants cultivated in accordance with this disclosure, typically generate a harvestable total biomass that is at least about 15%, at least about 20%, at least about 25%, at least about 50%, or at least about 100%, or greater, than a counterpart wild type plant monoculture grown under the same high-density monoculture cultivation conditions.

[0031] In certain embodiments of the invention, the plant is corn, switchgrass, sorghum, miscanthus, sugarcane, alfalfa, wheat, soy, cotton, barley, turf grass, tobacco, potato, bamboo, rape, sugar beet, sunflower, millet, bean, tomato, canola, or grape vine.

EXAMPLES Materials and Methods

Plan t Materia!

[0032] Nicotiana tabacum, cv John William's Broadieaf, and the yellow-green mutant Su/su (Homann and Schmid 1967; Okabe et al. 1977) were grown in the greenhouse under ambient sunlight conditions. Seeds were kindly provided by Dr. Georg H. Schmid, University of Bielefeld, Germany. Prior analysis has shown the yellow-green Sw'su mutant to have a substantially smaller than wild type light-harvesting antenna size (Melis and Thielen 1980; Thielen and van Gorkom 1981). Hence, the yellow-green mutant Sw'su mutant will be referred to as Truncated Light-harvesting Antenna tobacco (TLA tobacco).

Pigment determination and leaf protein analysis

[0033] The chlorophyll and carotenoid concentration of leaves was determined

spectrophotometrically in 100% methanol extracts of tobacco leaves according to Lichtenthaler (1987). Protein analyses were conducted with leaf extracts resolved in precast SDS-PAGE gels (BIO-RAD). Loading of samples was based on chlorophyll content.

Chloroplast and thylakoid membrane isolation

[0034] Leaves were homogenized in ice-cold chloroplast isolation buffer containing 0.4 M sucrose, 50 mM Tricine (pH 7.8), 10 mM NaCl, 5 mM MgCi ₂ , 0.2% polyvinylpyrrolidone 40, 1% sodium ascorbate, 1 mM aminocaproic acid, 1 mM aminobenzamidme and 100 μΜ phenylmethylsulfonyl fluoride (PMSF). The suspension was filtered to separate unbroken leaf pieces from the cell lysate. Chloroplasts were pelleted by centrifugation at 5,000 g for 10 min and washed twice with chilled chloroplast isolation buffer. For thylakoid membrane isolation, chloroplasts were lysed by re-suspension in a glass homogenizer in hypotonic buffer containing 50 mM Tricine (pH 7.8), 10 mM NaCl, 5 mM MgC , 0.2% polyvinylpyrrolidone 40, 1% sodium ascorbate, 1 mM aminocaproic acid, 1 mM aminobenzamidine and 100 μΜ

phenylmethylsulfonyl fluoride. Thylakoid membranes were then pelleted by centrifugation at 75,000 g for 45 min at 4°C. Membranes were resuspended in 50 mM Tricine (pH 7.8), 10 mM NaCl, 5 mM MgC for spectrophotometric analysis.

Spectrophotometric and kinetic analyses [0035] The light-wiiwwi-dark absorbance difference signal at 700 nm (P700) for PSI, and 320 nm (QA) for PSII (Melis and Brown 1980; Melis 1989) were measured with a laboratory constructed sensitive absorbance difference spectrophotometer. Photosystems quantification estimates were then based on the measurements of P700 and QA. The functional light- harvesting chlorophyll antenna size of PSI and PSII was estimated from the rate constant of P700 photo-oxidation and QA photoreduction, measured upon weak green actinic illumination of isolated and DCMU-poisoned thylakoid membranes (Melis 1989).

Measurement of photosynthesis

[0036] Photosynthetic gas exchange measurements were made using a portable open gas analysis device (Ll-6400, Li-Cor Inc., Lincoln, NE, USA). Light response curves were measured on the youngest and second youngest fully expanded and attached leaves of at least 2 different plants from the same canopy. Leaf temperature and COj concentration in the leaf chamber were 25°C and 400 μπιοΐ mo! ^" ' (ambient Ci¾ concentration), respectively. The vapor pressure deficit was maintained below 1 kPa. The adaxial side of the leaf was illuminated by the light source (10% blue, 90% red). The starting light intensity was 1500 μηιοΐ photons m ^" ' s ^"1 , then lowered to 1200, 1000, 800, 600, 400, 200, 100, 50, and 0. Measurements at various light intensities were recorded after the rate of photosynthesis reached steady state (after about 10 minutes).

Foliage density hiomass accumulation experiments [0037] Heterozygous (Su/su) Nicotiana tabacum seeds were sprinkled on soil in seed pots in the greenhouse nursery for germination. Wild type (dark green), TLA (light-green), and white/lethal phenotypes emanated from the heterozygous seeds. Viable seedlings were transferred to 4x4 inch peat pots (soil sourse: Sunshine Mix#I, Sun Gro Horticulture, McClelian Park, CA 95652) for primary growth as individual plants (2-to-3 weeks). They were then transferred into soil in 2-gallon pots having a 9-inch diameter. Twenty-five (25) such wild type or TLA pots were placed adjacent to each other in a 5x5 configuration for growth and biomass production measurements, comprising a 1.3 v (2,025 ineh ) plant monoculture. Such high- density monocultures with plants separated from one another by 9-inches were allowed to develop until about half of the plants showed signs of bolting. The biomass of the entire monoculture (all 25 plants) was then harvested and the fresh weight was recorded. Dry biomass weight was estimated by assuming a 12% wet-to-dry conversion factor. Experiments were conducted in pairs, with a wild type and TLA monoculture growing during the same period of time, in the same greenhouse and grown under identical ambient conditions. Results

Pigmentation characteristics qf WTand the TLA. tobacco

[0038] Leaves of the TLA tobacco displayed a distinct light-green coloration compared to the dark green wild type strain (Fig. 1). Analysis of the pigment content showed significantly lower total chlorophyll per leaf area in the TLA leaves compared to the wild type. T he total chlorophyll (Chi) content of wild type leaves was about 24.9 mg cm ^" ', whereas the TLA strain contained only about 12.1 mg cm ^"2 (Table 1). The Chi b content of the TLA leaves was disproportionately lower relative to the lowering in Chi a. Measurements showed that Chi a content in the TL A leaves was lowered to 57% of the wild type, whereas Chi b content was lowered to about 23% of wild type. In consequence, the Chi a i Chi b ratio of the TLA was elevated to a ratio of 8: 1 , relative to the wild type that displayed a Chi a I Chi b ratio of about 3: 1. The latter is typical for the fully developed photosynthetic apparatus in the leaves of green plants (Anderson 1986), whereas a substantially greater Chi a I Chi b ratio of the TLA leaves indicates a truncated light- harvesting Chi antenna size. The carotenoid content also differed between wild type and TLA tobacco. Total carotenoid content in the TLA strain was slightly lower, about 85% of that in the wild type (Table 1 ), resulting in a Car / Chi ratio of 0.16 in the wild type, but 0.29 in the TLA mutant (Table 1). These results are consistent with the notion of a truncated light-harvesting Chi antenna size in the tobacco mutant.

Photochemical apparatus organization in wild type and TLA tobacco [0039] The concentration of the photosystems in isolated tobacco thylakoid membranes was measured using the sensitive absorbance difference spectrophotometric method (Melis and Brown 1980) from the amplitude of the light minus dark absorbance difference signal at 700 nm (P700) for PSI, and 320 nm (QA) for PSII. Isolated thylakoid membranes from the wild-type tobacco showed an overall Chl/QA ratio of 383; 1, whereas this ratio dropped to 129: 1 for the TLA mutant (Table 2). Moreover, the overall Chl/700 ratio was 412: 1 for the wild type, whereas this ratio dropped to 378: 1 for the TLA mutant. These quantifications translated into a photosystem (PSII:PSI) molar ratio of 1.08: 1 for the wild type and 2.93: 1 for the TLA mutant. Enhancement of the PSILPSi molar ratio in the TLA tobacco is a compensation response of the chioroplasts to the disproportionately smaller antenna size of PSII, resulting from the disproportionate lowering of Chi b over that for Chi a in these mutants (Greene et al. 1988). Functional light-harvesting Chi antenna size of the photosystems

[0040] Chlorophyll b pigments are exclusively present in the light-harvesting antenna proteins hut not in the core photosystem or reaction center complexes. The higher Chi a I Chi b ratio in the TLA strain compared to the wild type suggested a lower amount of Chi a-b light harvesting antenna proteins in the TLA chloroplasts. The functional light-harvesting Chi antenna size of PSI and PSII was measured from the kinetics of P700 photo-oxidation and QA photoreduction kinetics, respectively (Melis 1989). Results from these measurements are summarized in Table 2. In the wild type, due to the biphasic kinetics of QA photoreduction, two rates constants of PSII photochemistry were discerned, ¾ a [=8.4 s ^'1 ] and ¾ β [=5.1 s ^"1 ], reflecting the well- known PSII heterogeneity of PSII-a and PSII-β centers in chloroplasts (Melis and Homann 1976). Under the same weak actinic illumination conditions, the rate constant Kj of PSI photochemistry was measured to be equal to 7.2 s ^"1 . These rate constants are a measure of the rate of light absorption by the photosystems, and are directly proportional to the specific photosystem functional light-harvesting ChS (a + b) antenna size. The precise number of the functional PSH chlorophylls was derived (Melis 1989) as 242 Chi for PSII-a and 148 Chi for PSII-β. The average functional PSII chlorophyll antenna size in the wild type was estimated to be 191 molecules. Similarly, the precise number of the functional PSI chlorophylls was estimated to be 207 in the wild type (Table 2). In the TLA mutant, all photosystem light- harvesting Chi (a + b) antenna sizes were smaller with 1 19 Chi for PSII-a and 86 Chi for PSII-β (average PSII Chi antenna size of 93 molecules) and a PSI average functional Chi antenna size of 104 molecules (Table 2). These results clearly show the effect of Chi deficiency on the functional antenna size of the photosystems, and are consistent with measurements conducted earlier (Melis and Thielen 1980; Thielen and van Gorkom 1981).

Protein analysis [0041] isolated (intact) tobacco chloroplasts were solubilized and subjected to SDS-PAGE and Western blot analysis with specific polyclonal antibodies, cross-reacting with the PsaA-PsaB PSI RC. the 1 ⁾ 2 (PsbD) PSII-RC protein, the LHCB 1 light-harvesting protein, or the LHCB2 protein. Loaded on a Chi basis (0.5 to 2 μg), the SDS-PAGE Coomassie stain showed substantially lower levels of the LHC-II proteins in the TLA mutant than in the wild type (Fig. 2). At the same time, there was more Rubisco (RBCL) in the TLA mutant than in the wild type. Western blot analysis showed that the TLA chlorop!asts contain lower levels of the PsaA-PsaB PSI-RC proteins (Fig. 3), relative to the wild type, consistent with the greater PSIi PSI ratio measured spectrophotometrically (Table 2). Levels of the D2 (PsbD) PSII-RC protein were about the same, whereas levels of the LHCB 1 and LHCB2 proteins were measurably lower in the TL A than wild type. These protein measurements are consistent with the smaller (truncated) light- harvesting Chi antenna size of the TLA mutant and corroborate the results of the

spectrophotometric and kinetic analysis.

Light-saturation curves of photosynthesis

[0042] To investigate the functional properties of the photosynthetic apparatus in TLA tobacco relative to that in wild type, the light-saturation curve of photosynthesis was measured under in vivo conditions (Fig. 4). Results in Fig. 4 were normalized to the same light-saturated rate I i _x value to better illustrate differences in half-saturation intensity between the two samples. On a leaf surface area basis, the absolute dark respiration rate of wild type and TLA tobacco was about -2 μηιοΐ C0 ₂ m ^"2 s ^"1 , whereas the absolute light-saturated rate P _mas varied in different leaves between 20 and 35 μηιοΐ CO?, m ^"" s ^"! , depending on leaf development stage and probably other physiological conditions. On occasion, P _niax of the TLA tobacco exceeded that of the wild type, when measured under ambient (400 pptn) C0 ₂ conditions. The reasons for this variation were not investigated further in the experiment.

[0043] Interestingly, in all cases examined, and irrespective of the P _uiax value attained, the photosynthesis-saturation intensity, measured from the intercept of the initially linear increase of the light-saturation curve with the P _aax asymptotic line (dashed lines in Fig. 4) indicated a saturation intensity of 425 μιηοΐ photons m ^"2 s ^"1 for the wild type and 635 μπιοΐ photons m ^'1 s ^"f for the TLA mutant. This difference suggested a 1.5 -fold difference in the light-harvesting antenna size of the photosystems in the two strains. Measured in this way, the apparent difference in the light-harvesting antenna size of the photosystems between the two strains is not as pronounced as that measured from the spectrophotometric and kinetic analysis (Table 2). Part of the discrepancy could be attributed to the specifics of the two measurements. In the spectrophotometric and kinetic analysis, only Chi a and Chi h molecules are sensitized by the weak green actinic illumination, whereas in the light-saturation curv es of photosynthesis red and blue actinic light sensitizes both Chi and Car molecules in the light-harvesting antenna. TLA chloroplasts have a greater Car / Chi ratio than the wild type (Table 1 ), and Car excitation in the blue region of the spectrum may attenuate the difference in light harvesting between the two samples. In addition, increased penetration of light into the leaves of the TLA mutant might result in higher rates of photosynthesis because of improved light distribution within the leaf. Biomass accumulation under high folidage-density conditions

[0044] Three different monoculture sets comparing the growth and biomass accumulation of wild-type and TLA tobacco plants were investigated in canopy density experiments.

Comparative monoculture biomass accumulation measurements were conducted in the greenhouse during the natural tobacco growth season spanning the period from May to October. The layout of the monoculture entailed individual tobacco plants growing in 2-galion pots, having a rim diameter of 9-mches. Pots were placed against one-another in a 5x5 monoculture configuration, comprising 25 plants separated by a 9-inch distance from each other. Relative to the wild type, TLA monocultures developed with a lag of 1 -2 weeks, attributed to the slower development of seedlings in the greenhouse nursery. There were distinct visual differences in mature monoculutre appearance between the wild type and TLA tobacco. Wild-type plants in the monoculture tended to develop longer intemode distances with lower density of leaves in their upper canopy than the TLA plants (Fig. 5). There was an overall higher foliage density in the TLA canopy, compared to the wild type. Importantly, there were differences in the total biomass that was harvested from the two types of monoculutres. Wild-type biomass averaged 736+67 g dw m ^"2 of monoculture surface area, whereas TLA biomass yield averaged 928±47 g dw m ^'2 (Fig. 6A). There was variation among the absolute yields of the three separate pairs (Fig. 6B), depending on time of the year and greenhouse location, but in all cases the TLA

monoculutre produced more biomass than the corresponding wild type. In a one-tailed, paired statistical analysis of the biomass accumulation data, the calculated p-value was p=0.014, i.e., substantially lower than p=0.05, considered to be the threshold of statistically significant results. Thus, there was a notable 25% improvement in biomass accumulation, attributed to the better sunlight distribution through the TL A monoculture foliage, as compared to that of the wild type.

TLA-supported agronomic improvements

[0045] Application of the TLA concept to agriculture affords high density planting, thereby minimizing the surface area that is needed for the generation of a given amount of biomass. For example, traditional tobacco cultivation entails planting in rows with individual plants separated from each other by about 18 inches (Fig. 7). This practice translates into a density of 4.35 plants cultivated per nr. Application of the TLA concept to tobacco, as applied in this work, would permit cultivation in rows with individual plants separated from each other by about 9 inches (Fig. 8). This improvement would increase monoculutre density to 14.9 plants per nr with obvious benefits to yield per hectare.

[0046] Practical considerations often dictate spacing for access to the plants in the field. A schematic of alternative tobacco agronomy with rows of three plants with an 18-inch distance between the groups of three is shown in Fig. 9. A relatively narrow distance of 9-inch still separates plants from each other within the group of three. This configuration with the 18-inch gap between rows of three translates into 12.7 plants per m ² . This planting density also benefits from the Truncated Light-harvesting Antenna (TLA) size of the photosystems, and of the ensuing lower chlorophyll content per leaf area, properties that afford a greater transmittance of sunlight through the foliage, thereby enhancing monoculture productivity. [0047] Alternative planting configurations are also possible with two, four, or five rows of plants grouped together, with groups of rows separated by a space of 15, 18, or 23 inches to enable access to the plants throughout the field.

[0048] These agronomic improvements were exemplified in tobacco. However, the TLA principle can apply to other agricultural plants, as the TLA property is established in barley, soybean, corn, sugar beet, and possibly other crop species (Table 3). For example, favorable corn planting densities for high yield are in the range of 65,000 to 75,000 plants per ha (~7 plants per m ² ). On the basis of the considerations described in this invention, this planting density could double to 140,000 plants per ha (14 plants per m ² ), substantially increasing crop yield. Similarly, currently optimal sugar beet planting density is about ~100,000 plants per ha, (- 10 plants per m ² ), a density that could double to 200,000 plants per ha (20 plants per rn ² ) upon application of the TLA-mvention to this crop.

Discussion of examples

[0049] A challenge for plant scientists on a global scale is to generate enough food and feed for human and farm-animal nutrition, so as to meet the needs of an expanding world population (Godfray et al. 2010; Aiexandratos and Brumsma 2012). Several analyses of the photosynthetic solar-to-biomass energy conversion efficiency identified truncation of the light-harvesting antenna in photosynthesis as a potentially high dividend strategy for increasing crop productivity (Mehs et al. 1999; Nagajima and Ueda 1999; Melis 2009; Ort et al. 2011, 2015; Kirst and Melis 2014). While assembly of large arrays of light-harvesting antenna pigments is a critical evolutionary adaptation and a survival strategy for plants competing to grow in limited light in the wild, this is strongly counterproductive in crop monocultures, where growth takes place under direct and excess sunlight. The large arrays of light harvesting antenna complexes in crop plants cause the upper canopies to over-absorb solar irradiance, far in excess of what is needed to saturate photosynthesis, forcing them to engage in wasteful dissipation mechanisms to deal with the excess energy. This property further causes shading of the inner and lower foliage leaves preventing them from attaining their maximum photosynthetic potential. The net effect of this large antenna configuration in crop plants is to lower the overall solar-to-biomass energy conversion efficiency of photosynthesis from a theoretically possible 8-10% to less than 1 % (Mehs 2009).

[0050] This example showed that the TL A technology could be applied to a crop monoculture, resulting in measurable improvement in biomass yield. The model plant Nicoiiana iahacum (tobacco) was used in this example. However, the example is non-limiting, the TLA principle applies to other crop plants, promising to increase yields, while minimizing the space needed for cultivation. For example, soybeans are known to be subject to lower yields when planted at high densities

(http://www.sugairesearch.com.au/icn^

-get the rate right.pdf). The lower yields observed with excessive sowing densities has been attributed to several factors, some of which could be mitigated upon application of TLA technology to this important crop. Declining yields with plant density are also seen in the case of corn, as documented by the University of Wisconsin, Agronomy Department

(corn.agronomy.wisc.edu/AA/A062.aspx), a problem that could in part be alleviated with the application of TLA technology to this crop. Narrow rows of either soybean or corn offer additional advantages, as they achieve canopy closure more quickly, thus causing substantial shading of weed seedlings in the lower canopy. In addition, a high-density foliage can significantly reduce losses of soil moisture. [0051] All publications, accession numbers, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. References cited in the disclosure by au thor and year

[0052] Abadia J, Click RE, Taylor SE, Terry N and Melis A (1985) Photochemical apparatus organization in the chloroplasts of two Beta vulgaris genotypes. Plant Physiol. 79: 872-878

[0053] Alexandratos N, Brumsma J (2012) World Agriculture: Towards 2030/2050. The 2012 revision. ESA Working Paper No. 12-03 (Food Agric Org, Rome) [0054] Anderson JM (1986) Photoregulation of the composition, function, and structure of thyiakoid membranes. Annu Rev Plant Physiol 37:93-136

[0055] Anion DI (1949) Copper enzymes in isolated chloroplasts: Polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1-15

[0056] Fitzmaurice WP, Nguyen LV, Wernsman EA, Thompson WF, Conkling MA ( 1999) Transposon tagging of the sulfur gene of tobacco using engineered maize ac/ds elements.

Genetics 153: 1919-1928

[0057] Ghirardi ML, McCauley SW and Melis A (1986) Photochemical apparatus organization in the thyiakoid membrane of Hordeum vuigare wild type and chlorophyll b-less chlorina £2 mutant. Biochim. Biophys. Acta 851 : 331 -339 [0058] Ghirardi ML and Melis A (1988) Chlorophyll b-deficiency in soybean mutants. I. Effects on photosystem stoichiometry and chlorophyll antenna size. Biochim. Biophys. Acta 932: 130-137

[0059] Godfray HCJ, Beddington JR, Crute !R. Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S, Thomas SM, Toulmm C (2010) Food security: the challenge of feeding 9 billion people. Science 327:812-818 100601 Greene BA, Staehelin LA, Melis A (1988) Compensatory alterations in the photochemical apparatus of a photoregulatory, chlorophyll b-deficient mutant of maize. Plant Physiol 87: 365-370

[0061] Hansson A, Gamini Kannangara C, Von Wettstein D, Hansson M (1999) Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase. Proc. Natl. Acad. Sci. USA 96: 1744-1749

[0062] Kirst H, Garcia-Cerdan JG, Zurbriggen A, Ruehle T, Melis A (2012) Truncated photosystem chlorophyll antenna size in the green microalga Chlamydomonas reinhardtii upon deletion of the TLA3-CpSRP43 gene. Plant Physiol. 160(4):2251-2260 [0063] Kirst H, Melis A (2014) The ch!oroplast Signal Recognition Particle pathway (CpSRP) as a tool to minimize chlorophyll antenna size and maximize photosvnthetic productivity.

Biotech Advances 32: 66-72

[0064] Kirst H, Formighieri C, Melis A (2014) Maximizing photosynthetic efficiency and culture productivity in cyanobacteria upon minimizing the phycobilisome light-harvesting antenna size. Biochim Biophys Acta - Bioenergetics 1837: 1653-1664

[0065] Homann PH, Schmid GH (1967) Photosynthetic reactions of chloroplasts with unusual structures. Plant Physiol 42: 1619-1632

[0066] Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymoi 148: 350-382 [0067] Melis A, Homann PH (1976) Heterogeneity of the photochemical centers in system II of chloroplasts. Photochem Photobiol 23 : 343-350

[0068] Melis A, Thielen APGM (1980) The relative absorption cross-section of photosystem I and photosystem II in chloroplasts from three types of Nicotiana tabacum. Biochim Biophys Acta 589: 275-286 [0069| Melis A, Brown JS (1980) Stoichiometiy of system I and system II reaction centers and of plastoquinone in different photosynthetic membranes. Proc Natl Acad Sci USA 77: 4712- 4716 [0070] Melis A (1989) Spectroscopic methods in photosynthesis: photosystem stoichiometry and chlorophyll antenna size. Phil Trans R Soc Lond B 323 : 397-409

[0071] Melis A, Neidhardt J, Benemami JR (1999) Dunahella salina (Ch!orophyta) with small chlorophyll antenna sizes exhibit higher photosynthetic productivities and photon use efficiencies than normally pigmented cells. J Appl Phycol 10: 515-525

[0072] Melis A (2009) Solar energy conversion efficiencies in photosynthesis: minimizing the chlorophyll antennae to maximize efficiency. Plant Science 177: 272-280

[0073] M tiller P, Li X-P, Niyogi KK (2001) Non-photochemical quenching: a response to excess light energy. Plant Physiol 125: 1558-1566 [0074] Mussgnug JH, Thomas-Hall S, Rupprecht J, Foo A, Klassen V, McDowall A, Schenk PM, Kruse O, Hankamer B (2007) Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion. Plant Biotech J 5: 802- 814

[0075] Nakajima Y, Ueda R (1997) Improvement of photosynthesis in dense microalgal suspension by reduction of light harvesting pigments. J Appl Phycol 9: 503-510 [0076] Nakajima Y, Ueda R (1999) Improvement of microalgal photosynthetic productivity by reducing the content of light harvesting pigments. J Appl Phycol 11 : 195-201

[0077] Nakajima Y, Itayama T (2003) Analysis of photosynthetic productivity of microalgal mass cultures. J Appl Phycol 15:497-505.

[0078] Okabe K, Schmid GH, Straub J (1977) Genetic characterization and high efficiency photosynthesis of an aurea mutant of tobacco. Plant Physiol 60: 150-156.

[0079] Ort DR, Zhu XG, Melis A (201 1) Optimizing antenna size to maximize photosynthetic efficiency. Plant Physiol 155:79-85

[0080] Ort DR, Merchant SS, Alric J, Barkan A, Blankenship RE, Bock R, Croce R, Hanson MR, , Hibberd JM, Long SP, Moore TA, Moroney J, Niyogi KK, Parr MA, Peralta-Yahya PP, [0081] Prince RC, Redding KE, Spalding MIL van Wijk KJ, Vermaas WF, von Caemmerer S, Weber AP, Yeates TO, Yuan JS, Zhu XG (2015) Redesigning photosynthesis to sustamably meet global food and bioenergy demand. Proc Natl Acad Sci USA 112:8529-8536. [0082] Polle JEW, Benemann JR, Tanaka A and Mel is A (2000) Photosynthetic apparatus organization and function in wild type and a Chi b-!ess mutant of Chlamydomonas reinhardtii. Dependence on carbon source. Planta 211 : 335-344

[0083] Polle JE, anakagin SD, Melis A (2003) tlal , a DNA insertionai transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size. Planta 217:49-59

[0084] Ruban AV (2016) Non-photochemical chlorophyll fluorescence quenching: mechanism and effectiveness in protection against photodamage. Plant Physiol 170: 1903-1916

[0085] Thielen APGM, van Gorkom HL (1981 ) Quantum efficiency and antenna size of photosystem Il-alpha, Il-beta and I in tobacco chloroplasts. Biochim Biophys Acta 635 : 1 1 1 - 120

Table 1 Chlorophyll and carotenoid content per leaf area and pigment ratios of Nicotiana tabacum wild type and TLA plants (n=3-5; means ± SD)

WT TLA % change

Total Chi, mg cm ^"2 of leaf area 249 ± 2.1 12.1 ± 1.3 49 ± 6

Chi a, mg cm ^"2 of leaf area 18.8 ± 1.6 10.7 ± 1.0 57 ± 7

Chi b, mg cm ^'2 of leaf area 6.1 ± 0.6 1,4 ± 0.4 23 ± 11

Chi a 1 Chi h ratio, mol:mol 3.1 ± 0.1 8.1 ± 1.7 -

Car, mg cm ^"2 of leaf area 4.1 ± 0.4 3.5 ± 0.6 85 ± 21

Car / Chi ratio (w:w) 0.16 ± 0.02 0.29 ± 0.05 -

Table 2 Photochemical apparatus characteristics of Nicotiana tabacum wild type (WT) and TLA plants grown under ambient sunlight in high foliage density conditions. Photosystem Chi antenna size and reaction center concentrations were measured spectrophotometrically (Melis 1998). { ^■ ■) 3. means ± SD.)

Table 3 Chlorophyll composition and chlorophyll antenna size of the photosystems in selected Truncated Light-harvesting Antenna crop plants. Photosystem Chi antenna size and reaction center concentrations were measured spectrophotometrically (Melis 1998). (n=3: means ± SD.) Ail corresponding wild type strains showed a Chi a / Chi b ratio of about 3.0±0.5. They possessed an average PSII antenna size in the range of 215-230 chlorophyll (a and b) and a PSI antenna size in the range of 180-210 chlorophyll (a and b) molecules. Genes that are mutated that result in a small or truncated antenna size (TLA) property in green plants and algae are known in the art and include TLAl (US Patent 7,745,696), TLA2 (US Patent Application 20140295448), TLA3 (Kirst et al. 2012), CAO (Chlorophyllide a oxygenase; Ghirardi et al.

1986; Poile et al. 2000), Mg-chelatase subunit l, CHL-I (Hansson et ai. 1999; Fitzmaunce et al. 1999).

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