Patent Application
20190166774
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 (Müller et al. 2001; Ruban 2016).
In organisms of oxygenic photosynthesis, large arrays of light-harvesting pigment-protein complexes are peripheral components of photosystem-1 (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 chloroplasts 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.
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.
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.
A “broad” leaf in the context of this disclosure refers to a leaf having a length-to-width ratio of less than 5:1.
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.
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.
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.
As used herein “bright sunlight” refers to incident photosynthetically active radiation (PAR, or visible light) exceeding 500 μmol photons per m2 per s1.
As used herein, the term “about” means that a value may vary ±15%, ±10% or ±5% and remain within the scope of the invention.
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.
The present disclosure is based in part on the discovery 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.
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.) Illustrative results of antenna size measurements are described in the examples.
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 TLA1 (U.S. Pat. No. 7,745,696), TLA2 (US Patent Application 20140295448), TLA3 (Kirst et al. 2012), CAO (Chlorophyllide a oxygenase; Ghirardi et al. 1986; Polle et al. 2000), Mg-chelatase subunit I, and CHL-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 which is herein incorporated by reference for this purpose.
TLA plants that are planted at high density in accordance with the disclosure, exhibit a chlorophyll 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 chlorophyll b of at least 4±1, 6±1, 8±1, 12±1, or 20±1. In some embodiments, the chlorophyll a to chlorophyll b ratio is greater than 20±1, e.g., 25±1, or greater. In some embodiments, TLA plants do not have measureable amounts of chlorophyll h. 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.
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.
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,
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.
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.
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.
Nicotiana tabacum, cv John William's Broadleaf, 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 Su/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 Su/su mutant will be referred to as Truncated Light-harvesting Antenna tobacco (TLA tobacco).
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.
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 MgCl2, 0.2% polyvinylpyrrolidone 40, 1% sodium ascorbate, 1 mM aminocaproic acid, 1 mM aminobenzamidine and 100 μM 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 MgCl2, 0.2% polyvinylpyrrolidone 40, 1% sodium ascorbate, 1 mM aminocaproic acid, 1 mM aminobenzamidine and 100 μM 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 MgCl2 for spectrophotometric analysis.
The light-minus-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).
Photosynthetic gas exchange measurements were made using a portable open gas analysis device (LI-6400, Li-Cor Inc., Lincoln, Nebr., 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 CO2 concentration in the leaf chamber were 25° C. and 400 μmol mol−1 (ambient CO2 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 μmol photons m−2 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).
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 4×4 inch peat pots (soil course: Sunshine Mix #1, Sun Gro Horticulture, McClellan Park, Calif. 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 5×5 configuration for growth and biomass production measurements, comprising a 1.3 m2 (2.025 inch) 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.
Leaves of the TLA tobacco displayed a distinct light-green coloration compared to the dark green wild type strain (
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 PSII:PSI molar ratio in the TLA tobacco is a compensation response of the chloroplasts to the disproportionately smaller antenna size of PSII, resulting from the disproportionate lowering of Chl b over that for Chl a in these mutants (Greene et al. 1988).
Chlorophyll b pigments are exclusively present in the light-harvesting antenna proteins but not in the core photosystem or reaction center complexes. The higher Chl a/Chl b ratio in the TLA strain compared to the wild type suggested a lower amount of Chl a-b light harvesting antenna proteins in the TLA chloroplasts. The functional light-harvesting Chl 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, KII α[=8.4 s−1] and KII β[=5.1 reflecting the well-known PSII heterogeneity of PSII-α and PSII-β centers in chloroplasts (Melis and Homann 1976). Under the same weak actinic illumination conditions, the rate constant K1 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 Chl (a+b) antenna size. The precise number of the functional PSII chlorophylls was derived (Melis 1989) as 242 Chl for PSII-α and 148 Chl 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 Chl (a+b) antenna sizes were smaller with 119 Chl for PSII-α and 86 Chl for PSII-β (average PSII Chl antenna size of 93 molecules) and a PSI average functional Chl antenna size of 104 molecules (Table 2). These results clearly show the effect of Chl 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).
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 D2 (PsbD) PSII-RC protein, the LHCB1 light-harvesting protein, or the LHCB2 protein. Loaded on a Chl 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 (
Western blot analysis showed that the TLA chloroplasts contain lower levels of the PsaA-PsaB PSI-RC proteins (
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 (
Interestingly, in all cases examined, and irrespective of the Pmax value attained, the photosynthesis-saturation intensity, measured from the intercept of the initially linear increase of the light-saturation curve with the Pmax asymptotic line (dashed lines in
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-gallon pots, having a rim diameter of 9-inches. Pots were placed against one-another in a 5×5 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 monoculture appearance between the wild type and tobacco. Wild-type plants in the monoculture tended to develop longer internode distances with lower density of leaves in their upper canopy than the TLA plants (
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 (
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
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.
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 m2). On the basis of the considerations described in this invention, this planting density could double to 140,000 plants per ha (14 plants per m2), substantially increasing crop yield. Similarly, currently optimal sugar beet planting density is about ˜100,000 plants per ha, (˜10 plants per m2), a density that could double to 200,000 plants per ha (20 plants per m2) upon application of the TLA-invention to this crop.
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; Alexandratos and Bruinsma 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 (Melis 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% (Melis 2009).
This example showed that the TLA technology could be applied to a crop monoculture, resulting in measurable improvement in biomass yield. The model plant Nicotiana tabacum (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.sugarresearch.com.au/icms_docs/157723_Bulletin_Vol_24_Sowing_soybean_seeds-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.
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.
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Nicotiana
tabacum wild type and TLA plants (n = 3-5; means ± SD)
Hordeum vulgare
Glycine max
Glycine max
Zea mays
Beta vulgaris
This application claims benefit of priority to U.S. Provisional Application No. 62/356,185, filed Jun. 29, 2016, which is herein incorporated by reference for all purposes.
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.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/039685 | 6/28/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62356185 | Jun 2016 | US |
