METHODS FOR IMPROVING PHOTOSYNTHETIC ORGANISMS
20220290174 · 2022-09-15
Inventors
- Nicholas John Roberts (Feilding, NZ)
- Somrutai Winichayakul (Palmerston North, NZ)
- ZACHARIA D'ARCY BEECHEY-GRADWELL (Palmerston North, NZ)
- LUKE JAMES COONEY (Waitarere Beach, NZ)
Cpc classification
C12N9/1029
CHEMISTRY; METALLURGY
C12Y203/0102
CHEMISTRY; METALLURGY
International classification
Abstract
The invention provides a method for reducing water soluble carbohydrate (WSC) in a photosynthetic cells and plants, the method comprising the step of genetically modifying the photosynthetic cells and plants to express a modified oleosin including at least one artificially introduced cysteine to reduce WSC. The applicants have shown that in such cells and plants, there is a strong correlation between between reduced WSC and elevated photosynthesis and low. In addition WSC is significantly simpler to measure that than the other typically measured characteristics when selecting cells or plants with the most favourable characteristics.
Claims
1-18. (canceled)
19: A method for reducing water-soluble carbohydrate (WSC) in a photosynthetic cell or plant, the method comprising the step of genetically modifying the photosynthetic cell or plant to express a modified oleosin including at least one artificially introduced cysteine to reduce WSC.
20: The method of claim 19 in which reducing WSC leads to increased CO.sub.2 assimilation in the cell.
21: The method of claim 19 in which the photosynthetic cell or plant is also modified to express at least one triacylglycerol (TAG) synthesising enzyme.
22: The method of claim 21 in which expression of the modified oleosin including at least one artificially introduced cysteine and the TAG synthesising enzyme leads to the reducing WSC.
23: The method of claim 19 in which the method includes the step of measuring WSC in the photosynthetic cell or plant.
24: The method of claim 23 in which measurement of a reduction in WSC is indicative of increased CO.sub.2 assimilation in the photosynthetic cell or plant.
25: A method for producing a photosynthetic cell or plant with increased CO.sub.2 assimilation, the method comprising modifying the photosynthetic cell or plant to reduce WSC.
26: The method of claim 25 in which the method comprises the step of genetically modifying the photosynthetic cell or plant to express a modified oleosin including at least one artificially introduced cysteine to reduce WSC.
27: The method of claim 25 in which reducing WSC leads to increased CO.sub.2 assimilation in the cell or plant.
28: The method of claim 25 in which the photosynthetic cell or plant is also modified to express at least one triacylglycerol (TAG) synthesising enzyme.
29: The method of claim 28 in which expression of the modified oleosin including at least one artificially introduced cysteine and the TAG synthesising enzyme leads to the reducing WSC.
30: The method of claim 25 in which the method includes the step of measuring WSC in the photosynthetic cell or plant.
31: The method of claim 30 in which measurement of a reduction in water soluble carbon is indicative of increased CO.sub.2 assimilation in the photosynthetic cell or plant.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
[0490] This invention will now be illustrated with reference to the following non-limiting examples.
Example 1: Construct Designs
[0491] The Garden Nasturtium (Tropaeolum majus) DGAT1 peptide sequence (GenBank AAM03340) with the single point mutation of serine at 197 amino acid sequence to alanine as described by Xu et al. (2008), linked with V5 epitope tag (GKPIPNPLLGLDST) at the C-terminal (DGAT1-V5), and the 15-kD sesame L-oleosin (accession no. AAD42942) with three engineered cysteine residues on each N- and C-terminal amphipathic arms (Cys-OLE; Winichayakul et al., 2013) were custom synthesized by GeneAR™ for expression in L. perenne (sequences 1-4) Both DGAT1-V5 and Cys-OLE coding sequences were optimized for expression in monocot grass and placed into the designed Gene Gun compatible construct. The resulting construct, labelled as LpDlo3-3, contained the DGAT1-V5 gene regulated by the rice ribulose-1, 5-bisphosphate carboxylase small subunit promoter (RuBisCO-Sp, GenBank AY583764) back-to-back with the Cys-OLE gene regulated by the rice chlorophyll a/b binding protein promoter (CABp; GenBank APO14965—region: 10845004-10845835).
[0492] The same peptide sequences were optimized for expression in Glycine max and were placed under a variety of promoter combinations including but not limited to: [0493] Phaseolus vulgaris ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS2) promoter, Accession number AF028707 [0494] Pisum sativum small subunit ribulose bisphosphate carboxylase (rbcS-3A) promoter, Accession numbers M21356; M27973 [0495] Pisum sativum CAB promoter, Accession number M64619 [0496] Glycine max Subunit-1 ubiquitin promoter, Accession number D16248 [0497] Arabidopsis thaliana polyubiquitin 10 promoter, Accession number L05399 [0498] Cauliflower mosaic virus 35s promoter, Accession numbers V00141; J02048
[0499] These were subcloned into binary vectors for Agrobacterium tumefaciens assisted transformation.
[0500] The same peptide sequences were optimized for expression in Cannabis sativa (sequences 9-12) and were placed under a variety of promoter combinations including but not limited to: [0501] Phaseolus vulgaris ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS2) promoter, Accession number AF028707 [0502] Pisum sativum small subunit ribulose bisphosphate carboxylase (rbcS-3A) promoter, Accession numbers M21356; M27973 [0503] Pisum sativum CAB promoter, Accession number M64619 [0504] Glycine max Subunit-1 ubiquitin promoter, Accession number D16248 [0505] Arabidopsis thaliana polyubiquitin 10 promoter, Accession number L05399 [0506] Cauliflower mosaic virus 35s promoter, Accession numbers V00141; J02048
[0507] These were subcloned into binary vectors for Agrobacterium tumefaciens assisted transformation.
Example 2: Lolium Perenne Transformation, Selection and Growth Conditions
[0508] Plants over-expressing the LpD1o3-3 construct were generated by microprojectile bombardment using a method adapted from Altpeter et al. (2000). Briefly, calli for transformation were induced from immature inflorescences harvested from a single transformation-competent genotype of cvr. Impact by culture on a Murashige Skoog basal medium supplemented with 2,4-dichlorophenoxyacetic acid. Plasmids for transformation were prepared using the Invitrogen Pure Link Hi Pure Plasmid Maxiprep Kit. The plasmid pAcH1, which contains an expression cassette comprising a chimeric hygromycin phosphotransferase (HPH) gene (Bilang et al., 1991) expressed from the rice actin promoter, was used for selection and mixed in a 1:1 molar ratio with LpDlo3-3. Plasmid DNA's were coated onto M17 tungsten particles using the method of Sanford et al. (1993) and co-transformed into target tissues using a DuPont PDS-1000/He Biolistic Particle Delivery System. Multiple independent heterozygous ryegrass transformants were generated, including transgenic plants transformed with pAcH1 as a vector control (VC). Transformed plants were transferred to a contained greenhouse environment (22/17° C. diurnal cycle and 12 hour photoperiod under supplementary LED lighting providing 1000 μM/sec/m.sup.2 PAR) for further analysis.
[0509] PCR analysis using primer pair's specific to the HPH and DGAT genes was performed to confirm stable integration of the transgenes into the genome of plants recovered from transformation experiments, and Southern blot hybridization was used to estimate the number of transgene copies per line. Leaves from these plants were initially analysed for total fatty acid content and recombinant DGAT1-V5 and Cys-OLE proteins.
Example 3: Glycine max Transformation
[0510] Glyine max can be transformed and selected essentially as described in Zeng, P. et al 2004, Plant Cell Reports, 22:478-482, and Paz N, M. et al., 2004, Euphytica, 136:167-179.
Example 4: Cannabis sativa Transformation
[0511] Cannabis sativa can be transformed and selected essentially as described in Feeney and Punja (2003).
Example 5: Reduction of Water Soluble Carbohydrate in Lolium Perenne
[0512] Plant Material and Experimental Layout
[0513] Plant material was transformed with cysteine-oleosin and DGAT1 under the control of the Oryza sativa CAB and RuBisCo promoters respectively as described in Roberts et al 2010; Roberts et al 2011; Beechy-Gradwell et al (2018);
[0514] The untransformed wild type (WT) control genotype ‘IMPACT 566’ used throughout this work was derived from the perennial ryegrass (Lolium perenne) cultivar ‘Grasslands Impact’ which was selected for its amenability to transformation and regeneration. Replicate plants in all experiments consisted of vegetative clonal ramets of WT or independent WT transformation events. Therefore, the transgenic genotypes differed genetically from the WT only in the presence of the cys-OLE/DGAT construct, while the transgenic genotypes differed genetically from one another only in the position and copy number of the cys-OLE/DGAT construct in the genome.
[0515] Experiments were conducted either in the glasshouse or in controlled environment growth chambers. Total leaf fatty acid (FA) and recombinant protein content were initially determined for WT, a vector control (VC) and 12 independent transgenic cys-OLE/DGAT genotypes, grown in the glasshouse under regular mechanical defoliation. WT, VC, and the transgenic genotypes ‘3501’ and ‘3807’ were also analysed for leaf TAG and root FA content, with samples taken approximately three weeks after defoliation (n=6-8). WT and the transgenic genotypes ‘3501’ and ‘6205’ were used in a preliminary growth trial at ambient and elevated [CO.sub.2] across two growth chambers. Then, in the main experiment described in this study, the same growth chambers (with identical settings, described below) were used for a detailed physiological comparison of WT and the high-expressing genotype ‘6205’ (HL), in a formal regrowth trial at ambient and elevated atmospheric [CO.sub.2] under different levels of NO.sub.3 and NH.sub.4.sup.+ supply.
[0516] Gas-Exchange Analysis
[0517] Rates of CO.sub.2 assimilation were measured from plants growing 3-WAC using an infrared gas analyzer (Li6400; Li-Cor Inc.) fitted with a standard 2×3-cm.sup.2 leaf chamber, a leaf thermocouple, and a blue-red light-emitting diode light source at 1500 μmol-m.sup.−2.Math.s.sup.−1 photosynthetically active radiation. Intrinsic water-use efficiency (iWUE) was estimated from the ratio of photosynthesis/stomata conductance (Osmond et al., 1980). Block temperature was held at 20° C., stomata ratio was set at 1, and the vapour pressure deficit was between 0.8 and 1.3 kPa.
[0518] SDS-PAGE Analysis of DGAT1 and Cys-OLE
[0519] Protein samples were prepared by collecting fresh 4 ryegrass leaf blades (approximately 2 cm long) or 10 mg DW finely ground leaf in a 2-mL screw cap micro tube containing 150 s L of sterile H.sub.2O, 200 μL of 2× protein loading buffer (1:2 diluted 4× lithium dodecyl sulfate (LDS) sample buffer [Life Technologies], 8 M urea, 5% [v/v]-mercaptoethanol, and 0.2 M dithiothreitol). The mixtures were homogenised using the Omni Bead Ruptor 24 model setting at speed level 5 until totally homogenised. The samples were heated at 70° C. for 10 min, centrifuged at 20,000 g for 30 sec and collected for the soluble protein suspension. Equal quantities of proteins were determined and separated by SDS-PAGE (Mini-PROTEAN® TGX stain-free™ precast gels; Bio-Rad) and blotted onto Bio-Rad polyvinylidene difluoride (PVDF) membrane for the DGAT1-V5 immunoblotting. Equivalent amounts of proteins were separated on gradient 4-12% Bis-Tris gel (NUPAGE; Life Technologies) and blotted onto nitrocellulose membrane for the Cys-OLE immunoblotting. Immunoblotting was performed as described previously in Winichayakul et al. (2013). Chemiluminescent activity was developed using Advansta WesternBright ECL spray and visualised by Bio-Rad ChemiDoc™ imaging system. To prepare protein samples for the LD fraction analysis, an equal volume of LD was mixed to the 2× protein loading buffer and heated at 70° C. for 10 min.
[0520] Ribulose 1, S-Bisphosphate Carboxylase Large Subunit (RuBisCO-L) Extraction and Analysis
[0521] Approximately 10 mg of freeze-dried finely ground leaf material was accurately weighed and extracted in 0.5 mL of phosphate buffer saline (PBS) pH 7.4. The extract was centrifuged at 10,000 g for 5 min at 22° C. and the soluble fraction was determined for protein content using Qubit Protein Assay Kits/Qubit 2.0 Fluorometer (ThermoFisher). Protein samples were prepared by mixing similar volumes of extract with 2× sample loading buffer (1:2 diluted 4×LDS sample buffer [Life Technologies], 5% [v/v] β-mercaptoethanol, and 0.2 M dithiothreitol) and heated at 70° C. for 10 min. Equal quantities of proteins were separated by SDS-PAGE. The amount of RuBisCo-L protein was visualised directly from the gels and confirmed by immunoblotting using anti-RuBisCo-L (1:5000 dilution; Agrisera AS03 037).
[0522] Chlorophyll Extraction
[0523] Approximately 10-15 mg of freeze-dried finely ground leaf material was accurately weighed and extracted with 2 mL of ethanol (95% v/v) in sealed glass tubes kept at 22° C. in the dark. Extraction was regularly mixed thoroughly for 3 h or until the leaf materials turned white. Chlorophyll a and b content in the extracts was measured spectrophotometrically for the absorbance at 648 and 664 nm and calculated as described by Lichtenthaler and Buschmann (2001) using the following equations chlorophyll a=(13.36 A.sub.664−5.19 A.sub.648), chlorophyll b=(27.43 A.sub.648−8.12 A.sub.664).
[0524] Stomatal Aperture Bioassays
[0525] Plants were watered well at beginning of the day light. After 3 h, leaves were harvested and immediately fixed in cold 4% (w/v) paraformaldehyde in 1×PBS with 10 min vacuum treatment and incubated in the fixing agent at 4° C. for at least overnight. Fixed leaves were washed twice with 1×PBS and stained with 20 μL of SlowFade®Gold Anti-Fade Mountant with 4′, 6-diamidino-2-phenylindole (DAPI; Life Technologies S36938) for fluorescence imaging and visualized using confocal microscopy with the excitation/emission max (Ex/Em) set at 359/461 nm for DAPI fluorescence. Measurements of stomatal aperture were carried out on at least 60 stomatal apertures (5 images taken from one leaf abaxial epidermis, 12 biological repeats) as described previously by Merlot et al. (2001) using the Olympus Fluoview FV10-ASW 3.1 Software.
[0526] Establishment Phase for Ryegrass Clones
[0527] In the main experiment described in this study, WT and HL clones were made from established plants by splitting them into ramets consisting of 3-4 tillers and cutting to 10 cm of combined root and shoot length. The ramets were placed in individual cylindrical plastic pots containing washed sand (1.6 L). Approximately 200 clones of each genotype were generated, of which 140 were selected (based on a uniform leaf DW) for the experiment. Following propagation, the ramets were given 23 days to establish a root system in a Conviron BDW 120 plant growth room at ambient CO.sub.2 (Thermo-Fisher, Auckland, NZ). Metal halide bulbs (400 W Venture Ltd., Mount Maunganui, NZ) and soft tone, white incandescent bulbs (100 W, Philips, Auckland, NZ) provided ˜500±50 μmol photosynthetically active radiation (PAR) m.sup.−2 s.sup.−1 as white light, under a 12 hour photoperiod, with light levels ramping at dawn/dusk for 60 minutes. The day/night temperature and humidity were 20/15° C. and 60/68% RH, respectively. A top-down airflow pattern, with a controlled flow of outdoor air, maintained ambient CO.sub.2 conditions (˜400 ppm. CO.sub.2). During the establishment period, pots were flushed with 100 ml of basal nutrient media described in (Andrews et al., 1989) containing 2 mM KNO.sub.3, three times per week. We found that supplying sub-optimal NO.sub.3.sup.− limited establishment phase growth enough to avoid ‘pot-limited’ conditions (Poorter et al., 2012) early in the subsequent regrowth phase, while also avoiding severe ‘transplanting shock’. At the end of the establishment phase, plants were defoliated and the DW of leaf clippings from 5 cm above the pot media surface were determined after oven-drying at 80° C. overnight. These averaged 0.118±0.036 g for the WT genotype and 0.113 g 0.020 for the HL genotype (Mean±SD, n=140). A subset of defoliated plants (n=5) were destructively sampled at this time, oven dried and weighed for ‘sheath’ (0-5 cm from the pot surface) and root DW, enabling the later calculation of relative growth rate (RGR).
[0528] Regrowth Phase for Ryegrass Clones
[0529] Following defoliation of the established plants, half of the material was moved into a second high CO.sub.2 Conviron BDW 120 plant growth room, with identical settings to those described above, except that the CO.sub.2 level was maintained at 760 ppm with G214 food grade CO.sub.2 (BOC, Auckland, NZ). The two cabinets were previously tested for uniformity (Andrews et al., 2018). The CO.sub.2 levels in both growth rooms were measured continuously using PP Systems WMA-4 Gas Analysers (John Morris Scientific, Auckland, NZ). Pots were randomly allocated to different N treatments (n=5) then flushed with 150 ml of basal nutrient media containing either 1, 2, 3, 4, 5, 7.5 or 10 mM of N as either NO.sub.3.sup.− or NH.sub.4.sup.+ every two days for the regrowth phase. The pH of the nutrient media solutions was in the range of 5.4-5.6. Potassium concentrations were balanced in all cases with the highest potassium treatment (10 mM) with K.sub.2SO.sub.4 but sulphate was not balanced.
[0530] Harvest of Ryegrass Clones
[0531] Plants were destructively harvested after 29-30 days regrowth and divided into ‘leaf’ (5 cm above the pot surface), ‘sheath’ (0-5 cm from the pot surface) and roots. Leaf subsamples were taken from plants treated with 3, 5, 7.5 and 10 mM N and snap frozen in liquid N, then stored at −80° C. The remaining material was oven dried at 65° C. for 4-6 days then weighed. Roots were cleaned and oven dried at 65° C. for 4-6 days before weighing. The fraction of biomass allocated to leaves (LMF) was calculated by dividing leaf DW by total plant DW. RGR was calculated from differences in paired plant DW, determined after defoliation (
[0532] Lipid and Carbohydrate Analyses
[0533] The frozen leaf material was later freeze-dried and ground to a powder and analysed for fatty acids (FA) and water-soluble carbohydrates (WSC). FA were extracted from 10-15 mg of ground sample and methylated in hot methanolic HCl, then quantified against a C15:0 internal standard by GC-MS (Browse et al., 1986). Total FA concentration was calculated as the sum of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) concentration in the leaves. The protocol for TAG extraction was as described in Winichayakul et al. (2013) without modification. For WSC, a 25 mg sample of ground material was mixed twice with 1 ml 80% ethanol and incubated at 65° C. for 30 min. After each extraction the homogenate was centrifuged at 13,000 rpm for 10 min and the supernatant containing low molecular weight (LMW) WSC was removed. High molecular weight (HMW) WSC were extracted by twice mixing the remaining insoluble residue with 1 ml of water, then incubating, centrifuging and removing the supernatant. Aliquots of these extracts were diluted then reacted with 1.25% anthrone in a mixture of H.sub.2SO.sub.4 and ethanol (3:5 V: V). The blue-green colour produced from the reaction was read at 620 nm. LMW and HMW WSC were calibrated against a series of sucrose and inulin standards, respectively.
[0534] Statistical Analysis
[0535] A complete randomised study design was used to investigate the relationship between genotype, CO.sub.2, N form and N concentration on various growth parameters, leaf FA and leaf WSC. Two or three-way ANOVA were used to compare the gas exchange, leaf structure and fluorescence data (collected at a single N concentration). For growth parameters, N concentration was treated as a continuous variable. For leaf FA and leaf WSC, N concentration was treated as a factor. A forward stepwise procedure was used for selecting variables. Variables and interaction terms with a p-value of <0.05 were retained in the final model. Due to residual heteroskedasticity, total plant DW data was log-transformed before modelling. Treatment means were compared and post hoc multiple comparison p-values were adjusted using the Benjamini-Hochberg (BH) method. Raw means and SE values are presented in the tables and figures, while p-values in the tables and text were obtained from the final statistical models. All statistical analyses were performed in R (Version 3.4.3, R foundation).
[0536] Leaf Fatty Acid and Protein Expression
[0537] In an initial screen of the transgenic material, there was no significant difference between WT and vector control leaf FA, while the cys-OLE/DGAT lines contained 23-100% more leaf FA (4.3-7.0% DW) than the WT (3.5% DW) (
TABLE-US-00004 TABLE 3 Total leaf FA Leaf TAG Total root FA Genotype (% DW) (% DW) (% DW) WT 3.49 ± 0.07 A 0.18 ± 0.03 A 0.66 ± 0.01 A VC 3.50 ± 0.13 A 0.23 ± 0.02 A 0.73 ± 0.01 B 3501 5.56 ± 0.06 B 2.20 ± 0.06 B 0.99 ± 0.01 C 3807 6.69 ± 0.07 C 2.47 ± 0.06 C 0.97 ± 0.01 C p value ** *** ***
[0538] Leaf C Storage
[0539] In the main experiment described in this study, the high expressing cys-OLE/DGAT genotype ‘6205’ (HL) had a substantially higher (67-96%) leaf FA concentration than the WT under two CO.sub.2 levels and 1-10 mM N supply (Genotype effect p<0.001) (
[0540] Growth
[0541] After 28-29 days regrowth under the different [CO.sub.2] and N treatments, total plant dry biomass (DW) increased by 7 to 23-fold. For both WT and HL, DW was greater under e[CO.sub.2] than a[CO.sub.2] and increased with N supply up until 4-10 mM (N concentration effect p<0.001), then stabilized or decreased thereafter (Quadratic N concentration effect p<0.001). The DW of (defoliated) plants at the end of the establishment phase was 18% greater for WT than for HL plants (p<0.01 student's t-test,
[0542] Morphology
[0543] The fraction of biomass allocated to leaves (LMF) increased with increasing N supply up until 5-7.5 mM, then stabilized thereafter (Quadratic N concentration effect p<0.001) (
[0544] Gas Exchange
[0545] HL displayed a higher A.sub.sat than WT at a[CO.sub.2] (Genotype effect p<0.001). Similar results were also obtained when A was measured at growth room irradiance (˜500 μmol m.sup.−2 s.sup.−1) (data not shown). For both WT and HL, A.sub.sat increased and stomatal conductance (g.sub.x) decreased at e[CO.sub.2] (CO.sub.2 effect, p<0.001), however the increase in A.sub.sat at e[CO.sub.2] compared to a[CO.sub.2] was greater for HL than for WT (Genotype×CO.sub.2 interaction, p<0.01) (Table 1). Relative to NO.sub.3.sup.− supply, NH.sub.4.sup.+ increased HL A.sub.sat (by 9%) and decreased WT A.sub.sat (by 29%) (Genotype×N form interaction p<0.001). Within [CO.sub.2]treatments, light saturated g.sub.x and A.sub.area correlated well (R.sup.2=0.79 under a[CO.sub.2] and 0.74 e[CO.sub.2], respectively) (Figure S3) and the ratio of leaf intracellular CO.sub.2 to ambient CO.sub.2 concentration (C/CG) did not differ between WT and HL, regardless of [CO.sub.2] level or N form (Table 4 below).
[0546] A/Ci analysis, determined for plants supplied with NO.sub.3.sup.− only, showed that HL had a substantially higher A.sub.sat at low (rubisco-limited) C.sub.i (68-83% at 69-72 ppm C.sub.i) compared to WT. This difference became smaller at high (RuBP regeneration-limited) C.sub.i (10-12% at 1023-1099 ppm C.sub.i) (
TABLE-US-00005 TABLE 4 SLA A.sub.sat g.sub.s A.sub.mass CO.sub.2 N form Genotype (cm.sup.2.g.DW.sup.−1) (μmol CO.sub.2.m.sup.−2.s.sup.−1) (CO.sub.2.m.sup.−2.s.sup.−1) (μmol CO.sub.2.gDW.sup.−1.s.sup.−1) C.sub.i/C.sub.a Ambient NO.sub.3.sup.− WT 211 ± 9 C 19.1 ± 0.9 D 0.32 ± 0.03 B 0.41 ± 0.03 D 0.71 ± 0.01 AB HL 290 ± 8 A 23.3 ± 0.2 C 0.40 ± 0.01 A 0.68 ± 0.02 B 0.71 ± 0.01 AB NH.sub.4.sup.+ WT 155 ± 3 DE 15.6 ± 0.6 E 0.22 ± 0.01 D 0.24 ± 0.01 E 0.67 ± 0.01 BC HL 244 ± 9 B 24.8 ± 1.2 C 0.36 ± 0.02 AB 0.60 ± 0.02 C 0.66 ± 0.01 C Elevated NO.sub.3.sup.− WT 174 ± 11 D 25.3 ± 0.9 C 0.23 ± 0.02 D 0.44 ± 0.04 D 0.72 ± 0.02 A HL 277 ± 9 A 30.8 ± 0.6 B 0.30 ± 0.02 BC 0.85 ± 0.01 A 0.73 ± 0.02 A NH.sub.4.sup.+ WT 150 ± 7 E 18.8 ± 0.9 D 0.13 ± 0.01 E 0.29 ± 0.03 E 0.67 ± 0.02 BC HL 231 ± 3 BC 34.6 ± 1.1 A 0.25 ± 0.02 CD 0.80 ± 0.03 A 0.66 ± 0.02 C G *** *** *** *** — N *** — *** *** *** CO.sub.2 ** *** *** *** — ANOVA G × N — *** — ** — G × CO.sub.2 — ** — *** — N × CO.sub.2 — — — — — Data points represent the raw averages of plants regrown under NO.sub.3.sup.− or NH.sub.4.sup.+ (n = 5) ± S.E. G = genotype effect, N = N form effect, CO.sub.2 = CO.sub.2 effect significant in a three-way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Different letters indicate statistically significant differences in predicted means obtained from three-way ANOVA, with p values adjusted according to BH method.
TABLE-US-00006 TABLE 5 % inhibition in CO.sub.2 N form Genotype Φ PSII Vo/Vc A.sub.amb at 20% O.sub.2 Ambient NO.sub.3.sup.− WT 0.42 ± 0.02 0.35 ± 0.02 34 ± 1 HL 0.54 ± 0.01 0.29 ± 0.01 29 ± 1 NH.sub.4.sup.+ WT 0.40 ± 0.02 0.41 ± 0.02 37 ± 2 HL 0.54 ± 0.01 0.31 ± 0.03 30 ± 2 Elevated NO.sub.3.sup.− WT 0.40 ± 0.01 0.18 ± 0.01 15 ± 2 HL 0.55 ± 0.01 0.13 ± 0.01 8 ± 2 NH.sub.4.sup.+ WT ND ND ND HL ND ND ND G *** *** *** N — * — ANOVA CO.sub.2 — *** *** G × N — — — G × CO.sub.2 — — Data points represent the raw averages of plants regrown under NO.sub.3.sup.− or NH.sub.4.sup.+ (n = 5) ± S.E. A.sub.amb = photosynthesis at growth room irradiance. G = genotype effect, N = N form effect, CO.sub.2 = CO.sub.2 effect significant in a three-way ANOVA. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. ND = Not determined.
[0547] The applicant has demonstrated that Cys-OLE/DGAT expression can be used to reduce water soluble carbohydrate and thereby confers a growth advantage with increased SLA and A.sub.area that improved yield. In addition the photosynthesis was more responsive to e[CO.sub.2] at high N.
[0548] Without wishing to be bound by theory, the applicant postulate that production of a lipid carbon microsink leads to reduction in water soluble carbohydrate.
[0549] By modifying two genes involved in lipid biosynthesis and storage (cys-OLE/DGAT) the accumulation of stable lipid droplets in perennial ryegrass (Lolium perenne) leaves was achieved. Growth, biomass allocation, leaf structure, gas exchange parameters, fatty acids and water-soluble carbohydrates were quantified for a high-expressing cys-OLE/DGAT ryegrass transformant (HL) and a wild type (WT) control grown in controlled conditions under 1-10 mM N supply at ambient and elevated atmospheric CO.sub.2. A dramatic shift in leaf C storage occurred in HL leaves, away from readily mobilizable carbohydrates and towards stable lipid droplets. Our results show that under ideal growing conditions, the manipulation of lipid biosynthesis and storage, and the resulting reduction in water soluble carbohydrate, can drive greater C assimilation. The applicant considers that lowering of WSC has a direct influence on the activity of photosynthetic machinery. The applicant's data predicate the present invention thus providing a more robust way of determining the influence on CO.sub.2 assimilation as compared to measuring either accumulation of the cysteine oleosin protein or the accumulation of additional lipids within the leaf both of which have indirect influences on photosynthesis.
Example 6: Elevated Fatty Acids Over a Range of Levels in Leaves Comes at the Expense of Leaf Sugar and Coincides with Increase Carbon Assimilation and Growth
[0550] Plant Material
[0551] Lolium perenne, transformed with DGAT+Cysteine Oleosin (CO) using both agro-bacterium and gene-gun mediated transformation were used in these comparisons.
[0552] Relative Growth Rates
[0553] Five Lolium perenne lines containing DGAT+CO (labelled DGAT+CO1, DGAT+CO2, DGAT+CO3, DGAT+CO4, DGAT+CO5) were selected from three genetic backgrounds (Table 6). Three Lolium perenne containing DGAT+CO lines contained a single loci with the Lolium perenne containing transgenes and two containing multiple-loci (see Table 6). To eliminate growth form or tiller age differences between ramets, all Lolium perenne lines, and respective WT controls, underwent three rounds of propagation over 4 months. During each round, 5 ramets of five tillers each were potted and grown for 4 weeks. All plants were grown in a controlled temperature room with 600 μmol photons m.sup.−2 s.sup.−1 red/blue light provided by **, 20° C./15° C. day/night temperature and 12 h day length. After the final round of propagation 40×5-tiller ramets were produced for each line, 10 of which were immediately harvested to confirm comparable starting weights (Table 7 below). The remaining 30 were transplanted into 1.3 L sand and flushed thrice weekly with 100 ml 2 mM KNO.sub.3 in a complete nutrient solution. Three weeks after propagation, shoot material was harvested 5 cm above sand, and used to rank plants from smallest to largest. The five smallest and five largest plants per line were discarded and 10 of the remaining 20 plants per line were randomly selected and harvested (post-establishment harvest). The remaining ten plants per line were grown for another three weeks, with 8 mM KNO.sub.3 applied as described above, and harvested (final harvest). Relative growth weight was calculated as per Poorter (1989a); RGR=(ln W.sub.2−In W.sub.1)/(t.sub.2−t.sub.1) where W.sub.1=post-establishment dry weight, W.sub.2=final harvest dry weight, t.sub.1=day 22 and t.sub.2=day 43.
TABLE-US-00007 TABLE 7 Propagation DW (mg) Post-Establishment DW (g) WT1 92.8 (±5.1) 0.82 (±0.04) DGAT + CO1 101.7 (±5) 0.7 * (±0.02) DGAT + CO2 101.8 (±4.7) 0.82 (±0.04) WT2 107 (±7.7) 0.57 (±0.03) DGAT + CO3 114.3 (±5.5) 0.73 ** (±0.03) DGAT + CO4 105.6 (±5) 0.72 * (±0.05) WT3 114.9 (±10.6) 0.55 (±0.02) DGAT + CO5 94.1 (±5.1) 0.65 (±0.05)
[0554] Photosynthetic Gas Exchange
[0555] One week prior to the final harvest, three tillers were selected per plant, and on the youngest fully expanded leaves, net photosynthesis per unit leaf area (A), net photosynthesis per unit leaf mass (A.sub.mass) stomatal conductance (gsw) and transpiration (E) was analysed using a Licor 6800 infrared gas exchange system (Licor Biosciences Ltd, Nebraska, USA). Leaves were acclimated under growing conditions; 600 μmol photons m.sup.−2 s.sup.−1 red/blue light, at 400 ppm CO.sub.2, 70% relative humidity and 20° C. for 15 minutes prior to data-logging. The three leaves were then abscised, photographed, dried and weighed. Leaf area was calculated using GIMP 2.8.22 (GNU Image Manipulation Program, http://www.gimp.org) and specific leaf area was calculated as SLA=LA/DW.
[0556] Fatty Acid Analysis
[0557] Leaf material was collected on the final day of our growth trial, freeze dried and ground via bead mill. 10 mg was sub-sampled per plant and from this, fatty acids (FA) were extracted in hot methanolic HCl (modified after Browse et al., 1986). FA were quantified by GC-MS (QP 2010 SE, Shimadzu Corp., Kyoto, Japan) against an internal standard of 10 mg C15:0 and total FA was calculated as the sum of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3).
[0558] Sugar Quantification
[0559] Total water soluble carbohydrates (WSC) were analysed using the anthrone method (Hedge Hofreiter, 1962). Using 25 mg freeze-dried, ground leaf material, low molecular weight carbohydrates (LMW) were twice extracted in 1 ml, 4:1 EtOH: H.sub.2O at 65° C. for 30 mins, centrifuged and supernatant collected and combined at each extraction. Using the sample pellet, high molecular weight carbohydrates (HMW) were twice extracted in 1 ml H.sub.2O at 65° C. for 30 mins, centrifuged and supernatant collected and combined at each extraction. The soluble carbohydrate extracts were mixed with anthrone reagent (Sigma-Aldrich, St Louis, Mo., USA) for 25 mins at 65° C., A.sub.620 determined using a Versamax tunable plate reader (Molecular Devices Corporation, Sunnyvale, Calif., USA) and compared to LMW and HMW standards, prepared using sucrose and inulin respectively.
[0560] Chlorophyll Quantification
[0561] Using 15 mg freeze-dried, ground leaf material, chlorophylls were extracted in ethanol: H20 (19:1), clarified by centrifugation and absorbance peaks measured using a Versamax tunable plate reader (Molecular Devices Corporation, Sunnyvale, Calif., USA). Chlorophyll concentrations were determined from A.sub.664 and A.sub.648 using the formulae described by Lichtenthaler (1987).
[0562] Leaf Fatty Acid and Sugar Profiles
[0563] All HME lines displayed a significant increase in leaf fatty acids (
TABLE-US-00008 TABLE 6 Total FA (% C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 DW) WT1 11.52 (± 0.1) 2.22 (± 0.04) 1.04 (± 0.01) 1.66 (± 0.04) 12.8 (± 0.1) 70.76 (± 0.2) 4.04 (± 0.1) DGAT + CO1 10.41 ** (± 0.1) 1.96 ** (± 0.08) 1.05 (± 0.02) 5.8 ** (± 0.19) 19.95 ** (± 0.21) 60.83 ** (± 0.39) 5.12 ** (± 0.09) DGAT + CO2 10.67 ** (± 0.02) 1.99 ** (± 0.04) 0.91 ** (± 0.02) 5.29 ** (± 0.1) 18.56 ** (± 0.1) 62.58 ** (± 0.19) 4.78 ** (± 0.03) WT2 11.38 (± 0.1) 2.7 (± 0.05) 0.99 (± 0.02) 1.44 (± 0.05) 13.96 (± 0.3) 69.52 (± 0.3) 3.64 (± 0.1) DGAT + CO3 10.38 ** (± 0.1) 2.47 ** (± 0.05) 0.96 (± 0.03) 4.28 ** (± 0.05) 18.7 ** (± 0.09) 63.2 ** (± 0.13) 5.25 ** (± 0.06) DGAT + CO4 8.95 ** (± 1) 2.07 ** (± 0.23) 0.88 (± 0.11) 6.36 ** (± 0.16) 22.49 ** (± 0.08) 57.97 ** (± 0.21) 5.54 ** (± 0.08) WT3 13.03 (± 0.1) 2.2 (± 0.05) 0.93 (± 0.01) 1.05 (± 0.02) 14.62 (± 0.2) 68.17 (± 0.2) 2.92 (± 0.1) DGAT + CO5 12.29 ** (± 0.1) 2.01 ** (± 0.04) 0.97 (± 0.02) 3.63 ** (± 0.08) 22.47 ** (± 0.12) 58.61 ** (± 0.19) 5.11 ** (± 0.08)
[0564] Low molecular weight carbohydrates (LMW) and high molecular weight carbohydrates (HMW) were significantly lower in DGAT+CO3, DGAT+CO4 and DGAT+CO5, compared to respective WT lines (
[0565] Growth, Photosynthesis and Chlorophyll
[0566] Of the five DGAT+cys-ole lines examined here, two (DGAT+CO1 and DGAT+CO2) showed no significant difference in gas exchange, chlorophyll or biomass, compared to their respective WT control (Table 8 below). In contrast, after six weeks' growth, DGAT+CO3, DGAT+CO4 and DGAT+CO5 were between 59%-82% larger than their respective WT controls and displayed a significant increase in leaf dry weight (DW), total shoot DW, root DW (Table 8 below) chlorophyll a and chlorophyll b (
TABLE-US-00009 TABLE 8 Amass gsw E Leaf Root Shoot DW Total DW (μmol kg.sup.−1 (mol m.sup.−2 (mol m.sup.−2 DW (g) DW (g) (g) (g) LA (cm2) SLA DW s.sup.−1) s.sup.−1) s.sup.−1) WT1 1.6 (± 0.9 (± 2.8 (± 3.7 (± 444 (± 274 (± 475 (± 0.27 (± 2.1 (± 0.1) 0.06) 0.1) 0.1) 19) 8) 21) 0.01) 0.1) DGAT + CO1 1.6 (± 0.7 * (± 2.7 (± 3.4 (± 451 (± 283 (± 468 (± 0.26 (± 2 (± 0.04) 0.03) 0.1) 0.1) 15) 9) 28) 0.02) 0.2) DGAT + CO2 1.6 (± 0.9 (± 2.7 (± 3.6 (± 454 (± 284 (± 499 (± 0.28 (± 2.1 (± 0.05) 0.07) 0.1) 0.2) 11) 8) 29) 0.02) 0.2) WT2 0.8 (± 0.4 (± 1.4 (± 1.9 (± 206 (± 260 (± 301 (± 0.15 (± 1.2 (± 0.03) 0.03) 0.1) 0.1) 15) 15) 20) 0.01) 0.1) DGAT + CO3 1.2 ** (± 0.9 ** (± 2.4 ** (± 3.3 ** (± 359 ** (± 290 (± 536 ** (± 0.34 ** (± 2.5 ** (± 0.03) 0.05) 0.1) 0.1) 9) 5) 11) 0.01) 0.1) DGAT + CO4 1.4 ** (± 0.9 ** (± 2.5 ** (± 3.4 ** (± 415 ** (± 287 (± 497 ** (± 0.3 ** (± 2.2 ** (± 0.1) 0.06) 0.1) 0.1) 19) 8) 13) 0.01) 0.1) WT3 0.9 (± 0.5 (± 2.1 (± 2.5 (± 197 (± 213 (± 256 (± 0.2 (± 1.5 (± 0.1) 0.04) 0.1) 0.2) 17) 8) 17) 0.01) 0.1) DGAT + CO5 1.3 * (± 0.8 ** (± 3.2 ** (± 4 ** (± 433 ** (± 343 ** (± 493 ** (± 0.2 (± 1.5 (± 0.1) 0.06) 0.2) 0.3) 31) 9) 27) 0.01) 0.1)
[0567] Regardless, DGAT+CO3, DGAT+CO4 and DGAT+CO5 all displayed a significant increase in total leaf area, compared to respective WT controls, of 74/6, 101% and 120% respectively (Table 8 above).
[0568] DGAT+CO3, DGAT+CO4 and DGAT+CO5 displayed a significant increase in net photosynthesis (
[0569] The applicant has demonstrated the combination of DGAT+cysteine oleosin dramatically increased fatty acids in the leaves of Lolium perenne and coincided with several morphological, physiological and biochemical changes in the plant. FA correlated positively with DGAT expression and for those lines with the largest increase in fatty acids, we identified a significant reduction in leaf sugar, both LMW and HMW carbohydrates, and a significant increase in A, A.sub.mass and chlorophyll. For DGAT+CO5, the line with the largest relative increase in fatty acids, we also identified a significant increase in specific leaf area. Collectively, the applicant shown that the elevation of fatty acids in leaves, at the expense of leaf sugar, coincides with traits that increase carbon assimilation (primarily increased SLA and photosynthesis) and subsequently, increase relative growth rate. DGAT+CO ryegrass presents a novel opportunity to increase the quality and quantity of forage production and examine the regulation of photosynthesis and other traits related to carbon capture.
[0570] The applicant has identified a strong negative correlation between relative fatty acid accumulation and water-soluble carbohydrates. This observation is consistent with Vanhercke et al. (2019), who similarly identified a trade-off in carbon allocation between lipids and sugar. Regulation of photosynthetic capacity is determined by, among other things, the availability of carbon (source strength), to the demand for carbon (sink strength) (Paul and Foyer, 2001; Arp, 1991; Ainsworth et al 2004), and sugar plays a key role in signalling this relationship (Paul and Driscoll, 2004; Iglesias et al, 2002; Roitsch, 1999; Ainsworth and Bush, 2011; Rierio et al, 2017). Here, we observed distinct morphological and physiological changes (e.g. increased chlorophyll, photosynthesis and specific leaf area) following DGAT+CO transformation, but only in those lines that displayed the largest reduction in leaf sugar. The applicant suggests that a reduction in leaf sugar, as a result of an introduced lipid carbon sink, is directly responsible for inducing those physiological and morphological acclimations (e.g. increased photosynthesis and specific leaf area), that improved carbon assimilation and subsequent growth rate. As such according to the present invention, the correlation between reduced WSC is a more robust way of determining the influence on CO.sub.2 assimilation as compared to measuring either accumulation of the cysteine oleosin protein or the accumulation of additional lipids within the leaf both of which have indirect influences on photosynthesis.
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TABLE-US-00010 SUMMARY OF SEQUENCE LISTING SEQ ID NO: Type SPECIES COMMENTS 1 Polypeptide S. indicum AAG23840 2 Polypeptide S. indicum AAB58402 3 Polypeptide A. thaliana CAA44225 4 Polypeptide A. thaliana AAZ23930 5 Polypeptide H. annuus CAA44224.1 6 Polypeptide B. napus CAA57545.1 7 Polypeptide Z. mays NP_001147032.1 8 Polypeptide O. sativa AAL40177.1 9 Polypeptide B. oleracea AAD24547.1 10 Polypeptide C. arabica AAY14574.1 11 Polypeptide B. oleraceae CAA65272.1 12 Polypeptide S. indicum AAD42942 13 Polynucleotide S. indicum AF302907 14 Polynucleotide S. indicum U97700 15 Polynucleotide A. thaliana X62353 16 Polynucleotide A. thaliana BT023738 17 Polynucleotide H. annuus X62352.1 18 Polynucleotide B. napus X82020.1 19 Polynucleotide Z. mays NM_001153560.1 20 Polynucleotide O. sativa AAL40177.1 21 Polynucleotide B. oleracea AF117126.1 22 Polynucleotide C. arabica AY928084.1 23 Polynucleotide B. oleraceae X96409 24 Polynucleotide S. indicum AF091840 25 Polypeptide A. thaliana NP_179535 26 Polypeptide T. majus AAM03340 27 Polypeptide Z. mays ABV91586 28 Polypeptide A. thaliana NP_566952 29 Polypeptide B. napus AC090187 30 Polypeptide A. hypogaea AAX62735 31 Polypeptide A. thaliana NP_196868 32 Polypeptide R. communis XP_002521350 33 Polynucleotide A. thaliana NM_127503 34 Polynucleotide T. majus AY084052 35 Polynucleotide Z. mays EU039830 36 Polynucleotide A. thaliana NM_115011 37 Polynucleotide B. napus FJ858270 38 Polynucleotide A. hypogaea AY875644 39 Polynucleotide A. thaliana NM_121367 40 Polynucleotide R. communis XM_002521304 41 Polynucleotide O. sativa AY583764 42 Polynucleotide O. sativa AP014965 43 Polynucleotide P. vulgaris AF028707 44 Polynucleotide P. sativum M21356; M27973 45 Polynucleotide P. sativum M64619 46 Polynucleotide G. max D16248 47 Polynucleotide A. thaliana L05399 48 Polynucleotide Cauliflower V00141; J02048 mosaic virus 49 Polypeptide S. indicum Modified oleosin 50 Polypeptide T. majus DGAT1