Figure legends
Fig. 1 A proposed model of photosynthetic carbon flow inArabidopsis thaliana overexpressing FbβCA3 . The cytosolicFbβCA3 having low Km for CO2 increase the hydration of CO2. The dashed arrows indicate the diffusion of CO2 and HCO3- within the cytosol and the chloroplast. The relatively higher amounts of CO2 and HCO3- present in the mesophyll cells of overexpressor likely to increase the CO2 diffusion gradient into the chloroplast during day time. Utilization of diffused CO2 by rubisco to make 3-phosphoglycerate would accelerate the carbon reduction cycle favoring the carboxylation activity.FbβCA3 overexpression increased the flux of the carboxylic acid to the tricarboxylic acid cycle (TCA) in mitochondria to play an anaplerotic role to synthesize higher amounts of total amino acids and proteins that contribute to increase photosynthetic efficiency and biomass.
Fig. 2 A schematic representation of the transgene used forArabidopsis transformation, photographs and conformation ofFlaveria bidentis CA overexpressed in Arabidopsis.(a) : Flaveria bidentis βCA3 cloned to pCAMBIA1304 vector having CaMV35S-Ω-poly A promoter cassette; CaMV35S-npt, coding region of neomycin phosphotransferase gene with CaMV35S promoter; CaMV35SΩ, CaMV 35S promoter with omega (Ω) enhancer; FbβCA3 cDNA, coding region of Fb CA gene; Poly A, Poly A tail; (b):Arabidopsis vector control (VC) and CAx (CAx3 & CAx5) plants grown at 210C under 14h L / 10h D photoperiod in cool-white-fluorescent light (100 µmol photons m-2s-1) for 4 weeks in pots; (c): qRT-PCR ofFbβCA3 —relative gene expression of CA in VC and transgenic lines; (d): 15% SDS-PAGE- twenty five μg protein was loaded in each lane and SDS-PAGE was run to check equal loading; (e): Western blot- protein samples from the gel were transferred to nitrocellulose membrane and immunoblot analysis of CA protein was made using Flaveria bidentis CA antibodies; (f): Quantification of CA blot- relative expression of CA in transgenic lines; (g): CA enzymatic activity- the activity of CA in VC was 276 µmol CO2 hydrated (mg Chl)-1 min-1. CA activity ranged from 348 to 550 µmol CO2 hydrated (mg Chl)-1 min-1 in different transgenic lines (CAx1, CAx2, CAx3 and CAx5); VC- vector control plants containing the null vector pCAMBIA1304). Each data point is the average of five replicates, and error bars represents ±SD; asterisks indicate significant differences determined by t test (*P < 0.05)
Fig. 3 PEPC enzymatic activity in-vitro, electron transport reactions in isolated thylakoid membrane and electron transport rates (ETRII & ETRI) in the intact leaves. (a ): PEPC enzymatic activity- the activity of PEPC in transgenics was similar to vector control plants (~3 μmol/mg protein/hr); (b):Electron transport through PSII (oxygen evolution; water to PD); (c) : whole chain (water to MV; oxygen uptake); (d):PSI (ascorbate to MV; oxygen uptake) was measured polarographically, as described under “Materials and Methods”; (e): ETRII; (f): ETRI, VC- vector control; and 2 different transgenic lines, CAx3 and CAx5. Each data point is the average of five replicates and the error bars represent ±SE; asterisks indicate significant differences determined by t test (*P < 0.05)
Fig. 4 The OJIP curve of chlorophyll a fluorescence and the non-photochemical quenching (NPQ) of the excited state of chlorophyll a of the vector control and FbβCA3x plants grown in soil. (a): Chl a fluorescence transients, the OJIP curves normalized at the O level; (b): Variable fluorescence transients from the I to the P—double normalized between I (F I) and P (F p):V IP = (F t -F I)/(F P -F I); (c): Variable fluorescence transients from the I –single normalization; F, in the diagram, stands for fluorescence at time t (F t), andF o is for fluorescence at the O level;(d): NPQ of the excited state of chlorophyll at different light intensities. Each data point is an average of 8 replicates and error bars represent ±SE; asterisks indicate significant differences determined by t test (*P < 0.05)
Fig. 5 Relative gene expression and immunoblot analysis. Relative expression of genes related to (a): chlorophyll biosynthesis; (b): photosynthesis; (c): SDS-PAGE (12%) of protein (20 μg) isolated from VC and transgenic plants to check equal loading and the immunoblot to check the abundance of electron transport chain components; (d): Quantification of western blot by densitometry analysis using Alpha Ease FC software,;PBGS - porphobilinogen synthase; UROD- encoding uroporphyrinogen decarboxylase;PPOX1 - encoding protoporphyrinogen oxidase; CHLI - encoding protoporphyrin-IX Mg-chelatase; PORC - encoding the light-inducible protochlorophyllide oxidoreductase; Lhcb1 andLhcb2, encoding components of the light harvesting complex associated with PSII; Lhca1 and Lhca2, encoding components of the light harvesting complex associated with PSI; PsbA and PsbD, Core proteins, encoding photosystem II D1 and D2 proteins; PsbO , encoding for OEC33, the oxygen-evolving complex; LHCII- light-harvesting chlorophyll-binding proteins; Cytb6f and Cytf, psaE- PSIIV; rubisco LSU and SSU- rubisco large and small subunit. Western blot data is an average of three independent replicates. qRT-PCR data are expressed as the mean ± SE of three independent experiments performed in triplicate. Asterisks indicate significant differences determined by Student’s t-test compared to control (*P < 0.05, **P < 0.01)
Fig. 6 Photosynthesis (net CO2 assimilation rate) light response curve. (a) : Net CO2assimilation rates of vector control and CAx plants were monitored by IRGA (LiCor-6400/XT) in ambient CO2 at 400 µmol photons m-2 s-1 at 21ºC; (b) : Net CO2 assimilation rates upto 80 µmol photons m-2 s-1; (c): Net CO2 assimilation rates; (d): Stomatal conductance (gs); (e): Transpiration rate; (f): Water use efficiency (WUE) of vector control and transgenic Arabidopsisplants. Each data point is an average of five replicates and error bars represent SE. Asterisks indicate significant differences determined by t test (*P<0.05).
Fig. 7 Photosynthetic carbon fixation rate as a function of increasing intercellular [CO2] at 21% O2 and 2% O2. (a):A/C i curve; dashed lines represents to at 2% O2; (b): Dry weight; (c): Starch content, per g FW. Each data point is an average of five replicates and error bars represent SE. Asterisks indicate significant differences determined by t test (*P<0.05).