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).