various into ATP and NADPH. Out of 30

various
pathways in three tissues (CONTROL, TR15, TR30) of J. curcas.

Light
reaction plays an important role in carbon fixation through photosynthesis.
Photosynthesis is a tightly controlled process, in which light energy is
captured and converted into ATP and NADPH. Out of 30 genes identified for
enzymes coding for the photosynthetic pathway, genes coding for LHCI, LHCII, PSI, PSII and F-type H+-transporting ATPase
were upregulated in TR15 whereas showed downregulation in TR30. This might be
the consequence of less photosynthetic activity in TR30 as compared to TR15 due
to reduced floral biomass (Renau-Morata et al. 2012). In photosynthesis,
photosystems (PSI and PSII) regulate the primary
photochemistry of photosynthesis, transfer of energy and light absorption
(Caffarri et al. 2014). Photosynthetic
capacity mainly depends on photosynthetic pigments, cytokinin
application induced the photosynthetic activity by inducing the photosynthesis
genes and by increasing the transcript levels of PSI and PSII genes thus
increasing the photosynthetic pigment complexes in TR15, however their activity
was reduced in TR30 where the floral buds were reduced due to increased
abortion rate which can be correlated with reduced photosynthetic activity. Current
results are in accordance with findings of Liu et al. (2010), who reported the
increased photosynthetic activity initially in transgenic rice with overexpression
of genes associated with cytokinin biosynthesis but decreased later. These
observations indicated that cytokinin application did induce the photosynthetic
activity initially however its prolonged effect was not observed. Further, it
could not keep pace with the increased number of floral buds thus source to
sink ratio was weakened, causing the abortion/abscission of floral buds in TR30.

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Genes
encoding plastocyanin, ferredoxin-NADP+ reductase, F-type H+-transporting
ATPase showed decreased transcript levels in TR30 compared to TR15 whereas no
change was observed in TR15 as compared to control (Supplementary Table 3a). Apart
from reduction in photosynthetic pigments, the reduction in photosynthetic rate
might be due to the debilitating metabolic reactions such as RuBP synthesis,
ATP synthesis, and electron transfer (Lawlor 2002). As per our observations,
the decreased transcript abundance of gene encoding enzyme NADP+ reductase in
TR30 is associated with reduced photosynthetic electron flow, further limits
ATPs required for carbon fixation (Kramer and Evans 2011, Sood and Chauhan 2017).
Thus, from our findings, it is inferred that the reduced biomass in TR30 might
be due to decrease in overall photosynthetic capacity or availability of
photosynthates required for the proper development of increased number of
flowers and subsequent formation of fruits and seeds (Wang et al. 1996).

Effect of cytokinin application on
carbon fixation

 ATP and NADPH generated through photosynthesis
are utilized in carbon fixation through Calvin cycle. Among 18 genes identified
for the Calvin cycle, 12 were differentially expressed in CONTROL, TR15 and TR30.
Genes 6-phosphogluconolactonase, fructose-1,6-bisphosphatase I,
ribulose-bisphosphate carboxylase small chain, glyceraldehyde-3-phosphate
dehydrogenase,  exhibited higher
transcript abundance in TR15 compared to CONTROL and TR30 whereas genes phosphoglucomutase,
phosphoglycerate kinase, fructose-bisphosphate aldolase (FBP), ribose 5-phosphate isomerase A, ribulose-phosphate 3-epimerase
and transaldolase, sedoheptulose-bisphosphatase (SBP) showed higher transcript abundance in TR15 compared to TR30
but same to that of CONTROL (Supplementary Table 3b). FBP and SBP are considered
as the major control points in the Calvin cycle for CO2 fixation in
plants. Previous studies have reported that the overexpression of SBP/FBP in various plants resulted in
increased photosynthetic CO2 fixation and carbohydrate (starch and
sucrose) accumulation (Lefebvre et al. 2005; Tamoi et al. 2006; Gong et al.
2015).  It is implied that the decreased
transcript abundance of SBP in TR30
could be the possible reason for reduction in floral biomass (Harrison et al.
2001). We hereby inferred that SBP/FBP
could be targeted through genetic engineering approaches to enhance the carbon
flux and thus biomass and further fruit or seed yield in Jatropha.

Apart from genes involved in calvin cycle, other genes
associated with carbon fixation are 
glutamate–glyoxylate aminotransferase, phosphoenolpyruvate
carboxykinase, aspartate aminotransferase, 
malate dehydrogenase, pyruvate orthophosphate dikinase, acetyl-CoA
carboxylase carboxyl transferase subunit alpha, ATP citrate (pro-S)-lyase,
aconitate hydratase, methylenetetrahydrofolate reductase (NADPH), isocitrate dehydrogenase, acetyl-CoA carboxylase which were
largely influenced by cytokinin treatment as these were overexpressed in TR15
as compared to CONTROL and TR30. ATPs required for carbon fixation, are also
generated by oxidative phosphorylation from oxidation of NADH and FADH2 from
photosynthesis. Out of 69 genes identified for oxidative phosphorylation, 33
showed differential transcript abundance in three tissues. Genes encoding
enzymes inorganic pyrophosphatase, NADH dehydrogenase, NAD(P)H-quinone
oxidoreductase subunit 5, H+-transporting ATPase, ubiquinol-cytochrome c
reductase, cytochrome c oxidase subunit 6a showed higher transcript abundance in
TR15 compared to CONTROL and TR30. Also, genes like succinate dehydrogenase,
V-type H+-transporting ATPase, F-type H+-transporting ATPase subunit g, F-type
H+-transporting ATPase subunit beta, showed lower transcript abundance in TR30
as compared to TR15 (Supplementary Table 3c). 
Thus, our observations inferred that carbon fixation increased after 15
days but decreased after 30 days of BA treatment suggesting that reduced carbon
availability might be affecting the floral biomass in TR30 and the seed yield. The
current observations are in line with previous studies for plants like
Arabidopsis, tobacco and rice supporting the correlation of carbon fixation and
biomass accumulation (Kurek et al. 2007; Zhu et al. 2007; Raines 2011).

 

Effect of cytokinin application on carbohydrate
metabolism

Carbon
fixed through Calvin cycle is further utilized for the synthesis of
carbohydrates. In plants, starch and sucrose serve as the major carbohydrates
storage points (Berg et al. 2002; Kumar
et al. 2016). Based on KEGG pathway assignments, we identified 23 genes
involved in starch and sucrose metabolism out of which 18 genes were
downregulated in TR30 as compared to TR15. Transcript abundance showed that
genes encoding enzymes pectinesterase, sucrose synthase, trehalose 6-phosphate
phosphatase, beta-glucosidase, beta-fructofuranosidase, polygalacturonase,
endoglucanase, beta-D-xylosidase 4 and alpha-1, 4-galacturonosyltransferase
were upregulated in TR15 as compared to CONTROL and TR30. Genes encoding
enzymes linked to starch and sucrose metabolism such as glucose-1-phosphate
adenylyltransferase (AGPase), trehalose
6-phosphate synthase/phosphatase, 4-alpha-glucanotransferase,
phosphoglucomutase (PGM), hexokinase,
1, 4-alpha-glucan branching enzyme and sucrose-phosphate synthase were down
regulated in TR30 as compared to TR15. Transcript abundance of genes encoding PGM and AGPase decreased in TR30 as compared to TR15 suggesting a lower
rate of starch formation in TR30. In starch biosynthetic pathway PGM converts glucose-6-phosphate into
glucose-1-phosphate and its reduced activity has been reported to reduce the
rate of starch accumulation (Yeh et al. 2001). Further, the rate of starch
biosynthesis is regulated by AGPase,
which catalyzes an irreversible reaction and is strongly regulated through
allosteric (3PGA activator and orthophosphate
as an inhibitor) and redox control (George et al., 2010). The activity of this
enzyme probably determines the flux of carbon into starch as its reduced
expression might limit the carbon flux and starch synthesis (Vigeolas et al.
2004). Thus, it is implicit that the decreased floral biomass in TR30 is possibly
due to the reduction of carbohydrate synthesis and supply to the florets which
are required for the proper development of reproductive organs (Corbesier et
al. 1998). Apart from floral biomass reduction in TR30, the reduction in seed
size and weight might be due to less carbohydrate reserves and their translocation
to the reproductive sink tissue further affecting seed yield at later stages of
development. These observations suggest that cytokinin application initially
enhanced the carbon flux, resulting in increased biomass. However, after a
particular time, the required carbon flux, could not meet the required demand, due
to the reduced triose phosphate availability needed for carbohydrate synthesis
(Walters et al. 2004).

 During night hours, the carbon stored in the
form of starch is mobilized by conversion to sucrose in the cytoplasm. Triose
phosphate form fructose1, 6-bisphosphate which is then converted into
fructose-6 phosphate by fructose-1, 6-bisphosphatase (FBP). FBP regulates
sucrose metabolism pathway which was supported by its upregulation in TR15 as
compared to CONTROL and TR30. Sucrose phosphate synthase (SPS) which forms sucrose phosphate by utilizing UDP-glucose and
D-fructose-6-phosphate was downregulated in TR30 as compared to TR15.
(Supplementary Table 3d). SPS plays a
crucial role in carbohydrate metabolism by regulating the partitioning of
carbon between starch production and carbohydrate accumulation (Chen et al.
2005). Based on the above observations it is imperative that there was a
reduction in carbohydrate synthesis as well as flow to the sink organs
(florets), thus, weakened sink strength causing the reduction in biomass in
TR30 and further affected the seed yield (Koch 2004; Hikosaka and Sugiyama 2015).

Starch
phosphorylase, which transfers glucose from nonreducing end of a-1, 4-linked
glucan to orthophosphate and generates glucose 1-phosphate, was found to be
repressed in TR30. This glucose 1-phosphate enters glycolysis for sugar
breakdown. The pyruvate formed at the end of glycolysis enters the citric acid
cycle (TCA) providing precursors for carbon skeleton (Berg et al. 2002). TCA
cycle plays a central role in generating ATP and providing carbon skeletons to
many biosynthetic processes. Overall, the citric acid cycle has been affected
after the treatment thus reducing the carbon flow, as the enzyme ATP citrate
lyase has been downregulated (Daloso et al. 2015). The reduction in TCA
activity might have affected the biomass
after 30 days due to compromise in the efficient use of carbohydrates (reduced
starch and sucrose level), which has already been reduced as stated above
(Schroder et al 2014). Overall these observations suggest that the increased floral buds might produce more
photoassimilates i.e. carbon gain and carbon assimilation, however, premature
exhaustion of carbohydrate reserves might trigger starvation leading to
decreased biomass, further affecting yield (Buchanan-Wollaston et al. 2005). Thus, it is inferred that the pathways contributing towards carbon
accumulation were reduced and could not meet the increased demand due to high
flowering intensity, which in turn affected the development of fruits further
leading to reduction in seed weight and size, thereby overall compromising the
yield.

Effect of cytokinin application on
nitrogen assimilation and carbon to nitrogen (C/N) ratio

Nitrogen
assimilation depends upon a regular co-ordination between nitrogen and carbon,
due to the requirement of carbon in the form of 2-oxoglutarate and ATP (Lancien
et al. 2000). Therefore, certain metabolites such as sucrose and amino acids
play an important role in the regulation of enzymes involved in nitrogen
assimilation. Nitrogen plays a significant role in the formation of compounds
required for cellular activities. 9 genes associated with nitrogen metabolism
were identified, of which 6 were differentially expressed in TR15. Transcript
abundance of genes encoding enzymes glutamine synthetase, carbonic anhydrase,
formamidase, nitrate reductase and nitatre/nitrite transporters was repressed
in TR30 (Supplementary Table 3e) showing less rate of nitrogen metabolism.
Transcript abundance of gene encoding glutamine synthetase was (GS) decreased in TR30 as compared to
TR15 which indicates less nitrogen availability, as it is a key enzyme
assimilating inorganic nitrogen (Lea et al. 1990). Glutamate dehydrogenase (GDH) activity was decreased from TR15 to
TR30, thus reducing the release of carbon required under low C/N ratios as it
acts as a shunt to the glutamate synthase cycle releasing carbon from amino
compounds as keto-acids (Miflin and Habash 2002). GDH also catalyzes the amination of 2-oxoglutarate in glutamate
synthesis. This indicated that due to the repressed activity of this enzyme,
carbon availability has been decreased causing reduced floral biomass in TR30
and further yield. Many reports deliberating an increase in biomass and yield
with GS transformation in various plant
species exist (Gallardo et al. 1999; Harrison et al. 2000; Czyzewicz and Below
1994). The reduction in biomass after 30 days of cytokinin treatment (TR30) might
be due to reduced nitrogen availability, thus affecting the assimilate
partitioning between vegetative and reproductive organs (Czyzewicz and Below
1994). Availability of nitrogen has the potential to increase total yield and
genes encoding enzymes GS and GDH are hereby referred as potential
targets to increase yield of J. curcas
as they regulate the C/N ratio (Yong et al. 2010).