Higher photosynthetic efficiency in the transgenics
The increased ETR of PSII and PSI at lower light intensities (50-125
μmol photons m-2 s-1) in intact
leaves of CAx plants is suggested to be mostly due to efficient
energy capture by the larger antenna, which must have been due to
increased gene/protein expression of the light-harvesting components of
PSII and PSI i.e. Lhcb2.1 , LHCII, Lhcb1 , Lhca1 andLhca2 (Figs. 5b,c,d ). ETR measured at higher light
intensity (~550 μmol photons m-2s-1) was also higher (13%-19% for PSII; 17%-25%
for PSI) in CAx plants than in the VC (Fig. 3e,f ). These
values of PSI and PSII ETR of intact leaves, derived from Chl afluorescence parameters, were confirmed by actual measurements of
light-saturated PSII, whole-chain and PSI in isolated thylakoid
membranes (Figs. 3b,c,d ). We suggest that higher
light-saturated PSII- and PSI-dependent ETR in the intact leaves and
PSII and PSI activities of thylakoid membranes, isolated from
transgenics, are due to the increased gene expression/ protein abundance
of essential components of PSII (PsbA , PsbD ), oxygen
evolving complex (PsbO ), PSI subunit IV (PsaE), as well as a few
other thylakoid membrane-bound photosynthetic proteins. On the basis of
all the results, presented in this paper, we conclude that theCAx plants had (i ) a higher light-harvesting capacity
under limiting light intensities due to larger light-harvesting
apparatus and (ii ) a greater ability to use light at higher
light intensities due to more and efficient PSII, PSI and intersystem
electron transport components. It is known that OEC33, which is on the
electron donor (oxidising) side of PSII, binds calcium as well as GTP
(Suorsa and Aro, 2007); furthermore, this side has a carbonic anhydrase
activity (Lu et al., 2005). Increased protein abundance of OEC33 in the
transgenics may have been responsible for higher CA activity on the
oxidising side of PSII resulting in enhanced PSII-dependent ETR
especially under high light. The presence of CA in thylakoid
membranes (Stemler, 1997), generating
HCO3-, could have contributed to
increased PSII activity by binding at the reducing side of PSII (for a
review on the “bicarbonate” effect, see Shevela et al., 2012). Coupled
with higher PSI and PSII reactions, increased protein abundance of
intersystem electron transport components, i.e., cytochrome b6/f (Cyt
b6/f) complex, which includes cytochrome f, must have contributed to
higher whole chain electron transport in the transgenic lines. These
conclusions are consistent with earlier work, from our laboratory, where
higher PSI, PSII and whole chain electron transport activities were
observed in chlorophyllide a oxygenase (CAO ) overexpressor lines
of tobacco, which was also due to an increase in photosynthetic proteins
(Biswal et al., 2012); this finding is in complete agreement with the
data of Simkin et al. (2017), on Arabidopsis, overexpressing Rieske FeS
protein of the Cyt b6/f complex resulting in increase of components of
PSI, PSII, and the ATPase. The Cyt b6/f is known to mediate electron
transfer from the plastoquinol to plastocyanin, and is involved in
noncyclic electron flow (Wood and Bendall, 1976; Hurt and Hauska, 1981),
as well as in the cyclic electron flow around PSI (Lam and Malkin,
1982). In addition, PsaE protein on the electron- acceptor side of PSI
is involved in cyclic electron transport, and is responsible for
preventing electron leakage to molecular oxygen (Mehler reaction)
(Jeanjean et al., 2008). We speculate that the increased protein
abundance of PsaE (Fig. 5d ) in CAx plants may have minimized
the photoreduction of reactive oxygen species by PSI.
Chl fluorescence transient (or induction) curve includes what is known
as the OJIP rise, where “O” is the first measured point when a
dark-adapted plant is exposed to continuous light; then it rises to a
peak P with two inflection points J and I. In the OJIP curve, normalized
at the “O” level (F o), we observe, in the
transgenics, a faster JI rise as well as a faster IP rise. We interpret
this to suggest that there a faster efficiency of electron transport in
the transgenics (Fig. 4a ) (Kandoi et al., 2016;
Jiménez-Francisco et al., 2020). The higher amount of bicarbonate in the
transgenics might have possibly helped in keeping bicarbonate bound to
the non-heme iron on the reducing side of PSII (as mentioned above),
probably affecting OJIP kinetics indirectly. Further research is needed
to understand this phenomenon in terms of the various available models
(see e.g. Stirbet et al., 2020). However, the faster IP rise in the
transgenics suggests that they may be more efficient in photosynthesis,
as empirically found in other systems (Hamdani et al., 2015; Soda et
al., 2018). The “performance index” in the CAx plants was observed to
be better than in the VC plants; clearly indicating higher efficiency of
the photosynthetic machinery in the transgenics. The area over the OJIP
curve, between F o and F m,
which is proportional to the size of the pool of the electron acceptors
in PSII, mainly the plastoquinone molecules (Malkin and Kok, 1966) was
higher (19%-22%) in the CAx3 and CAx5 lines than in VC, once again
confirming the advantage of the transgenics, over the controls