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