Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F /F) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

The plastidic C4 Zea mays NADP-malate dehydrogenase (ZmNADP-MDH), responsible for catalysis of oxaloacetate to malate, was overexpressed in Arabidopsis thaliana to assess its impact on photosynthesis and tolerance to salinity stress. Different transgenic lines were produced having ~3-6-fold higher MDH protein abundance and NADP-MDH enzyme activity than vector control. The overexpressors had similar chlorophyll, carotenoid, and protein content as that of vector control. Their photosynthetic electron transport rates, carbon assimilation rate, and consequently fresh weight and dry weight were almost similar. However, these overexpressors were tolerant to salt stress (150 mM NaCl). In saline environment, the Fv/Fm ratio, yield of photosystem II, chlorophyll, and protein content were higher in ZmNADP-MDH overexpressor than vector control. Under identical conditions, the generation of reactive oxygen species (HO) and production of malondialdehyde, a membrane lipid peroxidation product, were lower in overexpressors. In stress environment, the structural distortion of granal organization and swelling of thylakoids were less pronounced in ZmNADP-MDH overexpressing plants as compared to the vector control. Chloroplastic NADP-MDH in consort with cytosolic and mitochondrial NAD-MDH plays an important role in exporting reducing power (NADPH) and exchange of metabolites between different cellular compartments that maintain the redox homeostasis of the cell via malate valve present in chloroplast envelope membrane. The tolerance of NADP-MDH overexpressors to salt stress could be due to operation of an efficient malate valve that plays a major role in maintaining the cellular redox environment.

Chl -chlorophyll; Fm -maximum Chl a fluorescence; Fo -initial (minimal) Chl a fluorescence; Fv (= Fm -Fo) -variable Chl a fluorescence; I-step -Chl a fluorescence at ~ 30 ms; J-step -Chl a fluorescence at ~ 2 ms; OJIP curve -the 'fast' phase of the fluorescence transient ['O' is for the initial fluorescence (at ~ zero time), 'P' is for peak, and 'J' and 'I' are inflection points between 'O' and 'P']; PQ -plastoquinone; QA and QB -the first and second plastoquinone electron acceptor of PSII. Acknowledgments: We thank Zuzana Benedikty (of Photon Systems Instruments, The Czech Republic) for the generous gift of FluorPen to one of us (G. Govindjee). We are grateful to Roland Valcke for his suggestions to relate our observations to those on fluorescence imaging, which led to substantial improvement of our paper. We are highly thankful to Hartmut Lichtenthaler, the one who we are honoring here, for reading our paper, and for making crucial suggestions, before its submission. We are also grateful to the two anonymous reviewers for their very helpful comments. Conflict of interest: The authors declare that they have no conflict of interest.