Silencing of Chloroplast Vesiculation (CV) Promotes the Tolerance of Rice to Water-deficit Stress and High CO

The stability of chloroplasts is of paramount importance for the maintenance of photosynthesis during stress conditions. Abiotic stress-induced senescence is a process affecting the photosynthetic apparatus of plants, decreasing photosynthesis and inducing chloroplast degradation. The CHLOROPLAST VESICULATION (CV) protein was proposed to mediate chloroplast degradation during stress and natural senescence in Arabidopsis thaliana. Here, we characterized CV function in crop plants and demonstrated the use of CV-silencing as a viable biotechnological strategy for the production of stress-tolerant crops. Using rice (Oryza sativa) as the model crop plant, we showed that the expression of OsCV was induced during plant senescence and under water-deficit conditions. Overexpression of OsCV caused leaf yellowing and a decrease in photosynthetic activity. Downregulation of OsCV contributed to the maintenance of chloroplast integrity, chloroplastic grana-thylakoid organization, higher photosynthesis, and improved nitrogen assimilation and photorespiration under stress, all processes leading to increased stress tolerance and less stress-induced yield penalties. The CV-mediated chloroplast degradation pathway plays a unique role in thylakoid protein turnover, affecting the remobilization of nitrogen from sources tissues to sink organs.;Interestingly, OsCV overexpression led to reduced expression of chloroplastic glutamine synthetase (OsGS2), an enzyme with important roles in ammonium assimilation and photorespiration. Co-immunoprecipitation and bimolecular fluorescence complementation demonstrated the direct interaction between OsCV and OsGS2. Using sub-organellar markers for pre-vacuolar compartments and vacuoles, we observed the OsCV/OsGS2 interaction throughout the protein turnover pathway. Our results supported the involvement of the OsCV-mediated chloroplast degradation pathway in the regulation of nitrogen assimilation and source/sink relationships during stress-induced plant senescence.;To down-regulate the expression of CV in transgenic rice, we isolated the promoter of a MYB transcription factor that co-expressed with OsCV and displayed low basal expression under natural senescence. Stress-induced CV-silencing, driven by the stress-induced MYB promoter, contributed to higher grain yield and photosynthetic rates under stress conditions. During water-deficit stress, the stomatal response of the CV-silenced rice plants was not affected and there were minimal photosynthetic biochemical limitations, leading to the enhancement of instantaneous water-use efficiency.;Elevated levels of atmospheric CO2 also caused significant decreases in nitrogen assimilation, coincident with the induction of OsCV. Notably, under high CO2, CV -silenced rice maintained photorespiration and nitrogen assimilation. Our results showed that under high CO2, OsCV interacted with PEROXISOME BIOGENESIS FACTOR 11-1 (OsPEX11-1) in peroxisomes, pre-vacuolar compartments and vacuoles, indicating a role for OsCV in OsPEX11-1 turnover, resulting in a lower number of peroxisomes under elevated CO2. These results supported the notion that in addition to mediating chloroplast degradation, OsCV is also associated with the regulation of peroxisome biogenesis. These processes result in an enhanced stability of photorespiration and nitrogen assimilation during elevated CO2 and water-deficit stress.