Circadian-Mediated Regulation of Growth, Chloroplast Proteome, Targeted Metabolomics and Gene Regulatory Network in Spinacia oleracea Under Drought Stress

The paramount objectives of this study were to analyze the beneficial role of the circadian clock in alleviating drought stress in an essential green leafy horticultural crop, spinach (Spinacia oleracea), and to attain knowledge on drought-stress adaptation for crop productivity. From dawn to dusk, a circadian core oscillator-based defense mechanism was noticed in relation to the strength of the chloroplast proteome and transcriptome, and the defense hormone fused it along with the molecular physiology using genotypes “Malav Jyoti” and “Delhi Green”. A photo-periodic rhythmicity containing a 4 h time interval (morning–evening loop) for 12 h in spinach was exhibited under drought-stressed (day-5) and drought re-irrigated (day-10) conditions. The circadian oscillator controlled 70% of the major part of growth and physiological measures such as the biomass, plant height, leaf-relative water content, and the shoot–root ratio under drought stress. Contrarily, drought stress resulted in the upregulation of antioxidative activities and stress markers, whereas it was diversified and maintained in the case of the re-irrigated state at certain rhythmic time intervals of the circadian clock. The physiological parameters we examined, such as net photosynthesis, transpiration, stomatal conductance, and antioxidative enzymes, exhibited the role of the circadian clock in drought stress by showing 80–90% improvements found in plants when they were re-watered after drought stress based on their circadian oscillations. Based on the physiological results, 10 a.m. and 2 p.m. were disclosed to be the rhythmic times for controlling drought stress. Moreover, an extensive study on a gene expression analysis of circadian clock-based genes (CCA1, LHY, TOC1, PRR3, PRR5, PRR7, PRR9, and RVE8) and drought-responsive genes (DREB1, DREB2, and PIP1) depicted the necessity of a circadian oscillator in alleviating drought stress. Hence, the findings of our study allowed for an intense understanding of photo-periodic rhythms in terms of the morning–evening loop, which is in line with the survival rate of spinach plants and occurs by altering cellular ROS-scavenging mechanisms, chloroplast protein profiles, gene regulation, and metabolite concentrations.