Foxtail millet (Setaria italica L.) known as a relatively drought-tolerant crop across the world is grown in arid and semi-arid regions. To the best of our knowledge, no systematic study on drought tolerance screening of foxtail millet germplasm being a drought-tolerant crop has been reported so far. To explore genetic diversity of drought-induced oxidative stress tolerance in foxtail millet, we employed lipid peroxidation measure to assess membrane integrity under stress as biochemical marker to screen 107 cultivars and classified the genotypes as highly tolerant, tolerant, sensitive, and highly sensitive. From this comprehensive screening, four cultivars showing differential response to dehydration tolerance were selected to understand the physiological and biochemical basis of tolerance mechanisms. The dehydration-tolerant cultivars (IC-403579 and Prasad) showed considerably lower levels of lipid peroxidation and electrolyte leakage as compared with dehydration-sensitive cultivars (IC-480117 and Lepakshi), indicating better cell membrane integrity in tolerant cultivars. Correspondingly, tolerant genotypes maintained higher activity of catalase (EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2) across different time-course period of polyethylene glycol (PEG) treatments in comparison to sensitive ones. The above biochemical results were further validated through quantitative real-time PCR analysis of APX and GR, whose transcripts were substantially induced by PEG treatments in tolerant cultivars. These results suggest that tolerant cultivars possess wider array of antioxidant machinery with efficient ascorbate-glutathione pathway to cope with drought-induced oxidative stress.

No abstract

Highlight:2 1 Expansion of MIR169 members by duplication and new mature forms, acquisition of new 2 2 promoters, differential precursor-miRNA processivity and engaging novel targets increases the 2 3 functional diversification of MIR169 in tomato. (29/30) 2 4 Abstract 2 5 MIR169 family is an evolutionarily conserved miRNA family in plants. A systematic in-depth 2 6analysis of MIR169 family in tomato is lacking. We report eighteen miR169 precursors, 2 7 annotating new loci for MIR169a, b and d, as well as four novel mature isoforms 2 8 (MIR169f/g/h/i). The family has expanded by both tandem-and segmental-duplication events 2 9 during evolution. A tandem-pair 'MIR169b/b-1 and MIR169b-2/h' is polycistronic in nature 3 0 coding for three MIR169b isoforms and a new variant miR169h, that is evidently absent in the 3 1 wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets including RNA-3 2 binding-protein, protein-phosphatase, aminotransferase, chaperone, tetratricopeptide-repeat-3 3 protein, and transcription factors ARF-9B and SEPELLATA-3 were established by efficient 3 4 target cleavage in presence of specific precursors as well as increased target abundance upon 3 5 miR169 chelation by short-tandem-target-mimic construct in transient assays. Comparative 3 6 antagonistic expression profiles of MIR169:target pairs suggest MIR169 family as ubiquitous 3 7 regulator of various abiotic stresses (heat, cold, dehydration and salt) and developmental 3 8 pathways. This regulation is partly brought about by acquisition of new promoters as 3 9demonstrated by promoterMIR169:GUS-reporter assays as well as differential processivity of 4 0 different precursors and miRNA cleavage efficiencies. Thus, the current study augments the 4 1 functional horizon of MIR169 family with applications for stress tolerance in crops. 4 2

Summary The footprint of tomato cultivation, a cool region crop that exhibits heat stress (HS) sensitivity, is increasing in the tropics/sub‐tropics. Knowledge of novel regulatory hot spots from varieties growing in the Indian sub‐continent climatic zones could be vital for developing HS‐resilient crops. Comparative transcriptome‐wide signatures of a tolerant (CLN1621L) and sensitive (CA4) cultivar pair shortlisted from a pool of varieties exhibiting variable thermo‐sensitivity using physiological‐, survival‐ and yield‐related traits revealed redundant to cultivar‐specific HS regulation. The antagonistically expressing genes encode enzymes and proteins that have roles in plant defence and abiotic stresses. Functional characterization of three antagonistic genes by overexpression and silencing established Solyc09g014280 (Acylsugar acyltransferase) and Solyc07g056570 (Notabilis) that are up‐regulated in tolerant cultivar, as positive regulators of HS tolerance and Solyc03g020030 (Pin‐II proteinase inhibitor), that are down‐regulated in CLN1621L, as negative regulator of thermotolerance. Transcriptional assessment of promoters of these genes by SNPs in stress‐responsive cis‐elements and promoter swapping experiments in opposite cultivar background showed inherent cultivar‐specific orchestration of transcription factors in regulating transcription. Moreover, overexpression of three ethylene response transcription factors (ERF.C1/F4/F5) also improved HS tolerance in tomato. This study identifies several novel HS tolerance genes and provides proof of their utility in tomato thermotolerance.

Expansion of MIR169 members by duplication and new mature forms, acquisition of new promoters, differential precursor-miRNA processivity and engaging novel targets increases the functional diversification of MIR169 in tomato. (29/30