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

The α-crystallin domain (ACD) is an ancient domain conserved among all kingdoms. Plant ACD proteins have roles in abiotic stresses, transcriptional regulation, inhibiting virus movement, and DNA demethylation. An exhaustive in-silico analysis using Hidden Markov Model-based conserved motif search of the tomato proteome yielded a total of 50 ACD proteins that belonged to four groups, sub-divided further into 18 classes. One of these groups belongs to the small heat shock protein (sHSP) class of proteins, molecular chaperones implicated in heat tolerance. Both tandem and segmental duplication events appear to have shaped the expansion of this gene family with purifying selection being the primary driving force for evolution. The expression profiling of the Acd genes in two different heat stress regimes suggested that their transcripts are differentially regulated with roles in acclimation and adaptive response during recovery. The co-expression of various genes in response to different abiotic stresses (heat, low temperature, dehydration, salinity, and oxidative stress) and phytohormones (abscisic acid and salicylic acid) suggested possible cross-talk between various members to combat a myriad of stresses. Further, several genes were highly expressed in fruit, root, and flower tissues as compared to leaf signifying their importance in plant development too. Evaluation of the expression of this gene family in field grown tissues highlighted the prominent role they have in providing thermo-tolerance during daily temperature variations. The function of three putative sHSPs was established as holdase chaperones as evidenced by protection to malate-dehydrogenase against heat induced protein-aggregation. This study provides insights into the characterization of the Acd genes in tomato and forms the basis for further functional validation in-planta.

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