Evolution of differential regulatory mechanisms can lead to quite distinct physiological attributes. In the present study, we have identified one such regulatory schema that regulates osa-miR408 and responds differentially in drought-sensitive and -tolerant indica rice varieties. A comparison of the drought stress response in drought-sensitive (Pusa Basmati 1 and IR64) and drought-tolerant (Nagina 22 and Vandana) indica rice varieties revealed that, during drought stress, levels of miR408 transcript decrease significantly in sensitive cultivars, whereas they remain elevated in the tolerant cultivars. The trend is reflected in young seedlings, as well as in flag leaf and spikelets of adult plants (heading stage). Members of the plastocyanin-like protein family targeted by miR408 also show the inverse expression profile and thus accumulate at a lower level in tolerant cultivars during drought. Interestingly, some members of this family are implicated in maintaining the cellular redox state and spikelet fertility in Arabidopsis. An investigation of miR408 loci (including promoter) in all four cultivars did not reveal any significant sequence variation indicating an involvement of the upstream regulatory schema. Indeed, a similar variety-specific stress response was found in the Oryza sativa squamosa promoter-binding-like 9 transcription factor that regulates miR408 expression. We further demonstrate that drought-mediated induction of miR408 in Nagina 22 is regulated by [Ca 2+ ] cyt levels. However, [Ca 2+ ] cyt does not appear to regulate miR408 levels in Pusa Basmati 1, suggesting a variety-specific evolution of regulatory schema in rice.

Comparative characterization of microRNA-mediated stress regulatory networks in contrasting rice cultivars is critical to decipher plant stress response. Consequently, a multi-level comparative analysis, using sRNA sequencing, degradome analysis, enzymatic and metabolite assays and metal ion analysis, in drought tolerant and sensitive rice cultivars was conducted. The study identified a group of miRNAs “Cultivar-specific drought responsive” (CSDR)-miRNAs (osa-miR159f, osa-miR1871, osa-miR398b, osa-miR408-3p, osa-miR2878-5p, osa-miR528-5p and osa-miR397a) that were up-regulated in the flag-leaves of tolerant cultivar, Nagina 22 (N22) and Vandana, but down-regulated in the sensitive cultivar, Pusa Basmati 1 (PB1) and IR64, during drought. Interestingly, CSDR-miRNAs target several copper-protein coding transcripts like plantacyanins, laccases and Copper/Zinc superoxide dismutases (Cu/Zn SODs) and are themselves found to be similarly induced under simulated copper-starvation in both N22 and PB1. Transcription factor OsSPL9, implicated in Cu-homeostasis also interacted with osa-miR408-3p and osa-miR528-5p promoters. Further, N22 flag leaves showed lower SOD activity, accumulated ROS and had a higher stomata closure. Interestingly, compared to PB1, internal Cu levels significantly decreased in the N22 flag-leaves, during drought. Thus, the study identifies the unique drought mediated dynamism and interplay of Cu and ROS homeostasis, in the flag leaves of drought tolerant rice, wherein CSDR-miRNAs play a pivotal role.

MicroRNAs regulate a spectrum of developmental and biochemical processes in plants and animals. Thus, knowledge of the entire miRNome is essential to understand the complete regulatory schema of any organism. The current study attempts to unravel yet undiscovered miRNA genes in rice. Analysis of small RNA libraries from various tissues of drought-tolerant ‘aus’ rice variety Nagina 22 (N22) identified 71 novel miRNAs. These were validated based on precursor hairpin structure, small RNA mapping pattern, ‘star’ sequence, conservation and identification of targets based on degradome data. While some novel miRNAs were conserved in other monocots and dicots, most appear to be lineage-specific. They were segregated into two different classes based on the closeness to the classical miRNA definition. Interestingly, evidence of a miRNA-like cleavage was found even for miRNAs that lie beyond the classical definition. Several novel miRNAs displayed tissue-enriched and/or drought responsive expression. Generation and analysis of the degradome data from N22 along with publicly available degradome identified several high confidence targets implicated in regulation of fundamental processes such as flowering and stress response. Thus, discovery of these novel miRNAs considerably expands the dimension of the miRNA-mediated regulation in rice.

Drought-tolerant rice variety, Nagina 22 (N22), has a unique spikelet miRNome during anthesis stage drought as well as transition from heading to anthesis. Molecular characterization of genetic diversity of rice is essential to understand the evolution and molecular basis of various agronomically important traits such as drought tolerance. miRNAs play an important role in regulating plant development as well as stress response such as drought. In this study, we characterized the yet unexplored dynamics of the spikelet miRNA population during developmental transition from 'heading' to 'anthesis' as well as anthesis stage drought stress in a drought-tolerant indica rice variety, N22. A significant proportion of miRNA population (~20 %) in N22 spikelets is modulated during transition from heading to anthesis indicating a unique miRNome at anthesis, a developmental stage highly sensitive to stress (drought/heat). Based on the analysis of degradome data, majority of differentially regulated miRNAs appear to regulate transcription factors, some of which are implicated in regulation of development and fertilization. Similarly, drought during anthesis leads to a global change in miRNA expression pattern including those which regulate ROS homeostasis. It was possible to identify several miRNAs that were not reported to be drought responsive in earlier studies. Interestingly, a significant proportion of the drought-regulated miRNAs co-localize within QTLs related to drought tolerance and associated traits. Comparison of the expression profiles between N22 and Pusa Basmati 1 (drought sensitive) identified miRNAs with variety-specific expression patterns during phase transition (miR164, miR396, miR812, and miR1881) as well as drought stress (miR1881) indicating an evolution of a distinct and variety-specific regulatory mechanism. The promoters of these miRNAs contain LREs (light-responsive elements) and are induced by dark treatment. It was also possible to identify 4 novel miRNAs including an intronic miRNA that was conserved in both rice varieties.

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