Identification of miRNA-mediated drought responsive multi-tiered regulatory network in drought tolerant rice, Nagina 22

Scientific Reports, Nov 2017

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.

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Identification of miRNA-mediated drought responsive multi-tiered regulatory network in drought tolerant rice, Nagina 22

Abstract 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. Introduction Rice production is severely constrained by drought, especially if encountered during panicle initiation and flowering. Several traditional rice cultivars have inherent tolerance mechanisms functioning in diverse environmental adversities1,2. The genetic and molecular characterization of these traditional tolerant germplasm at the level of genome, metabolome, transcriptome, proteome and miRNome is critical to understand the schemas and molecular networks regulating stress tolerance and to identify candidate genes involved in stress tolerance. Drought is a complicated phenomenon involving multiple components wherein a subtle fine-tuning of component(s) can set a chain of events that may offer advantage to the plant during stress. Thus, detailed studies that can identify and integrate information3 about such multi-dimensional networks wherein regulatory and metabolic components are critical for understanding complex traits. Subtle variations in the activity of any of the components of the network would have a cascading affect on the entire network. Plants have evolved several different strategies to counter stress conditions including root exploration, water conservation, osmotic adjustment, root-soil isolation, leaf orientation, root to shoot ratio, stress recovery4. MiRNAs define one such dimension since they have been implicated in regulation of diverse biological processes such as response to abiotic5,6,7,8,9 and biotic10 stress response, development11,12,13, hormone signaling14,15 and nutrient homeostasis16,17. Beside the identification of several drought-regulated miRNAs in rice, the functional implication of miRNA-mediated gene regulation was still not clear. Over-expression of osa-miR393 in rice leads to early flowering, increased tiller number, auxin hyposensitivity and reduced tolerance to salt and drought18. The heat stress transcription factor (TF):miR398:Cu/Zn SOD module is critical for heat stress tolerance in Arabidopsis 19. In rice, while miR164:NAC module plays important role in drought tolerance, over-expression of miR169 leads to enhance drought tolerance in tomato20,21. Similarly, miR319 acts as a positive regulator of cold stress tolerance in rice22. MiRNAs have also been shown to be involved in grain yield. Over-expression of miR397 increases rice yield by enhancing grain size and panicle branching23. Recent report suggests the critical role of miR529a against oxidative stress in rice24. Over-expression of osa-miR528 in bentgrass leads to the enhanced tolerance to salt stress and nitrogen starvation25. In recent years, reactive oxygen species (ROS) has emerged as a critical signaling molecule regulating plant drought tolerance via regulation of stomata opening26,27,28. Regulation of ROS homeostasis is critical because controlled ROS generation is important for signaling whereas, excess is deleterious to the system. The redox active micronutrient copper (Cu) plays an important role in ROS homeostasis due to its requirement in both ROS producing (e.g. electron transport) and scavenging mechanisms29,30. In addition, the miRNA-mediated regulation of plant Cu proteins is conserved among plants31. In the current study, by a molecular comparative analysis of drought tolerant, Nagina 22 (N22)32,33,34,35 and sensitive rice cultivar, Pusa Basmati 1 (PB1)36 we could delineate a multi-dimensional regulatory network wherein the miRNA nodes function as a junction a (...truncated)


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Sonia Balyan, Mukesh Kumar, Roseeta Devi Mutum, Utkarsh Raghuvanshi, Priyanka Agarwal, Saloni Mathur, Saurabh Raghuvanshi. Identification of miRNA-mediated drought responsive multi-tiered regulatory network in drought tolerant rice, Nagina 22, Scientific Reports, 2017, Issue: 7, DOI: 10.1038/s41598-017-15450-1