Delineating the tissue-mediated drought stress governed tuning of conserved miR408 and its targets in rice.

Engineering drought tolerance in rice needs to focus on regulators that enhance tolerance while boosting plant growth and vigor. The present study delineated the concealed function and tissue-mediated interplay of the miR408/target module in imparting drought stress tolerance in rice. The plant miR408 family comprises three dominant mature forms (21 nt), including a distinct monocot variant (F-7 with 5' C) and is divided into six groups. miR408 majorly cleaves genes belonging to the blue copper protein in addition to several other species-specific targets in plants. Comparative sequence analysis in 4726 rice accessions identified 22 sequence variants (SNP and InDELs) in its promoter (15) and pre-miR408 region. Haplotype analysis of the sequence variants indicated eight haplotypes (three: Japonica-specific and five: Indica-specific) of the miR408 promoter. In drought-tolerant Nagina 22, miR408 follows flag leaf preferential expression. Under drought conditions, its levels are upregulated in flag leaf and roots which seems to be regulated by a differential fraction of methylated cytosines (mCs) in the precursor region. The active pool of miR408 regulated targets under control and drought conditions is impacted by the tissue type. Comparative expression analysis of the miR408/target module under different sets of conditions features 83 targets exhibiting antagonistic expression in rice, out of which 12 genes, including four PLANTACYANINS (OsUCL6, 7, 9 and 30), PIRIN, OsLPR1, OsCHUP1, OsDOF12, OsBGLU1, glycine-rich cell wall gene, OsDUT, and OsERF7, are among the high confidence targets. Further, overexpression of MIR408 in drought-sensitive rice cultivar (PB1) leads to the massive enhancement of vegetative growth in rice with improved ETR and Y(II) and enhanced dehydration stress tolerance. The above results suggest that miR408 is likely to act as a positive regulator of growth and vigor, as well as dehydration stress, making it a potential candidate for engineering drought tolerance in rice.

Understanding the evolution of miRNA biogenesis machinery in plants with special focus on rice.

miRNA biogenesis process is an intricate and complex event consisting of many proteins working in a highly coordinated fashion. Most of these proteins have been studied in Arabidopsis; however, their orthologs and functions have not been explored in other plant species. In the present study, we have manually curated all the experimentally verified information present in the literature regarding these proteins and found a total of 98 genes involved in miRNA biogenesis in Arabidopsis. The conservation pattern of these proteins was identified in other plant species ranging from dicots to lower organisms, and we found that a major proportion of proteins involved in the pri-miRNA processing are conserved. However, nearly 20% of the genes, mostly involved in either transcription or functioning of the miRNAs, were absent in the lower organisms. Further, we manually curated a regulatory network of the core components of the biogenesis process and found that nearly half (46%) of the proteins interact with them, indicating that the processing step is perhaps the most under surveillance/regulation. We have subsequently attempted to characterize the orthologs identified in Oryza sativa, on the basis of transcriptome and epigenetic modifications under field drought conditions in order to assess the impact of drought on the process. We found several participating genes to be differentially expressed and/or epigenetically methylated under drought, although the core components like DCL1, SE, and HYL1 remain unaffected by the stress itself. The study enhances our present understanding of the biogenesis process and its regulation.

An Integrated Bioinformatics and Functional Approach for miRNA Validation.

MicroRNAs (miRNAs) are small (20-24 nucleotides) non-coding ribo-regulatory molecules with significant roles in regulating target mRNA and long non-coding RNAs at transcriptional and post-transcriptional levels. Rapid advancement in the small RNA sequencing methods with integration of degradome sequencing has accelerated the understanding of miRNA-mediated regulatory hubs in plants and yielded extensive annotation of miRNAs and corresponding targets. However, it is becoming clear that large numbers of such annotations are questionable. Therefore, it is imperative to adopt reliable and strict bioinformatics pipelines for miRNA identification. Furthermore, sensitive methods are needed for validation and functional characterization of miRNA and its target(s). In this chapter, we have provided a comprehensive and streamlined methodology for miRNA identification and its functional validation in plants. This includes a combination of various in silico and experimental methodologies. To identify miRNA compendium from large-scale Next-Generation Sequencing (NGS) small RNA datasets, the miR-PREFeR (miRNA PREdiction From small RNA-Seq data) bioinformatics tool has been described. Also, a homology-based search protocol for finding members of a specific miRNA family has been discussed. The chapter also includes techniques to ascertain miRNA:target pair specificity using in silico target prediction from degradome NGS libraries using CleaveLand pipeline, miRNA:target validation by in planta transient assays, 5' RLM-RACE and expression analysis as well as functional techniques like miRNA overexpression, short tandem target mimic and resistant target approaches. The proposed strategy offers a reliable and sensitive way for miRNA:target identification and validation. Additionally, we strongly promulgate the use of multiple methodologies to validate a miRNA as well as its target.

Cultivar-biased regulation of HSFA7 and HSFB4a govern high-temperature tolerance in tomato.

MAIN CONCLUSION: Cultivar-biased regulation of HSFB4a and HSFA7 mediates heat stress tolerance/sensitivity in tomato. Reduced HSFB4a repressor levels and enhanced HSFA7 activator levels govern thermo-tolerance in tolerant cultivars. Heat shock factors (HSFs) are at the core of heat stress (HS) response in plants. However, the contribution of HSFs governing the inherent thermo-tolerance mechanism in tomato from sub-tropical hot climates is poorly understood. With the above aim, comparative expression profiles of the HSF family in a HS-tolerant (CLN1621L) and -sensitive cultivars (CA4 and Pusa Ruby) of tomato under HS revealed cultivar-biased regulation of an activator (HSFA7) and a repressor (HSFB4a) class HSF. HSFA7 exhibited strong upregulation while HSFB4a showed downregulation in tolerant tomato cultivar upon HS. Functional characterization of HSFA7 and HSFB4a in a tolerant-sensitive cultivar pair by virus-induced gene silencing (VIGS)-based silencing and transient overexpression established them as a positive and a negative regulator of HS tolerance, respectively. Promoter:GUS reporter assays and promoter sequence analyses suggest heat-mediated transcriptional control of both the HSF genes in the contrasting cultivars. Moreover, degradome data highlighted HSFB4a is a probable target of microRNA Sly-miR4200. Transient in-planta Sly-MIR4200-effector:HSFB4a-reporter assays showed miRNA-dependent target down-regulation. Chelation of miRNA by short-tandem-target-mimic of Sly-miR4200 increased target abundance, highlighting a link between Sly-miR4200 and HSFB4a. This miRNA has induced several folds upon HS in the tolerant cultivar where HSFB4a levels are reduced, thus exhibiting the inverse miR:target expression. Thus, we speculate that the alleviation of HSFB4a and increased HSFA7 levels govern thermo-tolerance in the tolerant cultivar by regulating downstream heat stress-responsive genes.

Characterization of novel regulators for heat stress tolerance in tomato from Indian sub-continent.

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.

Novel insights into expansion and functional diversification of MIR169 family in tomato.

MAIN CONCLUSION: Expansion of MIR169 members by duplication and new mature forms, acquisition of new promoters, differential precursor-miRNA processivity and engaging novel targets increase the functional diversification of MIR169 in tomato. MIR169 family is an evolutionarily conserved miRNA family in plants. A systematic in-depth analysis of MIR169 family in tomato is lacking. We report 18 miR169 precursors, annotating new loci for MIR169a, b and d, as well as 3 novel mature isoforms (MIR169f/g/h). The family has expanded by both tandem- and segmental-duplication events during evolution. A tandem-pair MIR169b/b-1 and MIR169b-2/h is polycistronic in nature coding for three MIR169b isoforms and a new variant miR169h, that is evidently absent in the wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets including RNA-binding protein, protein-phosphatase, aminotransferase, chaperone, tetratricopeptide-repeat-protein, and transcription factors ARF-9B and SEPELLATA-3 were established by efficient target cleavage in the presence of specific precursors as well as increased target abundance upon miR169 chelation by short-tandem-target-mimic construct in transient assays. Comparative antagonistic expression profiles of MIR169:target pairs suggest MIR169 family as ubiquitous regulator of various abiotic stresses (heat, cold, dehydration and salt) and developmental pathways. This regulation is partly brought about by acquisition of new promoters as demonstrated by promoter MIR169:GUS reporter assays as well as differential processivity of different precursors and miRNA cleavage efficiencies. Thus, the current study augments the functional horizon of MIR169 family with applications for stress tolerance in crops.

Investigation into the miRNA/5' isomiRNAs function and drought-mediated miRNA processing in rice.

MicroRNAs lie at the core of biological regulatory networks in plants. The recent discovery of isomiRs that are length variants of the annotated mature miRNAs has further unveiled the complexity of miRNome. Delineation of their functional relevance is critical to understand the complete functional spectrum of the miRNome. To apprehend the role of 5' isomiRs in rice, we performed a comprehensive analysis of the annotated miRNA pool using 8 deep-sequencing datasets from flag leaf and spikelet tissues from two cultivars of rice viz. N22 and PB1 grown under control and drought conditions. The products of the 5' start site variability termed as "5' isomiRs" were found to be widespread in all the datasets. It was possible to identify several 5' isomiRs that were highly distinct and abundant and supported by more than 90% of the tags that map in the region. Majority of miRNA/5' isomiR pair share similar tissue and drought-mediated expression dynamics. Analysis of the degradome data identified targets for several of these 5' isomiRs, thereby confirming their biological activity. Since the isomiRs are length variants at the 5' end, the target sites were found to be accordingly shifted as compared to the target site of the annotated miRNA. Further we also observed that drought affects the processing accuracy of several miRNAs across all tissues of both the cultivars leading to differential accumulation of 5' isomiR/miRNA pair.

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

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.

Identification of novel miRNAs from drought tolerant rice variety Nagina 22.

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.

Unique miRNome during anthesis in drought-tolerant indica rice var. Nagina 22.

MAIN CONCLUSION: 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.

Evolution of variety-specific regulatory schema for expression of osa-miR408 in indica rice varieties under drought stress.

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.