Meta-QTL and ortho analysis unravels the genetic architecture and key candidate genes for cold tolerance at seedling stage in rice.

Rice, a critical cereal crop, grapples with productivity challenges due to its inherent sensitivity to low temperatures, primarily during the seedling and booting stages. Recognizing the polygenic complexity of cold stress signaling in rice, a meta-analysis was undertaken, focusing on 20 physiological traits integral to cold tolerance. This initiative allowed the consolidation of genetic data from 242 QTLs into 58 meta-QTLs, thereby significantly constricting the genetic and physical intervals, with 84% of meta-QTLs (MQTLs) being reduced to less than 2 Mb. The list of 10,505 genes within these MQTLs, was further refined utilizing expression datasets to pinpoint 46 pivotal genes exhibiting noteworthy differential regulation during cold stress. The study underscored the presence of several TFs such as WRKY, NAC, CBF/DREB, MYB, and bHLH, known for their roles in cold stress response. Further, ortho-analysis involving maize, barley, and Arabidopsis identified OsWRKY71, among others, as a prospective candidate for enhancing cold tolerance in diverse crop plants. In conclusion, our study delineates the intricate genetic architecture underpinning cold tolerance in rice and propounds significant candidate genes, offering crucial insights for further research and breeding strategies focused on fortifying crops against cold stress, thereby bolstering global food resilience. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01412-1.

The emerging role of epitranscriptome in shaping stress responses in plants.

KEY MESSAGE: RNA modifications and editing changes constitute 'epitranscriptome' and are crucial in regulating the development and stress response in plants. Exploration of the epitranscriptome and associated machinery would facilitate the engineering of stress tolerance in crops. RNA editing and modifications post-transcriptionally decorate almost all classes of cellular RNAs, including tRNAs, rRNAs, snRNAs, lncRNAs and mRNAs, with more than 170 known modifications, among which m6A, Ψ, m5C, 8-OHG and C-to-U editing are the most abundant. Together, these modifications constitute the "epitranscriptome", and contribute to changes in several RNA attributes, thus providing an additional structural and functional diversification to the "cellular messages" and adding another layer of gene regulation in organisms, including plants. Numerous evidences suggest that RNA modifications have a widespread impact on plant development as well as in regulating the response of plants to abiotic and biotic stresses. High-throughput sequencing studies demonstrate that the landscapes of m6A, m5C, Am, Cm, C-to-U, U-to-G, and A-to-I editing are remarkably dynamic during stress conditions in plants. GO analysis of transcripts enriched in Ψ, m6A and m5C modifications have identified bonafide components of stress regulatory pathways. Furthermore, significant alterations in the expression pattern of genes encoding writers, readers, and erasers of certain modifications have been documented when plants are grown in challenging environments. Notably, manipulating the expression levels of a few components of RNA editing machinery markedly influenced the stress tolerance in plants. We provide updated information on the current understanding on the contribution of RNA modifications in shaping the stress responses in plants. Unraveling of the epitranscriptome has opened new avenues for designing crops with enhanced productivity and stress resilience in view of global climate change.

Genome-wide identification, characterization, and expression analysis unveil the roles of pseudouridine synthase (PUS) family proteins in rice development and stress response.

Pseudouridylation, the conversion of uridine (U) to pseudouridine (Ñ°), is one of the most prevalent and evolutionary conserved RNA modifications, which is catalyzed by pseudouridine synthase (PUS) enzymes. Ñ°s play a crucial epitranscriptomic role by regulating attributes of cellular RNAs across diverse organisms. However, the precise biological functions of PUSs in plants remain largely elusive. In this study, we identified and characterized 21 members in the rice PUS family which were categorized into six distinct subfamilies, with RluA and TruA emerging as the most extensive. A comprehensive analysis of domain structures, motifs, and homology modeling revealed that OsPUSs possess all canonical features of true PUS proteins, essential for substrate recognition and catalysis. The exploration of OsPUS promoters revealed presence of cis-acting regulatory elements associated with hormone and abiotic stress responses. Expression analysis of OsPUS genes showed differential expression at developmental stages and under stress conditions. Notably, OsTruB3 displayed high expression in salt, heat, and drought stresses. Several OsRluA members showed induction in heat stress, while a significant decline in expression was observed for various OsTruA members in drought and salinity. Furthermore, miRNAs predicted to target OsPUSs were themselves responsive to variable stresses, adding an additional layer of regulatory complexity of OsPUSs. Study of protein-protein interaction networks provided substantial support for the potential regulatory role of OsPUSs in numerous cellular and stress response pathways. Conclusively, our study provides functional insights into the OsPUS family, contributing to a better understanding of their crucial roles in shaping the development and stress adaptation in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01396-4.

Know when and how to die: gaining insights into the molecular regulation of leaf senescence.

Senescence is the ultimate phase in the life cycle of leaves which is crucial for recycling of nutrients to maintain plant fitness and reproductive success. The earliest visible manifestation of leaf senescence is their yellowing, which usually commences with the breakdown of chlorophyll. The degradation process involves a gradual and highly coordinated disassembly of macromolecules resulting in the accumulation of nutrients, which are subsequently mobilized from the senescing leaves to the developing organs. Leaf senescence progresses under overly tight genetic and molecular control involving a well-orchestrated and intricate network of regulators that coordinate spatio-temporally with the influence of both internal and external cues. Owing to the advancements in omics technologies, the availability of mutant resources, scalability of molecular analyses methodologies and the advanced capacity to integrate multidimensional data, our understanding of the genetic and molecular basis of leaf ageing has greatly expanded. The review provides a compilation of the multitier regulation of senescence process and the interrelation between the environment and the terminal phase of leaf development. The knowledge gained would benefit in devising the strategies for manipulation of leaf senescence process to improve crop quality and productivity.

Abiotic stress-mediated modulation of the chromatin landscape in Arabidopsis thaliana.

Limited information is available on abiotic stress-mediated alterations of chromatin conformation influencing gene expression in plants. In order to characterize the effect of abiotic stresses on changes in chromatin conformation, we employed FAIRE-seq (formaldehyde-assisted isolation of regulatory element sequencing) and DNase-seq to isolate accessible regions of chromatin from Arabidopsis thaliana seedlings exposed to either heat, cold, salt, or drought stress. Approximately 25% of regions in the Arabidopsis genome were captured as open chromatin, the majority of which included promoters and exons. A large proportion of chromatin regions apparently did not change their conformation in response to any of the four stresses. Digital footprints present within these regions had differential enrichment of motifs for binding of 43 different transcription factors. Further, in contrast to drought and salt stress, both high and low temperature treatments resulted in increased accessibility of the chromatin. Also, pseudogenes attained increased chromatin accessibility in response to cold and drought stresses. The highly accessible and inaccessible chromatin regions of seedlings exposed to drought stress correlated with the Ser/Thr protein kinases (MLK1 and MLK2)-mediated reduction and increase in H3 phosphorylation (H3T3Ph), respectively. The presented results provide a deeper understanding of abiotic stress-mediated chromatin modulation in plants.

Arabidopsis Argonaute 2 regulates innate immunity via miRNA393(∗)-mediated silencing of a Golgi-localized SNARE gene, MEMB12.

Argonaute (AGO) proteins are critical components of RNA silencing pathways that bind small RNAs and mediate gene silencing at their target sites. We found that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Further genetic analysis demonstrated that AGO2 functions in antibacterial immunity. One abundant species of AGO2-bound small RNA is miR393b(∗), which targets a Golgi-localized SNARE gene, MEMB12. Pst infection downregulates MEMB12 in a miR393b(∗)-dependent manner. Loss of function of MEMB12, but not SYP61, another intracellular SNARE, leads to increased exocytosis of an antimicrobial pathogenesis-related protein, PR1. Overexpression of miR393b(∗) resembles memb12 mutant in resistance responses. Thus, AGO2 functions in antibacterial immunity by binding miR393b(∗) to modulate exocytosis of antimicrobial PR proteins via MEMB12. Since miR393 also contributes to antibacterial responses, miR393(∗)/miR393 represent an example of a miRNA(∗)/miRNA pair that functions in immunity through two distinct AGOs: miR393(∗) through AGO2 and miR393 through AGO1.

Class I and II Small Heat Shock Proteins Together with HSP101 Protect Protein Translation Factors during Heat Stress.

The ubiquitous small heat shock proteins (sHSPs) are well documented to act in vitro as molecular chaperones to prevent the irreversible aggregation of heat-sensitive proteins. However, the in vivo activities of sHSPs remain unclear. To investigate the two most abundant classes of plant cytosolic sHSPs (class I [CI] and class II [CII]), RNA interference (RNAi) and overexpression lines were created in Arabidopsis (Arabidopsis thaliana) and shown to have reduced and enhanced tolerance, respectively, to extreme heat stress. Affinity purification of CI and CII sHSPs from heat-stressed seedlings recovered eukaryotic translation elongation factor (eEF) 1B (α-, ß-, and γ-subunits) and eukaryotic translation initiation factor 4A (three isoforms), although the association with CI sHSPs was stronger and additional proteins involved in translation were recovered with CI sHSPs. eEF1B subunits became partially insoluble during heat stress and, in the CI and CII RNAi lines, showed reduced recovery to the soluble cell fraction after heat stress, which was also dependent on HSP101. Furthermore, after heat stress, CI sHSPs showed increased retention in the insoluble fraction in the CII RNAi line and vice versa. Immunolocalization revealed that both CI and CII sHSPs were present in cytosolic foci, some of which colocalized with HSP101 and with eEF1Bγ and eEF1Bß. Thus, CI and CII sHSPs have both unique and overlapping functions and act either directly or indirectly to protect specific translation factors in cytosolic stress granules.

Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea.

BACKGROUND: Brassica juncea var. Varuna is an economically important oilseed crop of family Brassicaceae which is vulnerable to abiotic stresses at specific stages in its life cycle. Till date no attempts have been made to elucidate genome-wide changes in its transcriptome against high temperature or drought stress. To gain global insights into genes, transcription factors and kinases regulated by these stresses and to explore information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de-novo assembly to discover B. juncea transcriptome associated with high temperature and drought stresses. RESULTS: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS). More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPdenovo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Many of these were responsive to high temperature, drought or both stresses. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families, respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. CONCLUSIONS: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resource generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity.

Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing. RESULTS: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions. CONCLUSIONS: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development.

Corrigendum: Integrated transcriptomics and miRNAomics provide insights into the complex multi-tiered regulatory networks associated with coleoptile senescence in rice.

[This corrects the article DOI: 10.3389/fpls.2022.985402.].

Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.

Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.

Integrated transcriptomics and miRNAomics provide insights into the complex multi-tiered regulatory networks associated with coleoptile senescence in rice.

Coleoptile is the small conical, short-lived, sheath-like organ that safeguards the first leaf and shoot apex in cereals. It is also the first leaf-like organ to senesce that provides nutrition to the developing shoot and is, therefore, believed to play a crucial role in seedling establishment in rice and other grasses. Though histochemical studies have helped in understanding the pattern of cell death in senescing rice coleoptiles, genome-wide expression changes during coleoptile senescence have not yet been explored. With an aim to investigate the gene regulation underlying the coleoptile senescence (CS), we performed a combinatorial whole genome expression analysis by sequencing transcriptome and miRNAome of senescing coleoptiles. Transcriptome analysis revealed extensive reprogramming of 3439 genes belonging to several categories, the most prominent of which encoded for transporters, transcription factors (TFs), signaling components, cell wall organization enzymes, redox homeostasis, stress response and hormone metabolism. Small RNA sequencing identified 41 known and 21 novel miRNAs that were differentially expressed during CS. Comparison of gene expression and miRNA profiles generated for CS with publicly available leaf senescence (LS) datasets revealed that the two aging programs are remarkably distinct at molecular level in rice. Integration of expression data of transcriptome and miRNAome identified high confidence 140 miRNA-mRNA pairs forming 42 modules, thereby demonstrating multi-tiered regulation of CS. The present study has generated a comprehensive resource of the molecular networks that enrich our understanding of the fundamental pathways regulating coleoptile senescence in rice.

Long-read-based draft genome sequence of Indian black gram IPU-94-1 'Uttara': Insights into disease resistance and seed storage protein genes.

Black gram [Vigna mungo (L.) Hepper var. mungo] is a warm-season legume highly prized for its protein content along with significant folate and iron proportions. To expedite the genetic enhancement of black gram, a high-quality draft genome from the center of origin of the crop is indispensable. Here, we established a draft genome sequence of an Indian black gram cultivar, 'Uttara' (IPU 94-1), known for its high resistance to mungbean yellow mosaic virus. Pacific Biosciences of California, Inc. (PacBio) single-molecule real-time (SMRT) and Illumina sequencing assembled a draft reference-guided assembly with a cumulative size of ∼454.4 Mb, of which, 444.4 Mb was anchored on 11 pseudomolecules corresponding to 11 chromosomes. Uttara assembly denotes features of a high-quality draft genome illustrated through high N50 value (42.88 Mb), gene completeness (benchmarking universal single-copy ortholog [BUSCO] score 94.17%) and low levels of ambiguous nucleotides (N) percent (0.0005%). Gene discovery using transcript evidence predicted 28,881 protein-coding genes, from which, ∼95% were functionally annotated. A global survey of genes associated with disease resistance revealed 119 nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, while 23 genes encoding seed storage proteins (SSPs) were discovered in black gram. A large set of microsatellite loci were discovered for marker development in the crop. Our draft genome of an Indian black gram provides the foundational genomic resources for the improvement of important agronomic traits and ultimately will help in accelerating black gram breeding programs.

Northern Blotting Technique for Detection and Expression Analysis of mRNAs and Small RNAs.

Northern analysis is a conventional but gold standard method for detection and quantification of gene expression changes. It not only detects the presence of a transcript but also indicates size and relative comparison of transcript abundance on a single membrane. In recent years it has been aptly adapted to validate and study the size and expression of small noncoding RNAs. Here, we describe protocols employed in our laboratory for conventional northern analysis with total RNA/mRNA to study gene expression and validation of small noncoding RNAs using low molecular weight fraction of RNAs. A brief account on the recent advancements for improving the sensitivity and efficiency of northern blot detection is also included in this chapter.

Discovery of pathogen-regulated small RNAs in plants.

Small RNAs have emerged as one of the most important regulators for gene expression in eukaryotes. Small RNA-mediated gene silencing has been shown to play an essential role in antiviral defense in both plant and animal systems (Li and Ding, 2005; Voinnet, 2005; Wang et al., 2006). These viral RNA-generated small interfering RNAs (siRNAs) are extragenomic in origin. Studies from our lab and others suggest that host-endogenous small RNAs also play an important role in plant defense in response to other pathogens besides viruses (Katiyar-Agarwal et al., 2006; Navarro et al., 2006). The methods described here provide an opportunity to identify many more novel pathogen-regulated small RNAs in plants, which will help in understanding the regulatory mechanism of plant immunity. Here, we introduce the approaches of powerful high-throughput parallel sequencing and hybridization-based technologies for the discovery and detection of pathogen-regulated small RNAs. We mainly compare and discuss the methods of low-molecular-weight (LMW) RNA extraction from pathogen-infected tissue and strategies for detecting endogenous small RNAs by Northern blot analysis.

Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress.

Tetraspanin family is comprised of evolutionarily conserved integral membrane proteins. The incredible ability of tetraspanins to form 'micro domain complexes' and their preferential targeting to membranes emphasizes their active association with signal recognition and communication with neighboring cells, thus acting as key modulators of signaling cascades. In animals, tetraspanins are associated with multitude of cellular processes. Unlike animals, the biological relevance of tetraspanins in plants has not been well investigated. In Arabidopsis tetraspanins are known to contribute in important plant development processes such as leaf morphogenesis, root, and floral organ formation. In the present study we investigated the genomic organization, chromosomal distribution, phylogeny and domain structure of 15 rice tetraspanin proteins (OsTETs). OsTET proteins had similar domain structure and signature 'GCCK/R' motif as reported in Arabidopsis. Comprehensive expression profiling of OsTET genes suggested their possible involvement during rice development. While OsTET9 and 10 accumulated predominantly in flowers, OsTET5, 8, and 12 were preferentially expressed in root tissues. Noticeably, seven OsTETs exhibited more than twofold up regulation at early stages of flag leaf senescence in rice. Furthermore, several OsTETs were differentially regulated in rice seedlings exposed to abiotic stresses, exogenous treatment of hormones and nutrient deprivation. Transient subcellular localization studies of eight OsTET proteins in tobacco epidermal cells showed that these proteins localized in plasma membrane. The present study provides valuable insights into the possible roles of tetraspanins in regulating development and defining response to abiotic stresses in rice. Targeted proteomic studies would be useful in identification of their interacting partners under different conditions and ultimately their biological function in plants.

De novo transcriptome profiling of cold-stressed siliques during pod filling stages in Indian mustard (Brassica juncea L.).

Low temperature is a major abiotic stress that impedes plant growth and development. Brassica juncea is an economically important oil seed crop and is sensitive to freezing stress during pod filling subsequently leading to abortion of seeds. To understand the cold stress mediated global perturbations in gene expression, whole transcriptome of B. juncea siliques that were exposed to sub-optimal temperature was sequenced. Manually self-pollinated siliques at different stages of development were subjected to either short (6 h) or long (12 h) durations of chilling stress followed by construction of RNA-seq libraries and deep sequencing using Illumina's NGS platform. De-novo assembly of B. juncea transcriptome resulted in 133,641 transcripts, whose combined length was 117 Mb and N50 value was 1428 bp. We identified 13,342 differentially regulated transcripts by pair-wise comparison of 18 transcriptome libraries. Hierarchical clustering along with Spearman correlation analysis identified that the differentially expressed genes segregated in two major clusters representing early (5-15 DAP) and late stages (20-30 DAP) of silique development. Further analysis led to the discovery of sub-clusters having similar patterns of gene expression. Two of the sub-clusters (one each from the early and late stages) comprised of genes that were inducible by both the durations of cold stress. Comparison of transcripts from these clusters led to identification of 283 transcripts that were commonly induced by cold stress, and were referred to as "core cold-inducible" transcripts. Additionally, we found that 689 and 100 transcripts were specifically up-regulated by cold stress in early and late stages, respectively. We further explored the expression patterns of gene families encoding for transcription factors (TFs), transcription regulators (TRs) and kinases, and found that cold stress induced protein kinases only during early silique development. We validated the digital gene expression profiles of selected transcripts by qPCR and found a high degree of concordance between the two analyses. To our knowledge this is the first report of transcriptome sequencing of cold-stressed B. juncea siliques. The data generated in this study would be a valuable resource for not only understanding the cold stress signaling pathway but also for introducing cold hardiness in B. juncea.

A comprehensive genome-wide study on tissue-specific and abiotic stress-specific miRNAs in Triticum aestivum.

Productivity of wheat crop is largely dependent on its growth and development that, in turn, is mainly regulated by environmental conditions, including abiotic stress factors. miRNAs are key regulators of gene expression networks involved in diverse aspects of development and stress responses in plants. Using high-throughput sequencing of eight small RNA libraries prepared from diverse abiotic stresses and tissues, we identified 47 known miRNAs belonging to 20 families, 49 true novel and 1030 candidate novel miRNAs. Digital gene expression analysis revealed that 257 miRNAs exhibited tissue-specific expression and 74 were associated with abiotic stresses. Putative target genes were predicted for miRNAs identified in this study and their grouping into functional categories indicated that the putative targets were involved in diverse biological processes. RLM-RACE of predicted targets of three known miRNAs (miR156, miR160 and miR164) confirmed their mRNA cleavage, thus indicating their regulation at post-transcriptional level by the corresponding miRNAs. Mapping of the sequenced data onto the wheat progenitors and closely related monocots revealed a large number of evolutionary conserved miRNAs. Additional expression profiling of some of these miRNAs in other abiotic stresses underline their involvement in multiple stresses. Our findings provide valuable resource for an improved understanding of the role of miRNAs in stress tolerance as well as plant development.

Identification and characterization of Wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing.

Chickpea (Cicer arietinum) is the second most widely grown legume worldwide and is the most important pulse crop in the Indian subcontinent. Chickpea productivity is adversely affected by a large number of biotic and abiotic stresses. MicroRNAs (miRNAs) have been implicated in the regulation of plant responses to several biotic and abiotic stresses. This study is the first attempt to identify chickpea miRNAs that are associated with biotic and abiotic stresses. The wilt infection that is caused by the fungus Fusarium oxysporum f.sp. ciceris is one of the major diseases severely affecting chickpea yields. Of late, increasing soil salinization has become a major problem in realizing these potential yields. Three chickpea libraries using fungal-infected, salt-treated and untreated seedlings were constructed and sequenced using next-generation sequencing technology. A total of 12,135,571 unique reads were obtained. In addition to 122 conserved miRNAs belonging to 25 different families, 59 novel miRNAs along with their star sequences were identified. Four legume-specific miRNAs, including miR5213, miR5232, miR2111 and miR2118, were found in all of the libraries. Poly(A)-based qRT-PCR (Quantitative real-time PCR) was used to validate eleven conserved and five novel miRNAs. miR530 was highly up regulated in response to fungal infection, which targets genes encoding zinc knuckle- and microtubule-associated proteins. Many miRNAs responded in a similar fashion under both biotic and abiotic stresses, indicating the existence of cross talk between the pathways that are involved in regulating these stresses. The potential target genes for the conserved and novel miRNAs were predicted based on sequence homologies. miR166 targets a HD-ZIPIII transcription factor and was validated by 5' RLM-RACE. This study has identified several conserved and novel miRNAs in the chickpea that are associated with gene regulation following exposure to wilt and salt stress.

A genome-wide perspective of miRNAome in response to high temperature, salinity and drought stresses in Brassica juncea (Czern) L.

Micro RNAs (miRNAs) are involved in diverse biological processes including adaptive response towards abiotic stresses. To unravel small RNAs and more specifically miRNAs that can potentially regulate determinants of abiotic stress tolerance, next generation sequencing of B. juncea seedlings subjected to high temperature, high salt and drought conditions was carried out. With the help of UEA sRNA workbench software package, 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions.