Small RNA profiling from meiotic and post-meiotic anthers reveals prospective miRNA-target modules for engineering male fertility in sorghum.

Understanding male gametophyte development is essential to augment hybrid production in sorghum. Although small RNAs are known to critically influence anther/pollen development, their roles in sorghum reproduction have not been deciphered yet. Here, we report small RNA profiling and high-confidence annotation of microRNAs (miRNAs) from meiotic and post-meiotic anthers in sorghum. We identified 262 miRNAs (82 known and 180 novel), out of which 58 (35 known and 23 novel) exhibited differential expression between two stages. Out of 35 differentially expressed known miRNAs, 13 are known to regulate anther/pollen development in other plant species. We also demonstrated conserved spatiotemporal patterns of 21- and 24-nt phasiRNAs and their respective triggers, miR2118 and miR2275, in sorghum anthers as evidenced in other monocots. miRNA target identification yielded 5622 modules, of which 46 modules comprising 16 known and 8 novel miRNA families with 38 target genes are prospective candidates for engineering male fertility in grasses.

Comparative transcriptome analysis in different tissues of a medicinal herb, Picrorhiza kurroa pinpoints transcription factors regulating picrosides biosynthesis.

Transcriptional regulation of picrosides biosynthesis, the iridoid glycosides of an endangered medicinal herb, Picrorhiza kurroa, is completely unknown. P. kurroa plants obtained from natural habitat accumulate higher picrosides than in-vitro cultured plants, which necessitates identification of transcription factors (TFs) regulating their differential biosynthesis. The current study investigates complete spectrum of different TF classes in P. kurroa transcriptomes and discerns their association with picrosides biosynthesis. Transcriptomes of differential picroside-I content shoots and picroside-II content roots were mined for seven classes of TFs implicated in secondary metabolism regulation in plants. Key TFs were identified through in silico transcript abundance and qPCR analysis was performed to confirm transcript levels of TFs under study in differential content tissues and genotypes. Promoter regions of key picrosides biosynthetic pathway genes were explored to hypothesize which TFs can possibly regulate target genes. A total of 131, 137, 107, 82 and 101 transcripts encoding different TFs families were identified in PKS-25, PKS-15, PKSS, PKR-25 and PKSR transcriptomes, respectively. ERF-18, bHLH-104, NAC-25, 32, 94 and SUF-4 showed elevated expression in roots (up to 37 folds) and shoots (up to 195 folds) of plants obtained from natural habitat, indicating their role as activators of picrosides biosynthesis whereas, elevated expression of WRKY-17, 40, 71 and MYB-4 in low picrosides content conditions suggested their down-regulatory role. In silico analysis of key picrosides biosynthetic pathway gene promoter regions revealed binding domains for ERF-18, NAC-25, WRKY-40 and MYB-4. Identification of candidate TFs contributing towards picrosides biosynthesis is a pre-requisite for designing appropriate metabolic engineering strategies aimed at enhancing picrosides content in vitro and in vivo.

Mining NGS transcriptomes for miRNAs and dissecting their role in regulating growth, development, and secondary metabolites production in different organs of a medicinal herb, Picrorhiza kurroa.

MAIN CONCLUSION: This study is the first endeavor on mining of miRNAs and analyzing their involvement in development and secondary metabolism of an endangered medicinal herb Picrorhiza kurroa (P. kurroa ). miRNAs are ubiquitous non-coding RNA species that target complementary sequences of mRNA and result in either translational repression or target degradation in eukaryotes. The role of miRNAs has not been investigated in P. kurroa which is a medicinal herb of industrial value due to the presence of secondary metabolites, picroside-I and picroside-II. Computational identification of miRNAs was done in 6 transcriptomes of P. kurroa generated from root, shoot, and stolon organs varying for growth, development, and culture conditions. All available plant miRNA entries were retrieved from miRBase and used as backend datasets to computationally identify conserved miRNAs in transcriptome data sets. Total 18 conserved miRNAs were detected in P. kurroa followed by target prediction and functional annotation which suggested their possible role in controlling various biological processes. Validation of miRNA and expression analysis by qRT-PCR and 5' RACE revealed that miRNA-4995 has a regulatory role in terpenoid biosynthesis ultimately affecting the production of picroside-I. miR-5532 and miR-5368 had negligible expression in field-grown samples as compared to in vitro-cultured samples suggesting their role in regulating P. kurroa growth in culture conditions. The study has thus identified novel functions for existing miRNAs which can be further validated for their potential regulatory role.

Non-coding RNAs-mediated environmental surveillance determines male fertility in plants.

Plants are continuously exposed to environmental stresses leading to significant yield losses. With the changing climatic conditions, the intensity and duration of these stresses are expected to increase, posing a severe threat to crop productivity worldwide. Male gametogenesis is one of the most sensitive developmental stages. Exposure to environmental stresses during this stage leads to male sterility and yield loss. Elucidating the underlying molecular mechanism of environment-affected male sterility is essential to address this challenge. High-throughput RNA sequencing studies, loss-of-function phenotypes of sRNA biogenesis genes and functional genomics studies with non-coding RNAs have started to unveil the roles of small RNAs, long non-coding RNAs and the complex regulatory interactions between them in regulating male fertility under different growth regimes. Here, we discuss the current understanding of the non-coding RNA-mediated environmental stress surveillance and regulation of male fertility in plants. The candidate ncRNAs emerging from these studies can be leveraged to generate environment-sensitive male sterile lines for hybrid breeding or mitigate the impact of climate change on male fertility, as the situation demands.

Comprehensive phylogenomic analysis of ERF genes in sorghum provides clues to the evolution of gene functions and redundancy among gene family members.

APETALA2/Ethylene-Responsive transcription factors (AP2/ERF), with their multifunctional roles in plant development, hormone signaling and stress tolerance, are important candidates for engineering crop plants. Here, we report identification and analysis of gene structure, phylogenetic distribution, expression, chromosomal localization and cis-acting promoter analysis of AP2/ERF genes in the C4 crop plant sorghum. We identified 158 ERF genes in sorghum with 52 of them encoding dehydration-responsive binding elements (DREB) while 106 code for ERF subfamily proteins. Phylogenetic analysis organized sorghum ERF proteins into 11 distinct groups exhibiting clade-specific expansion. About 68% ERF genes have paralogs indicating gene duplications as major cause of expansion of ERF family in sorghum. Analysis of spatiotemporal expression patterns using publicly available data revealed their tissue/genotype-preferential accumulation. In addition, 40 ERF genes exhibited differential accumulation in response to heat and/or drought stress. About 25% of the segmental gene pairs and eleven tandem duplicated genes exhibited high correlation (> 0.7) in their expression patterns indicating genetic redundancy. Comparative phylogenomic analysis of sorghum ERFs with 74 genetically characterized ERF genes from other plant species provided significant clues to sorghum ERF functions. Overall data generated here provides an overview of evolutionary relationship among ERF gene family members in sorghum and with respect to previously characterized ERF genes from other plant species. This information will be instrumental in initiating functional genomic studies of ERF candidates in sorghum.

Transcriptional trajectories of anther development provide candidates for engineering male fertility in sorghum.

Sorghum is a self-pollinated crop with multiple economic uses as cereal, forage, and biofuel feedstock. Hybrid breeding is a cornerstone for sorghum improvement strategies that currently relies on cytoplasmic male sterile lines. To engineer genic male sterility, it is imperative to examine the genetic components regulating anther/pollen development in sorghum. To this end, we have performed transcriptomic analysis from three temporal stages of developing anthers that correspond to meiotic, microspore and mature pollen stages. A total of 5286 genes were differentially regulated among the three anther stages with 890 of them exhibiting anther-preferential expression. Differentially expressed genes could be clubbed into seven distinct developmental trajectories using K-means clustering. Pathway mapping revealed that genes involved in cell cycle, DNA repair, regulation of transcription, brassinosteroid and auxin biosynthesis/signalling exhibit peak expression in meiotic anthers, while those regulating abiotic stress, carbohydrate metabolism, and transport were enriched in microspore stage. Conversely, genes associated with protein degradation, post-translational modifications, cell wall biosynthesis/modifications, abscisic acid, ethylene, cytokinin and jasmonic acid biosynthesis/signalling were highly expressed in mature pollen stage. High concurrence in transcriptional dynamics and cis-regulatory elements of differentially expressed genes in rice and sorghum confirmed conserved developmental pathways regulating anther development across species. Comprehensive literature survey in conjunction with orthology analysis and anther-preferential accumulation enabled shortlisting of 21 prospective candidates for in-depth characterization and engineering male fertility in sorghum.