Evolutionary insights into 3D genome organization and epigenetic landscape of Vigna mungo.

Eukaryotic genomes show an intricate three-dimensional (3D) organization within the nucleus that regulates multiple biological processes including gene expression. Contrary to animals, understanding of 3D genome organization in plants remains at a nascent stage. Here, we investigate the evolution of 3D chromatin architecture in legumes. By using cutting-edge PacBio, Illumina, and Hi-C contact reads, we report a gap-free, chromosome-scale reference genome assembly of Vigna mungo, an important minor legume cultivated in Southeast Asia. We spatially resolved V. mungo chromosomes into euchromatic, transcriptionally active A compartment and heterochromatic, transcriptionally-dormant B compartment. We report the presence of TAD-like-regions throughout the diagonal of the HiC matrix that resembled transcriptional quiescent centers based on their genomic and epigenomic features. We observed high syntenic breakpoints but also high coverage of syntenic sequences and conserved blocks in boundary regions than in the TAD-like region domains. Our findings present unprecedented evolutionary insights into spatial 3D genome organization and epigenetic patterns and their interaction within the V. mungo genome. This will aid future genomics and epigenomics research and breeding programs of V. mungo.

Transcriptome analysis and differential expression in Arabidopsis thaliana in response to rohitukine (a chromone alkaloid) treatment.

Rohitukine is a chromone alkaloid and precursor of potent anticancer drugs flavopiridol, P-276-00, and 2,6-dichloro-styryl derivative (11d) (IIIM-290). The metabolite is reported to possess anticancer, anti-inflammatory, antiadipogenic, immunomodulatory, gastroprotective, anti-implantation, antidyslipidemic, anti-arthritic, and anti-fertility properties. However, the physiological role of rohitukine in plant system is yet to be explored. Here, we studied the effect of rohitukine isolated from Dysoxylum gotadhora on Arabidopsis thaliana. The A. thaliana plants grown on a medium fortified with different rohitukine concentrations showed a significant effect on the growth and development. The root growth of A. thaliana seedlings showed considerable inhibition when grown on medium containing 1.0 mM of rohitukine. Transcriptomic analysis indicated the expression of 895 and 932 genes in control and treated samples respectively at a cut-off of FPKM ≥ 1 and P-value < 0.05. Gene ontology (GO) analysis revealed the upregulation of genes related to photosynthesis, membrane transport, antioxidation, xenobiotic degradation, and some transcription factors (TFs) in response to rohitukine. Conversely, rohitukine downregulated several genes including RNA helicases and those involved in nitrogen compound metabolism. The RNA-seq result was also validated by real-time qRT-PCR analysis. In light of these results, we discuss (i) likely ecological importance of rohitukine in parent plant as well as (ii) comparison between responses to rohitukine treatment in plants and mammals.

Integrated genomic approaches delineate a novel role of ROP1 ENHANCER1 in controlling seed protein content of chickpea.

Utilizing a combinatorial approach of quantitative trait locus (QTL)-Seq and candidate gene-based association mapping, the QTLs and genes responsible for seed protein content (SPC), a major quality trait in chickpea, were identified. Whole genome re-sequencing based QTL-Seq analysis of bulked recombinant inbred lines from a mapping population contrasting for SPC led to the identification of two QTLs [0.94 Mb on Linkage Group (LG)5 and 1.16 Mb on LG6] encompassing three SNPs, displaying the highest ΔSNP index. These highly significant SNPs and their associated genes were validated in 211 chickpea mini-core accessions varying in SPC, revealing a tightly associated marker affecting CaREN1 (ROP1 ENHANCER1) and explaining a phenotypic variation of 23%. This SNP was subsequently converted into a cost effective allele-specific PCR-based marker that could be utilized for rapid screening of SPC during marker assisted breeding. Furthermore, in planta functional validation via knockdown of CaREN1 transcripts led to significant reduction in SPC of chickpea. This decrease in seed protein is likely due to disruption in the formation of CaREN1 protein complexes comprising chaperones, phosphopeptide-binding proteins, and GTPases that mediate folding, transport and accumulation of seed storage proteins, as indicated through affinity purification-mass spectrometry. Taken together, our data will expedite tailoring of chickpea cultivars with augmented SPC.

Evolutionary fates of gene-body methylation and its divergent association with gene expression in pigeonpea.

Pigeonpea (Cajanus cajan L. Huth) is an agronomically important legume cultivated worldwide. In this study, we extensively analyzed gene-body methylation (GbM) patterns in pigeonpea. We found a bimodal distribution of CG and CHG methylation patterns. GbM features- slow evolution rate and increased length remained conserved. Genes with moderate CG body methylation showed highest expression where as highly-methylated genes showed lowest expression. Transposable element (TE)-related genes were methylated in multiple contexts and hence classified as C-methylated genes. A low expression among C-methylated genes was associated with transposons insertion in gene-body and upstream regulatory regions. The CG methylation patterns were found to be conserved in orthologs compared with non-CG methylation. By comparing methylation patterns between differentially methylated regions (DMRs) of the three genotypes, we found that variably methylated marks are less likely to target evolutionary conserved sequences. Finally, our analysis showed enrichment of nitrogen-related genes in GbM orthologs of legumes, which could be promising candidates for generating epialleles for crop improvement.

MEDIATOR SUBUNIT17 integrates jasmonate and auxin signaling pathways to regulate thermomorphogenesis.

Plant adjustment to environmental changes involves complex crosstalk between extrinsic and intrinsic cues. In the past two decades, extensive research has elucidated the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and the phytohormone auxin in thermomorphogenesis. In this study, we identified a previously unexplored role of jasmonate (JA) signaling components, the Mediator complex, and their integration with auxin signaling during thermomorphogenesis in Arabidopsis (Arabidopsis thaliana). Warm temperature induces expression of JA signaling genes including MYC2, but, surprisingly, this transcriptional activation is not JA dependent. Warm temperature also promotes accumulation of the JA signaling receptor CORONATINE INSENSITIVE1 (COI1) and degradation of the JA signaling repressor JASMONATE-ZIM-DOMAIN PROTEIN9, which probably leads to de-repression of MYC2, enabling it to contribute to the expression of MEDIATOR SUBUNIT17 (MED17). In response to warm temperature, MED17 occupies the promoters of thermosensory genes including PIF4, YUCCA8 (YUC8), INDOLE-3-ACETIC ACID INDUCIBLE19 (IAA19), and IAA29. Moreover, MED17 facilitates enrichment of H3K4me3 on the promoters of PIF4, YUC8, IAA19, and IAA29 genes. Interestingly, both occupancy of MED17 and enrichment of H3K4me3 on these thermomorphogenesis-related promoters are dependent on PIF4 (or PIFs). Altered accumulation of COI1 under warm temperature in the med17 mutant suggests the possibility of a feedback mechanism. Overall, this study reveals the role of the Mediator complex as an integrator of JA and auxin signaling pathways during thermomorphogenesis.

Expressivity of the key genes associated with seed and pod development is highly regulated via lncRNAs and miRNAs in Pigeonpea.

Non-coding RNA's like miRNA, lncRNA, have gained immense importance as a significant regulatory factor in different physiological and developmental processes in plants. In an effort to understand the molecular role of these regulatory agents, in the present study, 3019 lncRNAs and 227 miRNAs were identified from different seed and pod developmental stages in Pigeonpea, a major grain legume of Southeast Asia and Africa. Target analysis revealed that 3768 mRNAs, including 83 TFs were targeted by lncRNAs; whereas 3060 mRNA, including 154 TFs, were targeted by miRNAs. The targeted transcription factors majorly belong to WRKY, MYB, bHLH, etc. families; whereas the targeted genes were associated with the embryo, seed, and flower development. Total 302 lncRNAs interact with miRNAs and formed endogenous target mimics (eTMs) which leads to sequestering of the miRNAs present in the cell. Expression analysis showed that notably, Cc_lncRNA-2830 expression is up-regulated and sequestrates miR160h in pod leading to higher expression of the miR160h target gene, Auxin responsive factor-18. A similar pattern was observed for SPIKE, Auxin signaling F-box-2, Bidirectional sugar transporter, and Starch synthetase-2 eTMs. All the identified target mRNAs code for transcription factor and genes are involved in the processes like cell division, plant growth and development, starch synthesis, sugar transportation and accumulation of storage proteins which are essential for seed and pod development. On a combinatorial basis, our study provides a lncRNA and miRNA based regulatory insight into the genes governing seed and pod development in Pigeonpea.

Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene TaHsp90 in Indian wheat (Triticum aestivum L.) cv C306.

To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to Hsp90 was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of TaHsp90 (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of TaHsp90 was validated in E. coli BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene TaHsp90 isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.

Unravelling the epigenomic interactions between parental inbreds resulting in an altered hybrid methylome in pigeonpea.

DNA methylation is an important heritable landmark conferring epigenetic changes in hybrids and has fascinated biologists and plant-breeders over the years. Although epigenetic changes have been documented in rice and maize hybrids, such investigations have not been reported in pigeonpea. Here, we report genome-wide methylation profiles of pigeonpea sterile and fertile inbred lines and their fertile F1 hybrid at single base resolution. We found that pigeonpea genome is relatively enriched in CG methylation. Identification of differentially methylated regions (DMRs) in the sterile and fertile parent revealed remarkable differences between their methylation patterns. Investigation of methylation status of parental DMRs in hybrid revealed non-additive methylation patterns resulting from trans-chromosomal methylation and trans-chromosomal demethylation events. Furthermore, we discovered several DMRs negatively associated with gene expression in the hybrid and fertile parent. Interestingly, many of those DMRs belonged to transposable elements and genes encoding pentatricopeptide repeats associated proteins, which may mediate a role in modulating the genes impacting pollen fertility. Overall, our findings provide an understanding of two parental epigenomes interacting to give rise to an altered methylome in pigeonpea hybrids, from genome-wide point of view.