Arabidopsis BECLIN1-induced autophagy mediates reprogramming in tapetal programmed cell death by altering the gross cellular homeostasis.

In flowering plants, the tapetum degeneration in post-meiotic anther occurs through developmental programmed cell death (dPCD), which is one of the most critical and sensitive steps for the proper development of male gametophytes and fertility. Yet the pathways of dPCD, its regulation, and its interaction with autophagy remain elusive. Here, we report that high-level expression of Arabidopsis autophagy-related gene BECLIN1 (BECN1 or AtATG6) in the tobacco tapetum prior to their dPCD resulted in developmental defects. BECN1 induces severe autophagy and multiple cytoplasm-to-vacuole pathways, which alters tapetal cell reactive oxygen species (ROS)-homeostasis that represses the tapetal dPCD. The transcriptome analysis reveals that BECN1- expression caused major changes in the pathway, resulting in altered cellular homeostasis in the tapetal cell. Moreover, BECN1-mediated autophagy reprograms the execution of tapetal PCD by altering the expression of the key developmental PCD marker genes: SCPL48, CEP1, DMP4, BFN1, MC9, EXI1, and Bcl-2 member BAG5, and BAG6. This study demonstrates that BECN1-mediated autophagy is inhibitory to the dPCD of the tapetum, but the severity of autophagy leads to autophagic death in the later stages. The delayed and altered mode of tapetal degeneration resulted in male sterility.

Histone deacetylase 9 interacts with SiHAT3.1 and SiHDA19 to repress dehydration responses through H3K9 deacetylation in foxtail millet.

Climate change inflicts several stresses on plants, of which dehydration stress severely affects growth and productivity. C4 plants possess better adaptability to dehydration stress; however, the role of epigenetic modifications underlying this trait is unclear. In particular, the molecular links between histone modifiers and their regulation remain elusive. In this study, genome-wide H3K9 acetylation (H3K9ac) enrichment using ChIP-sequencing was performed in two foxtail millet cultivars with contrasting dehydration tolerances (IC403579, cv. IC4-tolerant, and IC480117, cv. IC41-sensitive). It revealed that a histone deacetylase, SiHDA9, was significantly up-regulated in the sensitive cultivar. Further characterization indicated that SiHDA9 interacts with SiHAT3.1 and SiHDA19 to form a repressor complex. SiHDA9 might be recruited through the SiHAT3.1 recognition sequence onto the upstream of dehydration-responsive genes to decrease H3K9 acetylation levels. The silencing of SiHDA9 resulted in the up-regulation of crucial genes, namely, SiRAB18, SiRAP2.4, SiP5CS2, SiRD22, SiPIP1;4, and SiLHCB2.3, which imparted dehydration tolerance in the sensitive cultivar (IC41). Overall, the study provides mechanistic insights into SiHDA9-mediated regulation of dehydration stress response in foxtail millet.

Reducing the biosynthesis of condensed tannin in winged bean (Psophocarpus tetragonolobus (L.) DC.) by virus-induced gene silencing of anthocyanidin synthase (ANS) gene.

The Underutilized legume-winged bean (Psophocarpus tetragonolobus (L.) DC.) and its various parts are infested with condensed tannin (CT) or proanthocyanidin (PA). CT has anti-nutritional effect as it adversely affects the digestion of proteins, minerals and vitamin among ruminants and humans. It is also responsible for low protein digestibility and decreased amino acid availability. One of the probable reasons of underutilization of P. tetragonolobus is due to its infestation with CT. Histochemical staining of various tissues of P. tetragonolobus with dimethylcinnmaldehyde (DMACA) developed a deep-blue colour indicating the presence of polyphenolic condensed tannin. Structural monomeric unit catechin and epi-catechin were reported to be responsible for biosynthesis of CT in P. tetragonolobus. The enzyme anthocyanidin synthase (ANS) and its corresponding transcripts were identified and phylogenetically mapped. The transcript was subjected to virus-induced gene silencing (VIGS) through agro-infiltration in P. tetragonolobus for reducing the CT-content. The WbANS-VIGS induced P. tetragonolobus resulted in four-fold decrease of CT as compared to the control P. tetragonolobus. A decrease of 73% of CT level was reported in VIGS silenced Wb-ANS line of P. tetragonolobus. This study resulted and confirmed that, the silencing of (ANS) gene in P. tetragonolobus has a regulatory effect on the condensed tannin biosynthesis. This study will pave way for further manipulation of ANS enzyme for reducing the biosynthesis of the anti-nutrient CT. Reducing the CT content will make this underutilized legume more acceptable. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03435-5.

Histone acetylation dynamics regulating plant development and stress responses.

Crop productivity is directly dependent on the growth and development of plants and their adaptation during different environmental stresses. Histone acetylation is an epigenetic modification that regulates numerous genes essential for various biological processes, including development and stress responses. Here, we have mainly discussed the impact of histone acetylation dynamics on vegetative growth, flower development, fruit ripening, biotic and abiotic stress responses. Besides, we have also emphasized the information gaps which are obligatory to be examined for understanding the complete role of histone acetylation dynamics in plants. A comprehensive knowledge about the histone acetylation dynamics will ultimately help to improve stress resistance and reduce yield losses in different crops due to climate changes.

Stress Conditions Modulate the Chromatin Interactions Network in Arabidopsis.

Stresses have been known to cause various responses like cellular physiology, gene regulation, and genome remodeling in the organism to cope and survive. Here, we assessed the impact of stress conditions on the chromatin-interactome network of Arabidopsis thaliana. We identified thousands of chromatin interactions in native as well as in salicylic acid treatment and high temperature conditions in a genome-wide fashion. Our analysis revealed the definite pattern of chromatin interactions and stress conditions could modulate the dynamics of chromatin interactions. We found the heterochromatic region of the genome actively involved in the chromatin interactions. We further observed that the establishment or loss of interactions in response to stress does not result in the global change in the expression profile of interacting genes; however, interacting regions (genes) containing motifs for known TFs showed either lower expression or no difference than non-interacting genes. The present study also revealed that interactions preferred among the same epigenetic state (ES) suggest interactions clustered the same ES together in the 3D space of the nucleus. Our analysis showed that stress conditions affect the dynamics of chromatin interactions among the chromatin loci and these interaction networks govern the folding principle of chromatin by bringing together similar epigenetic marks.

Role of GhHDA5 in H3K9 deacetylation and fiber initiation in Gossypium hirsutum.

Cotton fibers are single-celled trichomes that initiate from the epidermal cells of the ovules at or before anthesis. Here, we identified that the histone deacetylase (HDAC) activity is essential for proper cotton fiber initiation. We further identified 15 HDACs homoeologs in each of the A- and D-subgenomes of Gossypium hirsutum. Few of these HDAC homoeologs expressed preferentially during the early stages of fiber development [-1, 0 and 6 days post-anthesis (DPA)]. Among them, GhHDA5 expressed significantly at the time of fiber initiation (-1 and 0 DPA). The in vitro assay for HDAC activity indicated that GhHDA5 primarily deacetylates H3K9 acetylation marks. Moreover, the reduced expression of GhHDA5 also suppresses fiber initiation and lint yield in the RNA interference (RNAi) lines. The 0 DPA ovules of GhHDA5RNAi lines also showed alterations in reactive oxygen species homeostasis and elevated autophagic cell death in the developing fibers. The differentially expressed genes (DEGs) identified through RNA-seq of RNAi line (DEP12) and their pathway analysis showed that GhHDA5 modulates expression of many stress and development-related genes involved in fiber development. The reduced expression of GhHDA5 in the RNAi lines also resulted in H3K9 hyper-acetylation on the promoter region of few DEGs assessed by chromatin immunoprecipitation assay. The positively co-expressed genes with GhHDA5 showed cumulative higher expression during fiber initiation, and gene ontology annotation suggests their involvement in fiber development. Furthermore, the predicted protein interaction network in the positively co-expressed genes indicates HDA5 modulates fiber initiation-specific gene expression through a complex involving reported repressors.

Distinct role of core promoter architecture in regulation of light-mediated responses in plant genes.

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less (initiator-containing) promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr (Initiator) elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS (transcription start site), and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

Large-scale resource development in Gossypium hirsutum L. by 454 sequencing of genic-enriched libraries from six diverse genotypes.

The sequence information has been proved to be an essential genomic resource in case of crop plants for their genetic improvement and better utilization by humans. To dissect the Gossypium hirsutum genome for large-scale development of genomic resources, we adopted hypomethylated restriction-based genomic enrichment strategy to sequence six diverse genotypes. Approximately 5.2-Gb data (more than 18.36 million reads) was generated which, after assembly, represents nearly 1.27-Gb genomic sequences. We predicted a total of 93,363 gene models (21,399 full length) and identified 35,923 gene models which were validated against already sequenced plant genomes. A total of 1,093 transcription factor-encoding genes, 3,135 promoter sequences and 78 miRNA (including 17 newly identified in Gossypium) were predicted. We identified significant no. of molecular markers including 47,093 novel simple sequence repeats and 66,364 novel single nucleotide polymorphisms. In addition, we developed NBRI-Comprehensive Cotton Genomics database, a web resource to provide access of cotton-related genomic resources developed at NBRI. This study contributes considerable amount of genomic resources and suggests a potential role of genic-enriched sequencing in genomic resource development for orphan crop plants.

Analysis of histones and histone variants in plants.

Histone proteins are the major protein components of chromatin - the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demonstrated that dynamic association of histones and their variants to the genome plays a very important role in gene regulation. Histones are extensively modified during posttranslation viz. acetylation, methylation, phosphorylation, ubiquitylation, etc., and the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Different biochemical techniques have been developed to purify and separate histone proteins; here, we describe techniques for analysis of histones from plant tissues.

Analysis of chromatin structure in plant cells.

A vast body of evidence in the literature indicates that nucleosomes can act as barriers to transcriptional initiation. The nucleosome at the promoter inhibits association of transcription factors disallowing active transcription of the gene. We have found a nucleosome on tobacco pathogenesis-related gene-1a (PR-1a) core promoter and mapped its boundaries and extension to find its span. The nucleosome covers the TATA box and Inr region of the core promoter and gets disassembled upon induction. Prior to its removal, modifications (i.e., acetylation and methylation of histones) occur at the nucleosome, proving a role of epigenetic modifications in transcriptional regulation. We summarize here various methodologies to analyze promoter chromatin structure in plants using the PR-1a core promoter as an example.