Publications of NIPGR Scientists
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Item Zinc finger transcriptional repressor ZOS5-09 regulates grain filling and development in rice(John Wiley & Sons, 2025) Jaiswal, Priya; Qasim, Falah; Mahto, Arunima; Vichitra, Ankur; Das, Upasana; Tyagi, Akhilesh K.; Agarwal, PinkyGrain size is one of the key determinants of grain yield. Our study focuses on a novel seed-preferential C2H2 zinc finger transcription factor, ZOS5-09 (LOC_Os05g38600) that plays an important role in regulating rice grain traits. Rice plants with the ZOS5-09 promoter::GUS construct showed high expression of ZOS5-09 in rice endosperm. In planta reporter effector assays and localization studies showed that ZOS5-09 is a nuclear-localized transcriptional repressor. It has two C2H2 zinc finger domains and a C-terminal NoRS (nucleolar retention signal). Ectopic and seed-preferential overexpression of ZOS5-09 resulted in lethality. Seed-preferential overexpression without NoRS was detrimental to grain filling. Rice plants with knock-down or CRISPR-based knock-out of ZOS5-09 displayed reduced grain length and weight but increased grain width. Grain size change was due to lower cell proliferation and increased cell size in the transverse direction because of downregulation of cell cycle-related genes and increased expression of expansins. Decreased expression of ZOS5-09 also resulted in reduced total starch and protein content and higher endosperm chalkiness, thus negatively affecting grain quality. ZOS5-09 directly bound to a zinc finger–binding site and regulated a seed storage protein-encoding gene, GLU6. It acted as a repressor by promoting deacetylation upon interaction with a histone deacetylase. In summary, our results indicate that an optimum expression of ZOS5-09 is essential for proper rice grain development. Our study highlights the role of a transcriptional repressor in regulating rice grain traits and improves our understanding of the transcriptional regulatory networks affecting grain size.Item SUPER STARCHY1/ONAC025 participates in rice grain filling(American Society of Plant Biologists, 2020) Mathew, Iny Elizebeth; Priyadarshini, Richa; Mahto, Arunima; Jaiswal, Priya; Parida, Swarup K.; Agarwal, PinkyNAC transcription factors (TFs) are known for their role in development and stress. This article attempts to functionally validate the role of rice SS1/ ONAC025 (LOC_ Os11g31330) during seed development. The gene is seed-specific and its promoter directs reporter expression in the developing endosperm and embryo in rice transgenic plants. Furthermore, rice transgenic plants ectopically expressing SS1/ ONAC025 have a plantlet lethal phenotype with hampered vegetative growth, but increased tillers and an altered shoot apical meristem structure. The vegetative cells of these plantlets are filled with distinct starch granules. RNAseq analysis of two independent plantlets reveals the differential expression of reproductive and photosynthetic genes. A comparison with seed development transcriptome indicates differential regulation of many seed-related genes by SS1/ ONAC025. Genes involved in starch biosynthesis, especially amylopectin and those encoding seed storage proteins, and regulating seed size are also differentially expressed. In conjunction, SS1/ ONAC025 shows highest expression in japonica rice. As a TF, SS1/ ONAC025 is a transcriptional repressor localized to endoplasmic reticulum and nucleus. The article shows that SS1/ ONAC025 is a seed-specific gene promoting grain filling in rice, and negatively affecting vegetative growth.Item Mediator subunit OsMED14_1 plays an important role in rice development(John Wiley & Sons, 2020) Malik, Naveen; Ranjan, Rajeev; Parida, Swarup K.; Agarwal, Pinky; Tyagi, Akhilesh K.Mediator, a multi‐subunit coactivator complex, regulates transcription in eukaryotes and is involved in diverse processes in Arabidopsis through its different subunits. Here, we have explored developmental aspects of one of the rice Mediator subunit gene OsMED14_1. We analyzed its expression pattern through RNA in‐situ hybridization and pOsMED14_1:GUS transgenics which showed its expression in roots, leaves, anthers and seeds prominently at younger stages, indicating possible involvement of this subunit in multiple aspects of rice development. To understand developmental roles of OsMED14_1 in rice, we generated and studied RNAi based knockdown rice plants which showed multiple effects including less height, narrower leaves and culms with reduced vasculature, lesser lateral root branching, defective microspore development, reduced panicle branching and seed set, and smaller seeds. Histological analyses showed that slender organs were caused by reduction in both cell number and cell size in OsMED14_1 knockdown plants. Flow cytometric analyses and expression analyses of cell‐cycle related genes revealed that defective cell‐cycle progression led to these defects. Expression analyses of auxin related genes and IAA immuno‐localization study indicated altered auxin level in these knockdown plants. Reduction of lateral root branching in knockdown plants was corrected by exogenous IAA supplement. OsMED14_1 physically interacts with transcription factors YABBY5, TDR and MADS29, possibly regulating auxin homeostasis and ultimately leading to lateral organ/leaf, microspore and seed development.Item Analysis of rice proteins with DLN repressor Motif/S(MDPI AG, 2019) Singh, Purnima; Mathew, Iny Elizebeth; Verma, Ankit; Tyagi, Akhilesh K.; Agarwal, PinkyTranscriptional regulation includes both activation and repression of downstream genes. In plants, a well-established class of repressors are proteins with an ERF-associated amphiphilic repression/EAR domain. They contain either DLNxxP or LxLxL as the identifying hexapeptide motif. In rice (Oryza sativa), we have identified a total of 266 DLN repressor proteins, with the former motif and its modifications thereof comprising 227 transcription factors and 39 transcriptional regulators. Apart from DLNxxP motif conservation, DLNxP and DLNxxxP motifs with variable numbers/positions of proline and those without any proline conservation have been identified. Most of the DLN repressome proteins have a single DLN motif, with higher relative percentage in the C-terminal region. We have designed a simple yeast-based experiment wherein a DLN motif can successfully cause strong repression of downstream reporter genes, when fused to a transcriptional activator of rice or yeast. The DLN hexapeptide motif is essential for repression, and at least two “DLN” residues cause maximal repression. Comparatively, rice has more DLN repressor encoding genes than Arabidopsis, and DLNSPP motif from rice is 40% stronger than the known Arabidopsis SRDX motif. The study reports a straightforward assay to analyze repressor activity, along with the identification of a strong DLN repressor from rice.Item Decoding the transcriptome of rice seed during development(InTech, 2017) Mahto, Arunima; Mathew, Iny Elizebeth; Agarwal, PinkyRice seed development is a continuous process wherein it undergoes complex molecular and tissue reprogramming. It is a collective effect of embryo and endosperm development, each of which undertakes its own developmental paths, with endosperm development significantly affecting embryo. Understanding the mechanistics of the regulatory networks administrating this process is the building block for any future research on grain yield and quality. High-throughput transcript profiling and small RNA profiling studies have proved useful in providing information about the molecular changes occurring in various tissues associated with seed development. Transcriptome sequencing studies have highlighted the significant genes and pathways that are operating during seed development. The involvement of TFs and hormones has also been implicated in regulating key aspects of seed development, including embryo patterning and seed maturation. This chapter will review the information provided by high-throughput sequencing studies on various aspects of rice seed development, highlighting the developmental complexities of embryo and endosperm.Item Three rice NAC transcription factors heteromerize and are associated with seed size(Frontiers Media S.A., 2016) Mathew, Iny Elizebeth; Das, Sweta; Mahto, Arunima; Agarwal, PinkyNACs are plant-specific transcription factors (TFs) involved in multiple aspects of development and stress. In rice, three NAC TF encoding genes, namely ONAC020, ONAC026, and ONAC023 express specifically during seed development, at extremely high levels. They exhibit significantly strong association with seed size/weight with the sequence variations located in the upstream regulatory region. Concomitantly, their expression pattern/levels during seed development vary amongst different accessions with variation in seed size. The alterations in the promoter sequences of the three genes, amongst the five rice accessions, correlate with the expression levels to a certain extent only. In terms of transcriptional properties, the three NAC TFs can activate and/or suppress downstream genes, though to different extents. Only ONAC026 is localized to the nucleus while ONAC020 and ONAC023 are targeted to the ER and cytoplasm, respectively. Interestingly, these two proteins interact with ONAC026 and the dimers localize in the nucleus. Trans-splicing between ONAC020 and ONAC026 results in three additional forms of ONAC020. The transcriptional properties including activation, repression, subcellular localization and heterodimerization of trans-spliced forms of ONAC020 and ONAC026 are different, indicating toward their role as competitors. The analysis presented in this paper helps to conclude that the three NAC genes, which are associated with seed size, have independent as well as overlapping roles during the process and can be exploited as potential targets for crop improvement.Item An efficient strategy combining SSR markers- and advanced QTL-seq-driven QTL mapping unravels candidate genes regulating grain weight in rice(Frontiers Media S.A., 2016) Daware, Anurag; Das, Sweta; Srivastava, Rishi; Badoni, Saurabh; Singh, Ashok K.; Agarwal, Pinky; Parida, Swarup K.; Tyagi, Akhilesh K.Development and use of genome-wide informative simple sequence repeat (SSR) markers and novel integrated genomic strategies are vital to drive genomics-assisted breeding applications and for efficient dissection of quantitative trait loci (QTLs) underlying complex traits in rice. The present study developed 6244 genome-wide informative SSR markers exhibiting in silico fragment length polymorphism based on repeat-unit variations among genomic sequences of 11 indica, japonica, aus, and wild rice accessions. These markers were mapped on diverse coding and non-coding sequence components of known cloned/candidate genes annotated from 12 chromosomes and revealed a much higher amplification (97%) and polymorphic potential (88%) along with wider genetic/functional diversity level (16–74% with a mean 53%) especially among accessions belonging to indica cultivar group, suggesting their utility in large-scale genomics-assisted breeding applications in rice. A high-density 3791 SSR markers-anchored genetic linkage map (IR 64 × Sonasal) spanning 2060 cM total map-length with an average inter-marker distance of 0.54 cM was generated. This reference genetic map identified six major genomic regions harboring robust QTLs (31% combined phenotypic variation explained with a 5.7–8.7 LOD) governing grain weight on six rice chromosomes. One strong grain weight major QTL region (OsqGW5.1) was narrowed-down by integrating traditional QTL mapping with high-resolution QTL region-specific integrated SSR and single nucleotide polymorphism markers-based QTL-seq analysis and differential expression profiling. This led us to delineate two natural allelic variants in two known cis-regulatory elements (RAV1AAT and CARGCW8GAT) of glycosyl hydrolase and serine carboxypeptidase genes exhibiting pronounced seed-specific differential regulation in low (Sonasal) and high (IR 64) grain weight mapping parental accessions. Our genome-wide SSR marker resource (polymorphic within/between diverse cultivar groups) and integrated genomic strategy can efficiently scan functionally relevant potential molecular tags (markers, candidate genes and alleles) regulating complex agronomic traits (grain weight) and expedite marker-assisted genetic enhancement in rice.