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Item Comparative transcriptome profiling of two contrasting foxtail millet cultivars provides insights into molecular mechanisms underlying dehydration stress response(Springer Nature Publishing AG, 2023) Muthamilarasan, Mehanathan; Suresh, Bonthala Venkata; Singh, Roshan Kumar; Choudhary, Pooja; Aggarwal, Pooja Rani; Prasad, ManojFoxtail millet (Setaria italica L.) has emerged as a model system to understand its adaptation to environmental stresses in the past decade. However, studies on understanding the molecular mechanism underlying the adaptation to dehydration stress and the regulatory network involved in the process remain elusive. In the present study, RNA-seq was performed during dehydration stress in the tolerant (IC4) and sensitive (IC41) cultivars at different time points (0, 6, and 12 h). A total of 2467 and 3318 differentially expressed genes (DEGs) were identified in IC4, and 2535 and 5572 in IC41 at 6 h and 12 h compared to control (0 h), respectively. Gene ontology (GO) analysis revealed that the DEGs were enriched in water transport, response to water deprivation, oxidative stress, amino acid and sugar transport, lipid biosynthesis, and regulation of stomatal opening. Pathway analysis suggested a significant modulation of genes involved in the metabolism of glutathione and tryptophan and biosynthesis of flavonoid, ascorbate, arginine, and proline in IC4 compared to IC41. Genes encoding for DIVARICATA, SBP family protein (teosinte glume architecture 1), and SRS family proteins (LATERAL ROOT PRIMORDIUM 1 and SHI-RELATED SEQUENCE 1) were found to be exclusively upregulated in IC4 during dehydration stress. Gene co-expression networks constructed based on the expression data showed the key modules and hubs that play critical roles during dehydration stress. Altogether, the present study has identified key genes, pathways, and regulatory modules that would serve as a base for further studies to gain insights into the dehydration-responsive molecular circuitry in foxtail millet.Item De novo transcriptome analysis identifies key genes involved in dehydration stress response in kodo millet (Paspalum scrobiculatum L.)(Elsevier B.V., 2022) Suresh, Bonthala Venkata; Choudhary, Pooja; Aggarwal, Pooja Rani; Rana, Sumi; Singh, Roshan Kumar; Ravikesavan, Rajasekaran; Prasad, Manoj; Muthamilarasan, MehanathanKodo millet (Paspalum scrobiculatum L.) is a small millet species known for its excellent nutritional and climate-resilient traits. To understand the genes and pathways underlying dehydration stress tolerance of kodo millet, the transcriptome of cultivar ‘CO3’ subjected to dehydration stress (0 h, 3 h, and 6 h) was sequenced. The study generated 239.1 million clean reads that identified 9201, 9814, and 2346 differentially expressed genes (DEGs) in 0 h vs. 3 h, 0 h vs. 6 h, and 3 h vs. 6 h libraries, respectively. The DEGs were found to be associated with vital molecular pathways, including hormone metabolism and signaling, antioxidant scavenging, photosynthesis, and cellular metabolism, and were validated using qRT-PCR. Also, a higher abundance of uncharacterized genes expressed during stress warrants further studies to characterize this class of genes to understand their role in dehydration stress response. Altogether, the study provides insights into the transcriptomic response of kodo millet during dehydration stress.Item GWAS identifies genetic loci underlying nitrogen responsiveness in the climate resilient C4 model Setaria italica (L.)(Elsevier B.V., 2022) Bandyopadhyay, Tirthankar; Swarbreck, Stéphanie M; Jaiswal, Vandana; Maurya, Jyoti; Gupta, Rajeev; Bentley, Alison R.; Griffiths, Howard; Prasad, ManojIntroduction N responsiveness is the capacity to perceive and induce morpho-physiological adaptation to external and internal Nitrogen (N). Crop productivity is propelled by N fertilizer and requires the breeding/selection of cultivars with intrinsically high N responsiveness. This trait has many advantages in being more meaningful in commercial/environmental context, facilitating in-season N management and not being inversely correlated with N availability over processes regulating NUE. Current lack of its understanding at the physio-genetic basis is an impediment to select for cultivars with a predictably high N response. Objectives To dissect physio-genetic basis of N responsiveness in 142 diverse population of foxtail millet, Setaria italica (L.) by employing contrasting N fertilizer nutrition regimes. Methods We phenotyped S. italica accessions for major yield related traits under low (N10, N25) and optimal (N100) growth conditions and genotyped them to subsequently perform a genome-wide association study to identify genetic loci associated with nitrogen responsiveness trait. Groups of accessions showing contrasting trait performance and allelic forms of specific linked genetic loci (showing haplotypes) were further accessed for N dependent transcript abundances of their proximal genes. Results Our study show that N dependent yield rise in S. italica is driven by grain number whose responsiveness to N availability is genetically underlined. We identify 22 unique SNP loci strongly associated with this trait out of which six exhibit haplotypes and consistent allelic variation between lines with contrasting N dependent grain number response and panicle architectures. Furthermore, differential transcript abundances of specific genes proximally linked to these SNPs in same lines is indicative of their N dependence in a genotype specific manner. Conclusion The study demonstrates the value/ potential of N responsiveness as a selection trait and identifies key genetic components underlying the trait in S. italica. This has major implications for improving crop N sustainability and food security.Item Genetic determinants of drought stress tolerance in Setaria(Springer, 2017) Muthamilarasan, Mehanathan; Prasad, ManojCultivated foxtail millet (Setaria italica) and its wild progenitor (S. viridis) have collectively been considered as tractable model species for studying C4 photosynthesis, stress biology, and biofuel traits. Being cultivated in arid and semiarid tropics of the world, these species are well adapted to harsh environments such as drought, heat, and salinity. This adaptation or acclimation potential of Setaria spp. has drawn research interest, and attempts have been made to dissect the molecular mechanisms of stress tolerance. Compared to other stresses, drought response has been studied extensively in S. italica and many drought-responsive genes encoding for transcription factors, signaling molecules, and enzymes have been identified and characterized. Several genome-wide studies have reported on identification of stress-responsive gene family members, and speculated on the potential for expansion and neofunctionalization of paralogs in these gene families. In this context, this chapter discusses the key genetic determinants identified for stress tolerance in S. italica and demonstrates their use in improving drought tolerance. In addition, strategies for identification of genes underlying stress tolerance are also described. Little effort has so far been made towards understanding the stress-tolerance characteristics of Setaria as compared to studies reported in other crops. Comprehensive functional studies along with the use of integrated -omics approaches are required to elucidate the genetics and genomics of stress tolerance in Setaria, as it is important to develop climate change resilient crops to meet the growing demand for food and feed.Item Drought stress responses and signal transduction in plants(Springer, 2015) Lata, Charu; Muthamilarasan, Mehanathan; Prasad, ManojNature provides all necessary components for healthy growth and development of plants in the form of air, water, light, nutrients, and soil. Any imbalance in the environmental harmony may cause stress to them. Stresses encountered by plants can broadly be categorized into biotic and abiotic stresses. Biotic stresses are mainly caused by pathogens and herbivory, whereas abiotic stresses include the threat imposed by drought, salinity, and extremes of temperature, heavy metals, and pollution. Drought stress is a major cause of yield instability in crops across diverse eco-geographic regions worldwide. A variety of biochemical, molecular, and physiological changes are manifested by plants in response to drought stress. The cellular abscisic acid (ABA) concentration increases on water deficit leading to the activation of a number of stress-responsive genes and the patterns of expression of these genes are very complex, with some genes being induced early while others respond slowly. In general, drought-responsive genes respond to salt and cold stresses as well as to exogenous ABA treatment. However, there are several genes, which express themselves in an ABA-independent manner suggesting that both ABA-dependent and -independent signal transduction cascades exist for drought stress perception, response, and adaptation. Drought stress response and adaptation in plants involves an array of pathways for signal perception, transduction, gene expression and synthesis of proteins, and other stress metabolites. Drought-responsive genes can mainly be classified into two groups. First group constitutes genes whose products provide osmotolerance and protection to plants thus directly functioning in tolerance to stress, while the second group includes genes playing a role in signal transduction as well as regulation of gene expression. This chapter summarizes the complex molecular mechanisms of drought stress response and adaptation in plants, highlighting the transcriptional regulation of stress-responsive gene expression. It also focuses on the recent advances in analyzing various stress-responsive pathways with prime emphasis on ABA-dependent and -independent pathways.Item Dynamics of defense related components in two contrasting genotypes to tomato upon infection with tomato leaf curl New Delhi virus(Springer, 2012) Sahu, Pranav Pankaj; Rai, Neeraj Kumar; Puranik, Swati; Roy, Anirban; Khan, Moinuddin; Prasad, ManojTomato leaf curl virus (ToLCV) disease is a serious threat for tomato cultivation in the tropics and subtropics. Despite serious efforts no immune commercial varieties or F(1) hybrids are available till date. In this study, the interaction between Solanum lycopersicum and ToLCV was characterized on molecular and biochemical basis. RNA silencing mediated by short interfering RNA (siRNA) and reactive oxygen species (ROS) has been proposed as central components of plant adaptation to several stresses. A comparative RNA interference study between two contrasting tomato genotypes, LA1777 (tolerant) and 15SBSB (susceptible) infected with Tomato Leaf Curl New Delhi Virus (ToLCNDV) revealed relatively higher accumulation of siRNA in the leaves of tolerant genotype. In LA1777, ToLCNDV produced chlorotic as well as necrotic areas at the inoculation sites 5-10 days post-inoculation. Caspase-9- and caspase-3-like activities were significantly increased in response to ToLCNDV infection in LA1777 at inoculated region. Activities of antioxidant enzymes involved in the detoxification of ROS were examined in both systemic and localized area of infection, and their expression level was further validated through quantitative real-time PCR of the corresponding transcripts. Expression patterns of three genes encoding pathogenesis-related proteins showed higher accumulation in tolerant genotype. Tolerance against the ToLCNDV in LA1777 can be attributed to the higher siRNA accumulation, localized cell death, altered levels of antioxidant enzymes and activation of pathogenesis-related genes at different durations of virus infection. Based on these direct and indirect evidences, we have proposed a putative mechanism for ToLCNDV tolerance in the tolerant genotype.Item Tomato cultivar tolerant to tomato leaf curl New Delhi Virus infection induces virus-specific siRNA accumulation and defense associated host gene expression(Wiley-Blackwell, 2010) Sahu, Pranav Pankaj; Rai, Neeraj K.; Chakraborty, Supriya; Singh, Major; Chandrappa, Prasanna H.; Ramesh, Bandarupalli; Chattopadhyay, Debasis; Prasad, ManojTomato leaf curl New Delhi virus (ToLCNDV) infection causes significant yield loss in tomato. The availability of a conventional tolerance source against this virus is limited in tomato. To understand the molecular mechanism of virus tolerance in tomato, the abundance of viral genomic replicative intermediate molecules and virus-directed short interfering RNAs (siRNAs) by the host plant in a naturally tolerant cultivar H-88-78-1 and a susceptible cultivar Punjab Chhuhara at different time points after agroinfection was studied. We report that less abundance of viral replicative intermediate in the tolerant cultivar may have a correlation with a relatively higher accumulation of virus-specific siRNAs. To study defence-related host gene expression in response to ToLCNDV infection, the suppression subtractive hybridization technique was used. A library was prepared from tolerant cultivar H-88-78-1 between ToLCNDV-inoculated and Agrobacterium mock-inoculated plants of this cultivar at 21 days post-inoculation (dpi). A total of 106 nonredundant transcripts was identified and classified into 12 different categories according to their putative functions. By reverse Northern analysis and quantitative real-time polymerase chain reaction (qRT-PCR), we identified the differential expression pattern of 106 transcripts, 34 of which were up-regulated (>2.5-fold induction). Of these, eight transcripts showed more than four fold induction. qRT-PCR analysis was carried out to obtain comparative expression profiling of these eight transcripts between Punjab Chhuhara and H-88-78-1 on ToLCNDV infection. The expression patterns of these transcripts showed a significant increase in differential expression in the tolerant cultivar, mostly at 14 and 21 dpi, in comparison with that in the susceptible cultivar, as analysed by qRT-PCR. The probable direct and indirect relationship of siRNA accumulation and up-regulated transcripts with the ToLCNDV tolerance mechanism is discussed.Item Comparative transcriptome analysis of differentially expressed genes in foxtail millet (Setaria italica L.) during dehydration stress(Elsevier B.V., 2010) Lata, Charu; Sahu, Pranav Pankaj; Prasad, Manojhydration stress is one of the most important abiotic stresses that adversely influence crop growth and productivity. With the aim to understand the molecular mechanisms underlying dehydration stress tolerance in foxtail millet (Setaria italica L.), a drought tolerant crop, we examined its transcriptome changes at two time points (early and late) of dehydration stress. Two suppression subtractive hybridization (SSH) forward libraries were constructed from 21-day old seedlings of tolerant cv. Prasad at 0.5 and 6h PEG-induced dehydration stress. A total of 327 unique ESTs were identified from both libraries and were classified into 11 different categories according to their putative functions. The plant response against dehydration stress was complex, representing major transcripts involved in metabolism, stress, signaling, transcription regulation, translation and proteolysis. By Reverse Northern (RN) technique we identified the differential expression pattern of 327 transcripts, 86 (about 26%) of which showed > or = 1.7-fold induction. Further the obtained results were validated by quantitative real-time PCR (qRT-PCR) to have a comparative expression profiling of randomly chosen 9 up-regulated transcripts (> or =2.5 fold induction) between cv. Prasad (tolerant) and cv. Lepakshi (sensitive) upon dehydration stress. These transcripts showed a differential expression pattern in both cultivars at different time points of stress treatment as analyzed by qRT-PCR. The possible relationship of the identified transcripts with dehydration tolerance mechanism is discussed.Item cDNA-AFLP analysis reveals differential gene expression in response to salt stress in foxtail millet (Setaria italica L.)(Springer, 2008) Jayaraman, Ananthi; Puranik, Swati; Rai, Neeraj Kumar; Vidapu, Sudhakar; Sahu, Pranav Pankaj; Lata, Charu; Prasad, ManojPlant growth and productivity are affected by various abiotic stresses such as heat, drought, cold, salinity, etc. The mechanism of salt tolerance is one of the most important subjects in plant science as salt stress decreases worldwide agricultural production. In our present study we used cDNA-AFLP technique to compare gene expression profiles of a salt tolerant and a salt-sensitive cultivar of foxtail millet (Seteria italica) in response to salt stress to identify early responsive differentially expressed transcripts accumulated upon salt stress and validate the obtained result through quantitative real-time PCR (qRT-PCR). The expression profile was compared between a salt tolerant (Prasad) and susceptible variety (Lepakshi) of foxtail millet in both control condition (L0 and P0) and after 1 h (L1 and P1) of salt stress. We identified 90 transcript-derived fragments (TDFs) that are differentially expressed, out of which 86 TDFs were classified on the basis of their either complete presence or absence (qualitative variants) and 4 on differential expression pattern levels (quantitative variants) in the two varieties. Finally, we identified 27 non-redundant differentially expressed cDNAs that are unique to salt tolerant variety which represent different groups of genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, mRNA splicing, seed development and storage, etc. The expression patterns of seven out of nine such genes showed a significant increase of differential expression in tolerant variety after 1 h of salt stress in comparison to salt-sensitive variety as analyzed by qRT-PCR. The direct and indirect relationship of identified TDFs with salinity tolerance mechanism is discussed.