Bijayalaxmi Mohanty

Transcriptional regulatory network triggered by oxidative signals configures the early response mechanisms of japonica rice to chilling stress.

BackgroundThe transcriptional regulatory network involved in low temperature response leading to acclimation has been established in Arabidopsis. In japonica rice, which can only withstand transient exposure to milder cold stress (10°C), an oxidative-mediated network has been proposed to play a key role in configuring early responses and short-term defenses. The components, hierarchical organization and physiological consequences of this network were further dissected by a systems-level approach.ResultsRegulatory clusters responding directly to oxidative signals were prominent during the initial 6 to 12 hours at 10°C. Early events mirrored a typical oxidative response based on striking similarities of the transcriptome to disease, elicitor and wounding induced processes. Targets of oxidative-mediated mechanisms are likely regulated by several classes of bZIP factors acting on as1/ocs/TGA-like element enriched clusters, ERF factors acting on GCC-box/JAre-like element enriched clusters and R2R3-MYB factors acting on MYB2-like element enriched clusters.Temporal induction of several H2O2-induced bZIP, ERF and MYB genes coincided with the transient H2O2 spikes within the initial 6 to 12 hours. Oxidative-independent responses involve DREB/CBF, RAP2 and RAV1 factors acting on DRE/CRT/rav1-like enriched clusters and bZIP factors acting on ABRE-like enriched clusters. Oxidative-mediated clusters were activated earlier than ABA-mediated clusters.ConclusionGenome-wide, physiological and whole-plant level analyses established a holistic view of chilling stress response mechanism of japonica rice. Early response regulatory network triggered by oxidative signals is critical for prolonged survival under sub-optimal temperature. Integration of stress and developmental responses leads to modulated growth and vigor maintenance contributing to a delay of plastic injuries.

Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development

The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development.

Elucidating rice cell metabolism under flooding and drought stresses using flux-based modeling and analysis.

A metabolic/regulatory network of rice incorporates two important tissue types, germinating seeds and photorespiring leaves, is validated through experiments with rice suspension cultures, and applied to analyze metabolic capability under flooding and drought conditions. Rice (Oryza sativa) is one of the major food crops in world agriculture, especially in Asia. However, the possibility of subsequent occurrence of flood and drought is a major constraint to its production. Thus, the unique behavior of rice toward flooding and drought stresses has required special attention to understand its metabolic adaptations. However, despite several decades of research investigations, the cellular metabolism of rice remains largely unclear. In this study, in order to elucidate the physiological characteristics in response to such abiotic stresses, we reconstructed what is to our knowledge the first metabolic/regulatory network model of rice, representing two tissue types: germinating seeds and photorespiring leaves. The phenotypic behavior and metabolic states simulated by the model are highly consistent with our suspension culture experiments as well as previous reports. The in silico simulation results of seed-derived rice cells indicated (1) the characteristic metabolic utilization of glycolysis and ethanolic fermentation based on oxygen availability and (2) the efficient sucrose breakdown through sucrose synthase instead of invertase. Similarly, flux analysis on photorespiring leaf cells elucidated the crucial role of plastid-cytosol and mitochondrion-cytosol malate transporters in recycling the ammonia liberated during photorespiration and in exporting the excess redox cofactors, respectively. The model simulations also unraveled the essential role of mitochondrial respiration during drought stress. In the future, the combination of experimental and in silico analyses can serve as a promising approach to understand the complex metabolism of rice and potentially help in identifying engineering targets for improving its productivity as well as enabling stress tolerance.

Insights into the molecular mechanism of RGL2-mediated inhibition of seed germination in Arabidopsis thaliana

BackgroundSeed germination is of immense significance for agriculture and has been studied for centuries. Yet, our understanding of the molecular mechanisms underlying regulation of dormancy and germination is still in its infancy. Gibberellins are the key phytohormones that promote germination, and the DELLA protein RGL2 is the main signalling intermediate involved in this response. Germination is completely inhibited if functional RGL2 is overexpressed and/or stabilized; however, the molecular mechanisms of RGL2 function are still largely unknown. We therefore attempted to shed light onto some of the genetic events downstream of RGL2.ResultsGene ontology of the transcriptome differentially regulated by RGL2, as well as extensive cross-comparison with other available microarray data indicates that RGL2-mediated inhibition of germination causes seeds to enter a state of dormancy. RGL2 also appears to differentially regulate a number of transcription factors, many of which are known to be involved in light- or phytohormone-mediated aspects of germination. A promoter analysis of differentially expressed genes identified an enrichment of several motifs that can be bound by specific transcription factors, for example GAMYB, ARF1, or Dof-type zinc fingers. We show that Dof-binding motifs indeed play a role in RGL2-mediated transcription. Using Chromatin Immunoprecipitation (ChIP), we show that RGL2 directly downregulates at least one cell wall modifying enzyme, which is predicted to constrain cell growth thereby leading to inhibition of seed germination.ConclusionsOur results reveal that RGL2 controls various aspects of germination. Through the repression of cell wall modifying enzymes, cell growth is directly constrained to inhibit germination. Furthermore, RGL2 likely interacts with various types of proteins to regulate transcription, and differentially regulates several transcription factors. Collectively, our data indicate that gibberellins, acting via RGL2, control several aspects of seed germination.

Microarray analysis of laser-microdissected tissues indicates the biosynthesis of suberin in the outer part of roots during formation of a barrier to radial oxygen loss in rice (Oryza sativa)

Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long-distance oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin deposited in cell walls are thought to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcription factor genes containing WRKY, AP2, and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a major constituent of the ROL barrier in roots of rice.

Unraveling the Light-Specific Metabolic and Regulatory Signatures of Rice through Combined in Silico Modeling and Multiomics Analysis

Combined in silico modeling and multiomics data analysis elucidate the transcriptional control of rice cellular metabolism upon light signaling. Light quality is an important signaling component upon which plants orchestrate various morphological processes, including seed germination and seedling photomorphogenesis. However, it is still unclear how plants, especially food crops, sense various light qualities and modulate their cellular growth and other developmental processes. Therefore, in this work, we initially profiled the transcripts of a model crop, rice (Oryza sativa), under four different light treatments (blue, green, red, and white) as well as in the dark. Concurrently, we reconstructed a fully compartmentalized genome-scale metabolic model of rice cells, iOS2164, containing 2,164 unique genes, 2,283 reactions, and 1,999 metabolites. We then combined the model with transcriptome profiles to elucidate the light-specific transcriptional signatures of rice metabolism. Clearly, light signals mediated rice gene expressions, differentially regulating numerous metabolic pathways: photosynthesis and secondary metabolism were up-regulated in blue light, whereas reserve carbohydrates degradation was pronounced in the dark. The topological analysis of gene expression data with the rice genome-scale metabolic model further uncovered that phytohormones, such as abscisate, ethylene, gibberellin, and jasmonate, are the key biomarkers of light-mediated regulation, and subsequent analysis of the associated genes’ promoter regions identified several light-specific transcription factors. Finally, the transcriptional control of rice metabolism by red and blue light signals was assessed by integrating the transcriptome and metabolome data with constraint-based modeling. The biological insights gained from this integrative systems biology approach offer several potential applications, such as improving the agronomic traits of food crops and designing light-specific synthetic gene circuits in microbial and mammalian systems.

Identification of candidate network hubs involved in metabolic adjustments of rice under drought stress by integrating transcriptome data and genome-scale metabolic network

In this study, we have integrated a rice genome-scale metabolic network and the transcriptome of a drought-tolerant rice line, DK151, to identify the major transcriptional regulators involved in metabolic adjustments necessary for adaptation to drought. This was achieved by examining the differential expressions of transcription factors and metabolic genes in leaf, root and young panicle of rice plants subjected to drought stress during tillering, booting and panicle elongation stages. Critical transcription factors such as AP2/ERF, bZIP, MYB and NAC that control the important nodes in the gene regulatory pathway were identified through correlative analysis of the patterns of spatio-temporal expression and cis-element enrichment. We showed that many of the candidate transcription factors involved in metabolic adjustments were previously linked to phenotypic variation for drought tolerance. This approach represents the first attempt to integrate models of transcriptional regulation and metabolic pathways for the identification of candidate regulatory genes for targeted selection in rice breeding.

Patterns of cis-element enrichment reveal potential regulatory modules involved in the transcriptional regulation of anoxia response of japonica rice

Unlike other cereal species, rice is able to germinate and elongate under anoxia. The regulatory mechanism that configures the transcriptome of rice during anaerobic germination is yet to be established. In this study, the major regulatory modules among anoxia-responsive genes in rice identified from published microarray data were predicted by ab initio analysis of cis-regulatory information content. Statistically overrepresented sequence motifs were detected from bona fide promoter sequences [-1000 to +200], revealing various patterns of cis-element enrichment that are highly correlated with bZIP, ERF and MYB types of transcription factors. As implied by the cis-element enrichment patterns, combinatorial mechanisms configure the overall changes in gene expression during anoxic germination and coleoptile elongation. High enrichment of cis-elements associated with ARF, bZIP, ERF, MYB and WRKY (SUSIBA2) transcription factors was also detected among the glycolytic and fermentative associated genes that were upregulated during anoxia. The patterns established from the global analysis of cis-element distribution for upregulated and downregulated genes and their associations with potential cognate regulatory transcription factors indicate the significant roles of ethylene and abscisic acid mediated signaling during coleoptile elongation under anoxia. In addition, the regulation of genes encoding enzymes in the glycolytic and fermentative metabolism could be associated with abscisic acid and auxin in rice coleoptiles to maintain sugar and ATP levels for longer survival.

The Promoter Signatures in Rice LEA Genes Can Be Used to Build a Co-expressing LEA Gene Network

Coordinated transcriptional modulation of large gene sets depends on the combinatorial use of cis-regulatory motifs in promoters. We postulate that promoter content similarities are diagnostic for co-expressing genes that function coherently during specific cellular responses. To find the co-expressing genes we propose an ab initio method that identifies motif families in promoters of target gene groups, map these families to the promoters of all genes in the genome, and determine the best matches of each of the target group gene promoters with all other promoters. When the method was tested in rice starting from a group of co-expressing Late Embryogenesis Abundant (LEA) genes, we obtained a promoter similarity-based network that contained candidate genes that could plausibly complement the function of LEA genes. Importantly, 73.36% of 244 genes predicted by our method were experimentally confirmed to co-express with the LEA genes in maturing rice embryos, making this methodology a promising tool for biological systems analyses.

Identification of salt gland-associated genes and characterization of a dehydrin from the salt secretor mangrove Avicennia officinalis

BackgroundSalt stress is a major challenge for growth and development of plants. The mangrove tree Avicennia officinalis has evolved salt tolerance mechanisms such as salt secretion through specialized glands on its leaves. Although a number of structural studies on salt glands have been done, the molecular mechanism of salt secretion is not clearly understood. Also, studies to identify salt gland-specific genes in mangroves have been scarce.ResultsBy subtractive hybridization (SH) of cDNA from salt gland-rich cell layers (tester) with mesophyll tissues as the driver, several Expressed Sequence Tags (ESTs) were identified. The major classes of ESTs identified include those known to be involved in regulating metabolic processes (37%), stress response (17%), transcription (17%), signal transduction (17%) and transport functions (12%). A visual interactive map generated based on predicted functional gene interactions of the identified ESTs suggested altered activities of hydrolase, transmembrane transport and kinases. Quantitative Real-Time PCR (qRT-PCR) was carried out to validate the expression specificity of the ESTs identified by SH. A Dehydrin gene was chosen for further experimental analysis, because it is significantly highly expressed in salt gland cells, and dehydrins are known to be involved in stress remediation in other plants. Full-length Avicennia officinalis Dehydrin1 (AoDHN1) cDNA was obtained by Rapid Amplification of cDNA Ends. Phylogenetic analysis and further characterization of this gene suggested that AoDHN1 belongs to group II Late Embryogenesis Abundant proteins. qRT-PCR analysis of Avicennia showed up-regulation of AoDHN1 in response to salt and drought treatments. Furthermore, some functional insights were obtained by growing E. coli cells expressing AoDHN1. Growth of E. coli cells expressing AoDHN1 was significantly higher than that of the control cells without AoDHN1 under salinity and drought stresses, suggesting that the mangrove dehydrin protein helps to mitigate the abiotic stresses.ConclusionsThirty-four ESTs were identified to be enriched in salt gland-rich tissues of A. officinalis leaves. qRT-PCR analysis showed that 10 of these were specifically enriched in the salt gland-rich tissues. Our data suggest that one of the selected genes, namely, AoDHN1 plays an important role to mitigate salt and drought stress responses.