Charu Lata

Role of DREBs in regulation of abiotic stress responses in plants.

Abiotic stresses such as drought, high salinity, and cold are common adverse environmental conditions that significantly influence plant growth and productivity worldwide. The phytohormone abscisic acid (ABA) plays an important role in physiological and developmental responses as well as in co-ordinating various stress signal transduction pathways in plants. DREBs (dehydration responsive element binding) are important plant transcription factors (TFs) that regulate the expression of many stress-inducible genes mostly in an ABA-independent manner and play a critical role in improving the abiotic stress tolerance of plants by interacting with a DRE/CRT cis-element present in the promoter region of various abiotic stress-responsive genes. This review summarizes recent studies highlighting the role of the DRE-binding family of TFs in the adaptive responses to different abiotic stresses and their structural and functional characters with emphasis on the expression and regulation of DREBs. The practical and application value of DREBs in crop improvement, such as stress tolerance engineering as well as marker-assisted selection (MAS), has also been discussed.

Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses

Foxtail millet is one of the oldest domesticated diploid C4 Panicoid crops having a comparatively small genome size of approximately 515 Mb, short life cycle, and inbreeding nature. Its two species, Setaria italica (domesticated) and Setaria viridis (wild progenitor), have characteristics that classify them as excellent model systems to examine several aspects of architectural, evolutionary, and physiological importance in Panicoid grasses especially the biofuel crops such as switchgrass and napiergrass. Foxtail millet is a staple crop used extensively for food and fodder in parts of Asia and Africa. In its long history of cultivation, it has been adapted to arid and semi-arid areas of Asia, North Africa, South and North America. Foxtail millet has one of the largest collections of cultivated as well as wild-type germplasm rich with phenotypic variations and hence provides prospects for association mapping and allele-mining of elite and novel variants to be incorporated in crop improvement programs. Most of the foxtail millet accessions can be primarily abiotic stress tolerant particularly to drought and salinity, and therefore exploiting these agronomic traits can enhance its efficacy in marker-aided breeding as well as in genetic engineering for abiotic stress tolerance. In addition, the release of draft genome sequence of foxtail millet would be useful to the researchers worldwide in not only discerning the molecular basis of biomass production in biofuel crops and the methods to improve it, but also for the introgression of beneficial agronomically important characteristics in foxtail millet as well as in related Panicoid bioenergy grasses.

Comparative transcriptome analysis of differentially expressed genes in foxtail millet (Setaria italica L.) during dehydration stress.

Dehydration 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.

Genome-wide investigation and expression profiling of AP2/ERF transcription factor superfamily in foxtail millet (Setaria italica L.).

The APETALA2/ethylene-responsive element binding factor (AP2/ERF) family is one of the largest transcription factor (TF) families in plants that includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and RAV (Related to ABI3/VP). AP2/ERFs are known to play significant roles in various plant processes including growth and development and biotic and abiotic stress responses. Considering this, a comprehensive genome-wide study was conducted in foxtail millet (Setaria italica L.). A total of 171 AP2/ERF genes were identified by systematic sequence analysis and were physically mapped onto nine chromosomes. Phylogenetic analysis grouped AP2/ERF genes into six classes (I to VI). Duplication analysis revealed that 12 (∼7%) SiAP2/ERF genes were tandem repeated and 22 (∼13%) were segmentally duplicated. Comparative physical mapping between foxtail millet AP2/ERF genes and its orthologs of sorghum (18 genes), maize (14 genes), rice (9 genes) and Brachypodium (6 genes) showed the evolutionary insights of AP2/ERF gene family and also the decrease in orthology with increase in phylogenetic distance. The evolutionary significance in terms of gene-duplication and divergence was analyzed by estimating synonymous and non-synonymous substitution rates. Expression profiling of candidate AP2/ERF genes against drought, salt and phytohormones revealed insights into their precise and/or overlapping expression patterns which could be responsible for their functional divergence in foxtail millet. The study showed that the genes SiAP2/ERF-069, SiAP2/ERF-103 and SiAP2/ERF-120 may be considered as potential candidate genes for further functional validation as well for utilization in crop improvement programs for stress resistance since these genes were up-regulated under drought and salinity stresses in ABA dependent manner. Altogether the present study provides new insights into evolution, divergence and systematic functional analysis of AP2/ERF gene family at genome level in foxtail millet which may be utilized for improving stress adaptation and tolerance in millets, cereals and bioenergy grasses.

Role of Plant Transcription Factors in Abiotic Stress Tolerance

Plants are constantly exposed to a wide range of environmental stresses such as drought, high salt, heat and extremes of temperature. Growth constraints due to these abiotic stresses result in reduced productivity and significant crop losses globally. Drought and salinity affect more than 10% of arable land, which results in more than 50% decline in the average yields of important crops worldwide (Bray et al., 2000). Tolerance or susceptibility to these stresses is also a very intricate event as stress may affect multiple stages of plant development and often several stresses concurrently affect the plants (Chinnusamy et al., 2004). Therefore, the basic mechanisms of abiotic stress tolerance and adaptation have been the area of comprehensive research. Plants counter adverse environmental conditions in a complex, integrated way depending on the timing and length that allows them to respond and adapt to the existing constraints present at a given time. Plant stress tolerance involves changes at whole-plant, tissue, cellular, physiological and molecular levels. Exhibition of a distinct or a combination of intrinsic changes ascertains the capacity of a plant to sustain itself under unfavorable environmental conditions (Farooq et al., 2009). This comprises a range of physiological and biochemical adjustments in plants including leaf wilting, leaf area reduction, leaf abscission, root growth stimulation, alterations in relative water content (RWC), electrolytic leakage (EL), production of reactive oxygen species (ROS) and accumulation of free radicals which disturb cellular homeostasis ensuing lipid peroxidation, membrane damage, and inactivation of enzymes thus influencing cell viability (Bartels and Sunkar, 2005). Other than these, abscissa acid (ABA), a plant stress hormone, induces leaf stomata closure, thus reducing transpirational water loss and photosynthetic rate which improves the water-use efficiency (WUE) of the plant. Molecular responses to abiotic stress on the other hand include perception, signal transduction, gene expression and ultimately metabolic changes in the plant thus providing stress tolerance (Agarwal et al., 2006). Several genes are activated in response to abiotic stresses at the transcriptional level, and their products are contemplated to provide stress tolerance by the production of vital metabolic proteins and also in regulating the downstream genes (Kavar et al., 2007). Transcript profiling can be a significant tool for the characterization of stress-responsive genes. Extensive transcriptome analyses have divulged that these gene products can largely be classified into two groups (Bohnert et al., 2001; Seki et al., 2002; Fowler and Thomashow, 2002). First group comprises of genes that encode for proteins that defend the cells from the

Differential antioxidative responses to dehydration-induced oxidative stress in core set of foxtail millet cultivars [Setaria italica (L.)]

Foxtail millet (Setaria italica L.) known as a relatively drought-tolerant crop across the world is grown in arid and semi-arid regions. To the best of our knowledge, no systematic study on drought tolerance screening of foxtail millet germplasm being a drought-tolerant crop has been reported so far. To explore genetic diversity of drought-induced oxidative stress tolerance in foxtail millet, we employed lipid peroxidation measure to assess membrane integrity under stress as biochemical marker to screen 107 cultivars and classified the genotypes as highly tolerant, tolerant, sensitive, and highly sensitive. From this comprehensive screening, four cultivars showing differential response to dehydration tolerance were selected to understand the physiological and biochemical basis of tolerance mechanisms. The dehydration-tolerant cultivars (IC-403579 and Prasad) showed considerably lower levels of lipid peroxidation and electrolyte leakage as compared with dehydration-sensitive cultivars (IC-480117 and Lepakshi), indicating better cell membrane integrity in tolerant cultivars. Correspondingly, tolerant genotypes maintained higher activity of catalase (EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2) across different time-course period of polyethylene glycol (PEG) treatments in comparison to sensitive ones. The above biochemical results were further validated through quantitative real-time PCR analysis of APX and GR, whose transcripts were substantially induced by PEG treatments in tolerant cultivars. These results suggest that tolerant cultivars possess wider array of antioxidant machinery with efficient ascorbate–glutathione pathway to cope with drought-induced oxidative stress.

Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations

Pearl millet [Pennisetum glaucum (L.) R. Br.] a widely used grain and forage crop, is grown in areas frequented with one or more abiotic stresses, has superior drought and heat tolerance and considered a model crop for stress tolerance studies. Selection of suitable reference genes for quantification of target stress-responsive gene expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of improved stress tolerance. For precise normalization of gene expression data in pearl millet, ten candidate reference genes were examined in various developmental tissues as well as under different individual abiotic stresses and their combinations at 1 h (early) and 24 h (late) of stress using geNorm, NormFinder and RefFinder algorithms. Our results revealed EF-1α and UBC-E2 as the best reference genes across all samples, the specificity of which was confirmed by assessing the relative expression of a PgAP2 like-ERF gene that suggested use of these two reference genes is sufficient for accurate transcript normalization under different stress conditions. To our knowledge this is the first report on validation of reference genes under different individual and multiple abiotic stresses in pearl millet. The study can further facilitate fastidious discovery of stress-tolerance genes in this important stress-tolerant crop.

A study of the role of gene TaMYB2 and an associated SNP in dehydration tolerance in common wheat

The myeloblastosis oncogenes (MYB) are one of the important transcription factors that facilitate induction of various developmental and stress responsive genes. They are hence, emerging as key players in improving stress tolerance of plants in response to several abiotic stresses. Therefore, isolation and characterization of these genes, development of transgenics and functional molecular markers for useful alleles is central to various crop improvement programs. In this manuscript, we for the first time are reporting the identification of a synonymous single nucleotide polymorphism associated with dehydration tolerance at 458th bp (an A/G transition) in the TaMYB2 gene of wheat (Triticum aestivum L.) and development of an allele-specific marker (ASM) for dehydration tolerance for the same. Further we validated this TaMYB2-ASM in a core set of 28 wheat cultivars which can be used for marker-assisted selection for dehydration tolerance in plant breeding programs.

Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering

Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si) is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2, and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization.

Setaria genome sequencing: an overview

The genus Setaria includes two important C4 Panicoid grass species, namely S. italica (cultivated) and S. viridis (weed; wild ancestor), which together represent an appropriate model system for architectural, physiological, evolutionary, and genomic studies in related grasses. It is a diploid, inbreeder, self-fertile annual cereal grass having short life cycle and minimal growth requirements. There close relatedness to biofuel crops like switch grass and napier grass further signifies their importance. Further, foxtail millet is an important food and fodder grain crop grown in arid and semi-arid regions in many parts of the world. Therefore, an increasing interest in these species has led to a gradual accumulation and development of genomic data and genetic resources. Setaria genome sequencing is an outcome of such endeavors. These sequencing efforts uncovered several distinctive attributes of Setaria genome that may help in understanding its physiology, evolution and adaptation. This will not only aid in comparative genomics studies of Setaria and related crops including bioenergy grasses but also help in rapid advancements of genomics information for developing varieties with superior traits either through marker-assisted selection (MAS) or using transgenic approaches in these crops.