Monika Dalal

Abiotic stress and ABA-inducible Group 4 LEA from Brassica napus plays a key role in salt and drought tolerance

Late-embryogenesis abundant (LEA) proteins are a family of hydrophilic proteins that form an integral part of desiccation tolerance of seeds. LEA proteins have been also postulated to play a protective role under different abiotic stresses. Their role in abiotic stress tolerance has been well documented for Group 1, 2 and 3 LEAs among the nine different groups. The present study evaluates the functional role of a Group 4 LEA protein, LEA4-1 from Brassica napus. Expression analysis revealed that abscisic acid, salt, cold and osmotic stresses induce expression of LEA4-1 gene in leaf tissues in Brassica species. Conversely, reproductive tissues such as flowers and developing seeds showed constitutive expression of LEA4, which was up-regulated in flowers under salt stress. For functional evaluation of LEA4-1 with regard to stress tolerance, LEA4-1 cDNA was cloned from B. napus, and overexpressed in both Escherichia coli and transgenic Arabidopsis plants. Overexpression of BnLEA4-1 cDNA in E. coli conferred salt and extreme temperature tolerance to the transformed cells. Furthermore, transgenic Arabidopsis plants overexpressing BnLEA4-1 either under constitutive CaMV35S or abiotic stress inducible RD29A promoter showed enhanced tolerance to salt and drought stresses. These results demonstrate that LEA4-1 plays a crucial role in abiotic stress tolerance during vegetative stage of plant development.

Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat

Small Heat Shock Proteins (sHSPs)/HSP20 are molecular chaperones that protect plants by preventing protein aggregation during abiotic stress conditions, especially heat stress. Due to global climate change, high temperature is emerging as a major threat to wheat productivity. Thus, the identification of HSP20 and analysis of HSP transcriptional regulation under different abiotic stresses in wheat would help in understanding the role of these proteins in abiotic stress tolerance. We used sequences of known rice and Arabidopsis HSP20 HMM profiles as queries against publicly available wheat genome and wheat full length cDNA databases (TriFLDB) to identify the respective orthologues from wheat. 163 TaHSP20 (including 109 sHSP and 54 ACD) genes were identified and classified according to the sub-cellular localization and phylogenetic relationship with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica). Spatio-temporal, biotic and abiotic stress-specific expression patterns in normalized RNA seq and wheat array datasets revealed constitutive as well as inductive responses of HSP20 in different tissues and developmental stages of wheat. Promoter analysis of TaHSP20 genes showed the presence of tissue-specific, biotic, abiotic, light-responsive, circadian and cell cycle-responsive cis-regulatory elements. 14 TaHSP20 family genes were under the regulation of 8 TamiRNA genes. The expression levels of twelve HSP20 genes were studied under abiotic stress conditions in the drought- and heat-tolerant wheat genotype C306. Of the 13 TaHSP20 genes, TaHSP16.9H-CI showed high constitutive expression with upregulation only under salt stress. Both heat and salt stresses upregulated the expression of TaHSP17.4-CI, TaHSP17.7A-CI, TaHSP19.1-CIII, TaACD20.0B-CII and TaACD20.6C-CIV, while TaHSP23.7-MTI was specifically induced only under heat stress. Our results showed that the identified TaHSP20 genes play an important role under different abiotic stress conditions. Thus, the results illustrate the complexity of the TaHSP20 gene family and its stress regulation in wheat, and suggest that sHSPs as attractive breeding targets for improvement of the heat tolerance of wheat.

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.

Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench)

Modification of lignin composition and content are important to enhance the saccharification potential of lignocellulosic biomass. Brown midrib (bmr) mutants with altered lignin and enhanced glucose yields are a valuable resource for modification of the lignin biosynthetic pathway in sorghum (Sorghum bicolor (L.) Moench). Of the 38 bmr mutants reported in sorghum, some have been classified into four independent groups, namely bmr2, bmr6, bmr12 and bmr19, based on the allelic test, and a few have been characterized at the molecular level. The bmr2, bmr6 and bmr12 groups have mutations that impair 4-coumarate:coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD2) and caffeic O-methyltransferase (COMT), respectively. The molecular basis of bmr19 is unknown. In the present study, four spontaneous bmr mutants of sorghum were analyzed for allelic variation at two candidate gene loci. cDNAs of CAD2 and COMT genes were cloned and sequenced from these mutants. Sequence analysis revealed that two of these mutants, IS23789 and IS23253, share a new allele of CAD2. These mutants have a G-to-C transversion at position 3699 of the genomic sequence that leads to glycine-to-arginine (G191R) substitution in the CAD2 protein sequence. This mutation lies in the highly conserved glycine-rich motif 188G(X)GGV(L)G193 that participates in the binding of the pyrophosphate group of NADP+ cofactor and hence might impair the activity of CAD2. Phloroglucinol staining of midribs of these mutants also showed a dark wine-red color that is characteristic of the bmr6 group. These two mutants can be distinguished by an intron length polymorphic marker developed based on the COMT gene sequence in this study. Mutant IS23549, which has also been assigned to the bmr6 group, was found to have another new allele with alanine-to-valine (A164V) substitution in CAD2. Alanine-164 is highly conserved among MDR proteins in plants and hence may be necessary for the activity of the enzyme. In mutant IS11861, there was no mutation that led to a change in amino acid in CAD2, while a threonine-to-serine (T302S) substitution was found in COMT. This single nucleotide polymorphism (SNP) at position 2645 in the COMT gene was converted into a cleaved amplified polymorphic sequence marker that can be used for its identification. In addition, additional SNP- and/or indel-based markers were developed, which can be used for exploiting these alleles in the molecular breeding of sorghum for dedicated bioenergy feedstock.

Differential Regulation of Genes Coding for Organelle and Cytosolic ClpATPases under Biotic and Abiotic Stresses in Wheat

A sub-group of class I Caseinolytic proteases (Clps) function as molecular chaperone and confer thermotolerance to plants. We identified class I Clp family consisting of five ClpB/HSP100, two ClpC, and two ClpD genes from bread wheat. Phylogenetic analysis showed that these genes were highly conserved across grass genomes. Subcellular localization prediction revealed that TaClpC and TaClpD subgroup proteins and TaClpB1 proteins are potentially targeted to chloroplast, while TaClpB5 to mitochondria, and TaClpB2, TaClpB3, and TaClpB4 to cytoplasm. Spatio-temporal expression pattern analysis revealed that four TaClpB and TaClpD2 genes are expressed in majority of all tissues and developmental stages of wheat. Real-time RT-PCR analysis of expression levels of Clp genes in seven wheat genotypes under different abiotic stresses revealed that genes coding for the cytosolic Clps namely TaClpB2 and TaClpB3 were upregulated under heat, salt and oxidative stress but were downregulated by cold stress in most genotypes. In contrast, genes coding for the chloroplastic Clps TaClpC1, TaClpC2, and TaClpD1 genes were significantly upregulated by mainly by cold stress in most genotypes, while TaClpD2 gene was upregulated >2 fold by salt stress in DBW16. The TaClpB5 gene coding for mitochondrial Clp was upregulated in all genotypes under heat, salt and oxidative stresses. In addition, we found that biotic stresses also upregulated TaClpB4 and TaClpD1. Among biotic stresses, Tilletia caries induced TaClpB2, TaClpB3, TaClpC1, and TaClpD1. Differential expression pattern under different abiotic and biotic stresses and predicted differential cellular localization of Clps suggest their non-redundant organelle and stress-specific roles. Our results also suggest the potential role of Clps in cold, salt and biotic stress responses in addition to the previously established role in thermotolerance of wheat.

Transcriptional regulation of ABA core signaling component genes in sorghum (Sorghum bicolor L. Moench)

Abscisic acid (ABA) plays an important role in growth, development and adaptation of plants to environmental stresses. The mechanism of ABA signal transduction involves three core components namely ABA receptors [pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory component of ABA receptor (RCAR)], clade A PP2Cs and Class III SnRK2 family proteins. In the present study, we identified and analyzed the core components of ABA signaling in sorghum, which is known for its drought tolerance. Genome wide in silico analysis led to the identification of eight PYL ABA receptors, nine clade A PP2Cs and three class III SnRK2 family members. Abiotic stresses and exogenous ABA-mediated transcriptional changes of the genes encoding ABA core signaling components were analyzed at seedling stage. All the members of SbPYL gene family were downregulated, except SbPYL1 and SbPYL7 which showed significant upregulation in leaf under drought stress. SbPYL1 and SbPYL5 were upregulated in response to ABA, cold, high salt and PEG-induced osmotic stress, while SbPYL4 showed significant upregulation only under cold stress. Expression levels of the SbPP2C genes were higher or unaffected in response to exogenous ABA and abiotic stresses in leaf except SbPP2C5, which decreased under cold stress. SbPP2C4, SbPP2C5 and SbPP2C6 were highly induced (up to 56-fold–99-fold increase) under different stresses. Expression of class III SbSnRK2 genes was either unaffected or downregulated under abiotic stresses and exogenous ABA. Heat stress downregulated the expression of all the ABA core signaling component genes except that of SbPP2C6 which was upregulated under heat stress. In general, abiotic stresses upregulated the expression of PP2Cs but downregulated the expression of SnRK2 in sorghum seedlings. Differential stress-responsive expression and less number of PYLs in sorghum as compared with Arabidopsis suggest that SbPYL family members might have acquired distinct functions during evolution.

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.

Ear culture as a technique to overcome hybrid necrosis in wheat

Hybrid necrosis in wheat is a problem for gene transfer in wheat breeding. Hybrid necrosis occurs due to dominant complementary interaction of two genes, Ne1 and Ne2. A cross between wheat (Triticum aestivum L.) varieties C306 (drought tolerant, Ne1 carrier) and WL711(high yielding, Ne2 carrier) produced necrotic F1 hybrids, which died before ear emergence and produced no seeds. To overcome the problem of hybrid necrosis, ears enclosed in the leaf sheath were taken and cultured to maturity in liquid medium containing 5% sucrose and 0.04% glutamine. The necrotic hybrids produced only a few seeds per ear compared to parents, but individual grain weight was similar in the hybrid and the parents. The F1 ear culture study has been repeated for three years and F2 seeds obtained. In 1996–97, the cultured ears of F1 hybrids produced 62 seeds, of which only 52 showed germination and were grown under normal field conditions. Out of the 52 seeds, 50% were non-necrotic and showed segregation for various physiological traits. The results reveal that hybrids ears had the potential to form viable seeds. Culturing of wheat ears before ear emergence and production of viable F2 seeds from necrotic hybrids is a simple and efficient method for overcoming the problem of hybrid necrosis.

ABA Receptors: Prospects for Enhancing Biotic and Abiotic Stress Tolerance of Crops

The plant stress hormone abscisic acid (ABA) regulates myriad of plant developmental programs such as germination, root development, vegetative growth, seed development, dormancy, and seed desiccation tolerance. ABA, the master controller of transpiration, regulates ion channels and gene expression that are necessary for abiotic stress tolerance of plants and hence popularly called as the plant stress hormone. In the past one decade, the role of ABA in regulation of biotic stress tolerance is emerging. In response to low leaf water status as well as pathogen-associated molecular pattern (PAMP) signaling, ABA induces closure of stomata, which are major gateways of pathogens entry into plant cells. Salicylic acid (SA) promotes systemic acquired resistance (SAR) to biotrophic lifestyle, while jasmonic acid (JA) and ethylene positively regulate induced systemic resistance (ISR) against necrotrophic pathogens and insects. In addition to its role in PAMP-mediated stomatal closure, ABA interacts synergistically or antagonistically with SA, JA, and ethylene to regulate disease resistance pathway. Intense efforts made since 1980s have unraveled the molecular details of ABA signaling, culminating with the breakthrough discovery of the START domain proteins PYR/PYL/RCAR as ABA receptors (ABAR) and identification of core components of ABA signaling in 2009. Recent studies have also revealed the critical role of ABA receptors in plant processes such as fruit ripening and secondary metabolite accumulation. Genetic manipulation of ABA signaling is envisaged as a potential tool for enhancing plant development and biotic and abiotic stress tolerance of crops. This chapter focuses on molecular and structural basis of ABA signaling. Further, it explores the potential of genetic engineering of core components, protein engineering to develop orthogonal receptor, and development of novel synthetic agonists of ABARs for improving crop yield, quality, and stress tolerance.

Biotechnological Applications for Improvement of Drought Tolerance

Biotechnology has contributed significantly toward understanding the fundamentals of plant biology. It is steadily making headway in to the agricultural sector for improving quality and sustaining yield under different environmental stress conditions. However, progress in developing transgenic plants with enhanced drought tolerance is relatively slow. Drought tolerance is a quantitative trait. Plants respond to water deficit by different physiological, molecular, and biochemical mechanisms. Several genes and gene networks have been identified and have been shown to confer drought tolerance in different plant species. However, most of these studies on drought tolerance are limited to controlled conditions of lab or greenhouses. There are a few studies where evaluation of transgenic plants has been carried out in field or near-field conditions, and yield advantage has been demonstrated under drought stress. Since performance of crop plants under field condition is essential, this article describes studies on evaluation of transgenic crop plants for drought tolerance under field conditions.