Abiotic Stresses Downregulate Key Genes Involved in Nitrogen Uptake and Assimilation in Brassica juncea L.
ABSTRACT: Abiotic stresses such as salinity, drought and extreme temperatures affect nitrogen (N) uptake and assimilation in plants. However, little is known about the regulation of N pathway genes at transcriptional level under abiotic stress conditions in Brassica juncea. In the present work, genes encoding nitrate transporters (NRT), ammonium transporters (AMT), nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagines synthetase (ASN) were cloned from Brassica juncea L. var. Varuna. The deduced protein sequences were analyzed to predict their subcellular localization, which confirmed localization of all the proteins in their respective cellular organelles. The protein sequences were also subjected to conserved domain identification, which confirmed presence of characteristic domains in all the proteins, indicating their putative functions. Moreover, expression of these genes was studied after 1h and 24h of salt (150 mM NaCl), osmotic (250 mM Mannitol), cold (4°C) and heat (42°C) stresses. Most of the genes encoding nitrate transporters and enzymes responsible for N assimilation and remobilization were found to be downregulated under abiotic stresses. The expression of BjAMT1.2, BjAMT2, BjGS1.1, BjGDH1 and BjASN2 was downregulated after 1hr, while expression of BjNRT1.1, BjNRT2.1, BjNiR1, BjAMT2, BjGDH1 and BjASN2 was downregulated after 24h of all the stress treatments. However, expression of BjNRT1.1, BjNRT1.5 and BjGDH2 was upregulated after 1h of all stress treatments, while no gene was found to be upregulated after 24h of stress treatments, commonly. These observations indicate that expression of most of the genes is adversely affected under abiotic stress conditions, particularly under prolonged stress exposure (24h), which may be one of the reasons of reduction in plant growth and development under abiotic stresses.
Project description:BACKGROUND:Abiotic stresses like drought, heat, cold and salinity cause major productivity loss in the rapeseed-mustard crops (Brassica). Major efforts have been made in the past to identify genes that provide resistance against such stresses. Superoxide dismutase (SOD) proteins, member of the metallo-enzyme family play vital role in protecting plants against abiotic stresses. In the present study, genome-wide analysis of abiotic stress responsive SOD gene family has been done in B. juncea and B. rapa. RESULTS:A total of 29 and 18 SOD genes were identified in B. juncea and B. rapa respectively and chromosome location mapping indicated their wide distribution across genome. On the basis of domain composition, the SODs were phylogenetically classified into sub-groups which was also substantiated by the gene structure and sub-cellular locations of SOD proteins. Functional annotation of SODs was also done by Gene Ontology (GO) mapping and the result was corroborated by the identified cis-regulatory elements in the promoter region of SOD genes. Based on FPKM analysis of SRA data available for drought, heat and salt stress, we identified 14 and 10 abiotic stress responsive SOD genes in B. rapa and B. juncea respectively. The differential expression analysis under drought and heat stress of identified abiotic-stress responsive SOD genes was done through quantitative Real Time PCR. CONCLUSION:We identified abiotic-stress responsive genes that could help in improving the plant tolerance against abiotic stresses. This was the first study to describe the genome-wide analysis of SOD gene family in B. rapa and B. juncea, and the results will help in laying basic ground for future work of cloning and functional validation of SOD genes during abiotic stresses leading to Brassica crop improvement.
Project description:Brassica juncea var. Varuna is an economically important oilseed crop of family Brassicaceae which is vulnerable to abiotic stresses at specific stages in its life cycle. Till date no attempts have been made to elucidate genome-wide changes in its transcriptome against high temperature or drought stress. To gain global insights into genes, transcription factors and kinases regulated by these stresses and to explore information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de-novo assembly to discover B. juncea transcriptome associated with high temperature and drought stresses.We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS). More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPdenovo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Many of these were responsive to high temperature, drought or both stresses. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families, respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes.Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resource generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity.
Project description:Micro RNAs (miRNAs) are involved in diverse biological processes including adaptive response towards abiotic stresses. To unravel small RNAs and more specifically miRNAs that can potentially regulate determinants of abiotic stress tolerance, next generation sequencing of B. juncea seedlings subjected to high temperature, high salt and drought conditions was carried out. With the help of UEA sRNA workbench software package, 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions.
Project description:Purpose: Brassica. juncea is vulnerable to abiotic stresses at specific stages in its life cycle. However, till date no attempts have been made to elucidate the genome-wide changes in the transcriptome of B. juncea subjected to either high temperature or drought stress. Hence, to gain global insights into genes, transcription factors and kinases regulated by these stresses and to provide basic information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de novo assembly to discover B. juncea transcriptome associated with high temperature and drought. Results: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS) from control, high temperature treated and drought treated seedlings of Brassica juncea. More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPde-novo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Investigation of identified transcription factors revealed that 92 responded to high temperature, 72 exhibited alterations in expression during drought stress, and 60 were commonly associated with both the stresses. Similarly, 217, 259 and 193 kinases were responsive to high temperature, drought or both stresses, respectively. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. Conclusions: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resources generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity. Total three RNA-Seq libraries were prepared and sequenced independently [B. juncea control (BC), B. juncea high temperature stressed (BHS) and B. juncea drought stressed (BDS) on Illumina GAIIx sequencer].
Project description:The AP2/ERF superfamily of transcription factors is one of the largest transcription factor families in plants and plays an important role in plant development processes and stress responses. In this study, BjABR1, an AP2/ERF superfamily gene, from tuber mustard (Brassica juncea var. tumida Tsen et Lee), sharing high amino acid sequence similarity with the AtABR1 (Arabidopsis thaliana AP2-like abscisic acid repressor 1) gene, were performed functional research, and the ABR1 homologous genes in Brassica species were identified and performed phylogenetic analysis. The promoter sequence of BjABR1 contained many phytohormone- and stress-related cis-elements; ABA (abscisic acid) and abiotic stresses can induce BjABR1 expression in tuber mustard; overexpression of BjABR1 in Arabidopsis can alleviate plant sensitivity to ABA and salt and osmotic stresses, and the alleviation may be due to changes in stress/ABA-induced gene expression. These results indicated that BjABR1 functions in ABA and abiotic stress responses. By BLAST searches against the genome database of five Brassica species (three diploids, B. rapa, B. nigra, and B. oleracea, and two allotetraploid, B. juncea and B. napus) using the protein sequence of AtABR1, 3, 3, 3, 6, and 5 ABR1 homologous genes in B. nigra, B. rapa, B. oleracea, B. juncea, and B. napus were identified, respectively, and they shared high sequence similarity. By sequence analysis, annotation mistakes of the protein-coding regions of two ABR1 homologous genes, GSBRNA2T00134741001 and BjuB007684, were found and corrected. Then, the evolution analysis of these ABR1 homologous genes showed that the ancestor of the three diploid species had three ABR1 homologous genes and each diploid inherited all the three genes from their ancestor; then, allotetraploid B. juncea inherited all the six genes from B. rapa and B. nigra with no gene lost, while allotetraploid B. napus inherited all the three genes from B. oleracea and two genes from B. rapa with one gene lost, indicating that ABR1 homologous genes possessed greater hereditary conservation in Brassica species. The ABR1 homologous genes between B. rapa and B. oleracea shared much higher sequence similarity compared to that of B. nigra in diploid species, indicating that ABR1 homologous genes in B. nigra had experienced more rapid evolution, and B. rapa and B. oleracea may share closer relationship compared to B. nigra. Moreover, the spatial and temporal expression analysis of six ABR1 homologous genes of tuber mustard showed that they possessed different expression models. These results imply that ABR1 homologous genes are important to Brassica plants, and they may possess similar function in ABA and abiotic stress responses but play a role in different tissues and growing stages of plant. This study will provide the foundation to the functional research of ABR1 homologous genes in the Brassica species and help to reveal and understand the evolution mechanisms of Brassica species.
Project description:Transport inhibitor response 1/auxin signaling f-box proteins (TIR1/AFBs) play important roles in the process of plant growth and development as auxin receptors. To date, no information has been available about the characteristics of the TIR1/AFB gene family in Brassica juncea var. tumida. In this study, 18 TIR1/AFB genes were identified and could be clustered into six groups. The genes are located in 11 of 18 chromosomes in the genome of B. juncea var. tumida, and similar gene structures are found for each of those genes. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses are found in the promoter of BjuTIR1/AFB genes. The results of qPCR analysis show that most genes have differential patterns of expression among six tissues, with the expression levels of some of the genes repressed by salt stress treatment. Some of the genes are also responsive to pathogen Plasmodiophora brassicae treatment. This study provides valuable information for further studies as to the role of BjuTIR1/AFB genes in the regulation of plant growth, development, and response to abiotic stress.
Project description:Abscisic acid (ABA) plays important roles in multiple physiological processes, such as plant response to stresses and plant development. The ABA receptors pyrabactin resistance (PYR)/ PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) play a crucial role in ABA perception and signaling. However, little is known about the details regarding PYL family genes in Brassica juncea var. tumida. Here, 25 PYL family genes were identified in B. juncea var. tumida genome, including BjuPYL3, BjuPYL4s, BjuPYL5s, BjuPYL6s, BjuPYL7s, BjuPYL8s, BjuPYL10s, BjuPYL11s, and BjuPYL13. The results of phylogenic analysis and gene structure showed that the PYL family genes performed similar gene characteristics. By analyzing cis-elements in the promoters of those BjuPYLs, several hormone and stress related cis-elements were found. The results of gene expression analysis showed that the ABA receptor homologous genes were induced by abiotic and biotic stress. The tissue-specific gene expression patterns of BjuPYLs also suggested those genes might regulate the stem swelling during plant growth. These findings indicate that BjuPYLs are involved in plant response to stresses and organ development. This study provides valuable information for further functional investigations of PYL family genes in B. juncea var. tumida.
Project description:The Salt Overly Sensitive (SOS) pathway in Arabidopsis thaliana plays important roles in maintaining appropriate ion homeostasis in the cytoplasm and regulating plant tolerance to salinity. However, little is known about the details regarding SOS family genes in the tuber mustard crop (Brassica juncea var. tumida). Here, 12 BjSOS family genes were identified in the B. juncea var. tumida genome including two homologous genes of SOS1, one and three homologs of SOS2 and SOS3, two homologs of SOS4, two homologs of SOS5 and two homologs of SOS6, respectively. The results of conserved motif analysis showed that these SOS homologs contained similar protein structures. By analyzing the cis-elements in the promoters of those BjSOS genes, several hormone- and stress-related cis-elements were found. The results of gene expression analysis showed that the homologous genes were induced by abiotic stress and pathogen. These findings indicate that BjSOS genes play crucial roles in the plant response to biotic and abiotic stresses. This study provides valuable information for further investigations of BjSOS genes in tuber mustard.
Project description:Abiotic stress is one of the major factors responsible for huge yield loss in crop plants. MicroRNAs play a key role in adaptive responses of plants under abiotic stress conditions through post-transcriptional gene regulations. In present study, 95 potential miRNAs were predicted in Brassica juncea using comparative genomics approach. It was noted that these miRNAs, target several transcription factors (TFs), transporter family proteins, signaling related genes, and protease encoding genes. Nineteen distinct miRNA-target regulatory networks were observed with significant involvement in regulation of transcription, response to stimulus, hormone and auxin mediated signaling pathway related gene ontology (GO) term. The sucrose-starch metabolism and pentose-gluconate interconversion pathways were found significantly enriched for these target genes. Molecular markers such as Simple Sequence Repeats (SSR) and Single Nucleotide Polymorphism (SNPs) were identified on miRNAs (miR-SSRs and miR-SNPs) and their target genes in B. juncea. Notably, one of the miR-SNP (C/T) was found at the 5th position on mature region of miR2926. This C/T transition led to the distorted and unstable hairpin structure of miR2926, consequently complete loss of target function. Hence, findings from this study will lay a foundation for marker assisted breeding for abiotic stress tolerant varieties of B. juncea.
Project description:Brassica juncea is promising for metal phytoremediation, but little is known about the functional role of most metal transporters in this plant. The functional characterization of two B. juncea cation-efflux family proteins BjCET3 and BjCET4 is reported here. The two proteins are closely related to each other in amino acid sequence, and are members of Group III of the cation-efflux transporters. Heterologous expression of BjCET3 and BjCET4 in yeast confirmed their functions in exporting Zn, and possibly Cd, Co, and Ni. Yeast transformed with BjCET4 showed higher metal resistance than did BjCET3 transformed. The two BjCET-GFP fusion proteins were localized to the plasma membrane in the roots when expressed in tobacco, and significantly enhanced the plants' Cd tolerance ability. Under Cd stress, tobacco plants transformed with BjCET3 accumulated significant amounts of Cd in shoots, while maintaining similar shoot biomass production with vector-control subjects. Transformed BjCET4 tobacco plants showed significantly enhanced shoot biomass production with markedly decreased shoot Cd content. The two transporter genes have a lower basal transcript expression in B. juncea seedling tissues when grown in normal conditions than under metal-stress, however, their transcripts levels could be substantially increased by Zn, Cd, NaCl or PEG, suggesting that BjCET3 and BjCET4 may play roles in several stress conditions, roles which appear to be different from those of previous characterized cation-efflux transporters, for example, AtMTP1, BjCET2, and BjMTP1.