Md. Abdul Kayum

Identification and expression analysis of WRKY family genes under biotic and abiotic stresses in Brassica rapa

WRKY proteins constitute one of the largest transcription factor families in higher plants, and they are involved in multiple biological processes such as plant development, metabolism, and responses to biotic and abiotic stresses. Genes of this family have been well documented in response to many abiotic and biotic stresses in many plant species, but not yet against Pectobacterium carotovorum subsp. carotovorum and Fusarium oxysporum f.sp. conglutinans in any of the plants. Moreover, potentiality of a specific gene may vary depending on stress conditions and genotypes. To identify stress resistance-related potential WRKY genes of Brassica rapa, we analyzed their expressions against above-mentioned pathogens and cold, salt, and drought stresses in B. rapa. Stress resistance-related functions of all Brassica rapa WRKY (BrWRKY) genes were firstly analyzed through homology study with existing biotic and abiotic stress resistance-related WRKY genes of other plant species and found a high degree of homology. We then identified all BrWRKY genes in a Br135K microarray dataset, which was created by applying low-temperature stresses to two contrasting Chinese cabbage doubled haploid (DH) lines, Chiifu and Kenshin, and selected 41 BrWRKY genes with high and differential transcript abundance levels. These selected genes were further investigated under cold, salt, and drought stresses as well as after infection with P. carotovorum subsp. carotovorum and F. oxysporum f.sp. conglutinans in B. rapa. The selected genes showed an organ-specific expression, and 22 BrWRKY genes were differentially expressed in Chiifu compared to Kenshin under cold and drought stresses. Six BrWRKY genes were more responsive in Kenshin compared to Chiffu under salt stress. In addition, eight BrWRKY genes showed differential expression after P. carotovorum subsp. carotovorum infection and five genes after F. oxysporum f.sp. conglutinans infection in B. rapa. Thus, the differentially expressed BrWRKY genes might be potential resources for molecular breeding of Brassica crops against abiotic and biotic stresses and several genes, which showed differential expressions commonly in response to several stresses, might be useful for multiple stress resistance. These findings would also be helpful in resolving the complex regulatory mechanism of WRKY genes in stress resistance and for this further functional genomics study of these potential genes in different Brassica crops is essential.

Genome-wide identification and characterization of MADS-box family genes related to organ development and stress resistance in Brassica rapa

BackgroundMADS-box transcription factors (TFs) are important in floral organ specification as well as several other aspects of plant growth and development. Studies on stress resistance-related functions of MADS-box genes are very limited and no such functional studies in Brassica rapa have been reported. To gain insight into this gene family and to elucidate their roles in organ development and stress resistance, we performed genome-wide identification, characterization and expression analysis of MADS-box genes in B. rapa.ResultsWhole-genome survey of B. rapa revealed 167 MADS-box genes, which were categorized into type I (Mα, Mβ and Mγ) and type II (MIKCc and MIKC*) based on phylogeny, protein motif structure and exon-intron organization. Expression analysis of 89 MIKCc and 11 MIKC* genes was then carried out. In addition to those with floral and vegetative tissue expression, we identified MADS-box genes with constitutive expression patterns at different stages of flower development. More importantly, from a low temperature-treated whole-genome microarray data set, 19 BrMADS genes were found to show variable transcript abundance in two contrasting inbred lines of B. rapa. Among these, 13 BrMADS genes were further validated and their differential expression was monitored in response to cold stress in the same two lines via qPCR expression analysis. Additionally, the set of 19 BrMADS genes was analyzed under drought and salt stress, and 8 and 6 genes were found to be induced by drought and salt, respectively.ConclusionThe extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of MADS-box genes in stress resistance in addition to their growth and developmental functions, which ultimately provides the basis for functional characterization and exploitation of the candidate genes for genetic engineering of B. rapa.

Genome-wide expression profiling of aquaporin genes confer responses to abiotic and biotic stresses in Brassica rapa

BackgroundPlants contain a range of aquaporin (AQP) proteins, which act as transporter of water and nutrient molecules through living membranes. AQPs also participate in water uptake through the roots and contribute to water homeostasis in leaves.ResultsIn this study, we identified 59 AQP genes in the B. rapa database and Br135K microarray dataset. Phylogenetic analysis revealed four distinct subfamilies of AQP genes: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs) and small basic intrinsic proteins (SIPs). Microarray analysis showed that the majority of PIP subfamily genes had differential transcript abundance between two B. rapa inbred lines Chiifu and Kenshin that differ in their susceptibility to cold. In addition, all BrPIP genes showed organ-specific expression. Out of 22 genes, 12, 7 and 17 were up-regulated in response to cold, drought and salt stresses, respectively. In addition, 18 BrPIP genes were up-regulated under ABA treatment and 4 BrPIP genes were up-regulated upon F. oxysporum f. sp. conglutinans infection. Moreover, all BrPIP genes showed down-regulation under waterlogging stress, reflecting likely the inactivation of AQPs controlling symplastic water movement.ConclusionsThis study provides a comprehensive analysis of AQPs in B. rapa and details the expression of 22 members of the BrPIP subfamily. These results provide insight into stress-related biological functions of each PIP gene of the AQP family, which will promote B. rapa breeding programs.

Characterization and stress-induced expression analysis of Alfin-like transcription factors in Brassica rapa

AbstractThe Alfin-like (AL) transcription factors (TFs) family is involved in many developmental processes, including the growth and development of roots, root hair elongation, meristem development, etc. However, stress resistance-related function and the regulatory mechanism of these TFs have yet to be elucidated. This study identified 15 Brassica rapa AL (BrAL) TFs from BRAD database, analyzed the sequences and profiled their expression first time in response to Fusarium oxysporum f. sp. conglutinans and Pectobacterium carotovorum subsp. carotovorum in fection, cold, salt and drought stresses in B. rapa. Structural and phylogenetic analyses of 15 BrAL TFs revealed four distinct groups (groups I–IV) with AL TFs of Arabidopsis thaliana. In the expression analyses, ten BrAL TFs showed responsive expression after F. oxysporum f. sp. conglutinans infection, while all BrAL TFs showed responses under cold, salt and drought stresses in B. rapa. Interestingly, ten BrAL TFs showed responses to both biotic and abiotic stress factors tested here. The differentially expressed BrAL TFs thus represent potential resources for molecular breeding of Brassica crops resistant against abiotic and biotic stresses. Our findings will also help to elucidate the complex regulatory mechanism of AL TFs in stress resistance and provide a foundation for further functional genomics studies and applications.

Research on biotic and abiotic stress related genes exploration and prediction in Brassica rapa and B. oleracea: a review.

Global population is increasing day-by-day, simultaneously, crop production need to increase proportionately. Whereas, increase crop production being restricted due to abiotic and biotic stresses. Abiotic stresses are adversely affected crop growth and development, leading to crop loss globally and thereby causing huge amount of economic loss as well. Contrary, pathogens are attacked the plants imposing biotic stress and severely hampers the yield. Therefore, it is prime need to understand the molecular mechanism and genes involved to minimize the biotic and abiotic stresses for mitigating the Brassica vegetable crop losses. The stress responsive, pathogens related genes are involved in tolerance or resistance to stress in plants that are cross-talk with different types of stress components in signal transduction pathways. The plants have their own mechanism to overcome biotic and abiotic stresses to follow the abscisic acid (ABA)-dependent and ABA-independent pathways. Several transcription factors such as WRKY, Alfin-like, MYB, NAC, DREB, CBF are integrating to various stress signals and controlling the gene expression through networking with their related cis-elements. To develop stress tolerance and/or resistant crops plants, there is need to realize both of the plant and pathogenic disease development mechanisms. Therefore, this article is focused on (i) major and devastating stresses on vegetable crops, (ii) role of genes to overcome the stresses, and (iii) differential genes expressed under biotic and abiotic stresses in Brassica oleracea and B. rapa for getting insight of the mechanisms of development of resistance lines.

A Genome-Wide Analysis Reveals Stress and Hormone Responsive Patterns of TIFY Family Genes in Brassica rapa.

The TIFY family is a plant-specific group of proteins with a diversity of functions and includes four subfamilies, viz. ZML, TIFY, PPD, and JASMONATE ZIM-domain (JAZ) proteins. TIFY family members, particularly JAZ subfamily proteins, play roles in biological processes such as development and stress and hormone responses in Arabidopsis, rice, chickpea, and grape. However, there is no information about this family in any Brassica crop. This study identifies 36 TIFY genes in Brassica rapa, an economically important crop species in the Brassicaceae. An extensive in silico analysis of phylogenetic grouping, protein motif organization and intron-exon distribution confirmed that there are four subfamilies of BrTIFY proteins. Out of 36 BrTIFY genes, we identified 21 in the JAZ subfamily, seven in the TIFY subfamily, six in ZML and two in PPD. Extensive expression profiling of 21 BrTIFY JAZs in various tissues, especially in floral organs and at different flower growth stages revealed constitutive expression patterns, which suggest that BrTIFY JAZ genes are important during growth and development of B. rapa flowers. A protein interaction network analysis also pointed to association of these proteins with fertility and defense processes of B. rapa. Using a low temperature-treated whole-genome microarray data set, most of the JAZ genes were found to have variable transcript abundance between the contrasting inbred lines Chiifu and Kenshin of B. rapa. Subsequently, the expression of all 21 BrTIFY JAZs in response to cold stress was characterized in the same two lines via qPCR, demonstrating that nine genes were up-regulated. Importantly, the BrTIFY JAZs showed strong and differential expression upon JA treatment, pointing to their probable involvement in JA-mediated growth regulatory functions, especially during flower development and stress responses. Additionally, BrTIFY JAZs were induced in response to salt, drought, Fusarium, ABA, and SA treatments, and six genes (BrTIFY3a, 3b, 6a, 9a, 9b, and 9c) were identified to have co-responsive expression patterns. The extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of TIFY genes in stress resistance and different developmental functions, which ultimately provides the basis for functional characterization and exploitation of the candidate TIFY genes for genetic engineering of B. rapa.

Genome-Wide Identification, Characterization, and Expression Profiling of Glutathione S-Transferase (GST) Family in Pumpkin Reveals Likely Role in Cold-Stress Tolerance

Plant growth and development can be adversely affected by cold stress, limiting productivity. The glutathione S-transferase (GST) family comprises important detoxifying enzymes, which play major roles in biotic and abiotic stress responses by reducing the oxidative damage caused by reactive oxygen species. Pumpkins (Cucurbita maxima) are widely grown, economically important, and nutritious; however, their yield can be severely affected by cold stress. The identification of putative candidate genes responsible for cold-stress tolerance, including the GST family genes, is therefore vital. For the first time, we identified 32 C. maxima GST (CmaGST) genes using a combination of bioinformatics approaches and characterized them by expression profiling. These CmaGST genes represent seven of the 14 known classes of plant GSTs, with 18 CmaGSTs categorized into the tau class. The CmaGSTs were distributed across 13 of pumpkin’s 20 chromosomes, with the highest numbers found on chromosomes 4 and 6. The large number of CmaGST genes resulted from gene duplication; 11 and 5 pairs of CmaGST genes were segmental- and tandem-duplicated, respectively. In addition, all CmaGST genes showed organ-specific expression. The expression of the putative GST genes in pumpkin was examined under cold stress in two lines with contrasting cold tolerance: cold-tolerant CP-1 (C. maxima) and cold-susceptible EP-1 (Cucurbita moschata). Seven genes (CmaGSTU3, CmaGSTU7, CmaGSTU8, CmaGSTU9, CmaGSTU11, CmaGSTU12, and CmaGSTU14) were highly expressed in the cold-tolerant line and are putative candidates for use in breeding cold-tolerant crop varieties. These results increase our understanding of the cold-stress-related functions of the GST family, as well as potentially enhancing pumpkin breeding programs.

Genome-wide characterization and expression profiling of PDI family gene reveals function as abiotic and biotic stress tolerance in Chinese cabbage (Brassica rapa ssp. pekinensis)

BackgroundProtein disulfide isomerase (PDI) and PDI-like proteins contain thioredoxin domains that catalyze protein disulfide bond, inhibit aggregation of misfolded proteins, and function in isomerization during protein folding in endoplasmic reticulum and responses during abiotic stresses.Chinese cabbage is widely recognized as an economically important, nutritious vegetable, but its yield is severely hampered by various biotic and abiotic stresses. Because of, it is prime need to identify those genes whose are responsible for biotic and abiotic stress tolerance. PDI family genes are among of them.ResultsWe have identified 32 PDI genes from the Br135K microarray dataset, NCBI and BRAD database, and in silico characterized their sequences. Expression profiling of those genes was performed using cDNA of plant samples imposed to abiotic stresses; cold, salt, drought and ABA (Abscisic Acid) and biotic stress; Fusarium oxysporum f. sp. conglutinans infection. The Chinese cabbage PDI genes were clustered in eleven groups in phylogeny. Among them, 15 PDI genes were ubiquitously expressed in various organs, while 24 PDI genes were up-regulated under salt and drought stress. By contrast, cold and ABA stress responsive gene number were ten and nine, respectively. In case of F. oxysporum f. sp. conglutinans infection 14 BrPDI genes were highly up-regulated. Interestingly, BrPDI1–1 gene was identified as putative candidate against abiotic (salt and drought) and biotic stresses, BrPDI5–2 gene for ABA stress, and BrPDI1–4, 6–1 and 9–2 were putative candidate genes for both cold and chilling injury stresses.ConclusionsOur findings help to elucidate the involvement of PDI genes in stress responses, and they lay the foundation for functional genomics in future studies and molecular breeding of Brassica rapa crops. The stress-responsive PDI genes could be potential resources for molecular breeding of Brassica crops resistant to biotic and abiotic stresses.

Molecular characterisation and expression profiling of calcineurin B-like (CBL) genes in Chinese cabbage under abiotic stresses

Calcium signals act as a second messenger in plant responses to various abiotic stresses, which regulate a range of physiological processes. Calcium-binding proteins, like calcineurin B-like (CBL) proteins, belong to a unique group of calcium sensors that play a role in calcium signalling. However, their identities and functions are unknown in Chinese cabbage. In this study, 17 CBL genes were identified from the Brassica rapa L. (Chinese cabbage) database and Br135K microarray datasets. They were used to construct a phylogenetic tree with known CBL proteins of other species. Analysis of genomic distribution and evolution revealed different gene duplication in Chinese cabbage compared to Arabidopsis. The microarray expression analysis showed differential expression of BrCBL genes at various temperatures. Organ-specific expression was observed by RT-PCR, and qRT-PCR analyses revealed responsiveness of BrCBL genes to cold, drought and salt stresses. Our findings confirm that CBL genes are involved in calcium signalling and regulate responses to environmental stimuli, suggesting this family gene have crucial role to play in plant responses to abiotic stresses. The results facilitate selection of candidate genes for further functional characterisation. In addition, abiotic stress-responsive genes reported in this study might be exploited for marker-aided backcrossing of Chinese cabbage.