Papers by Sangeeta Dhaubhadel
BMC Plant Biology, 2014
Cyclophilins (CYPs) belong to the immunophilin superfamily, and have peptidyl-prolyl cis-trans is... more Cyclophilins (CYPs) belong to the immunophilin superfamily, and have peptidyl-prolyl cis-trans isomerase (PPIase) activity. PPIase catalyzes cis- and trans-rotamer interconversion of the peptidyl-prolyl amide bond of peptides, a rate-limiting step in protein folding. Studies have demonstrated the importance of many PPIases in plant biology, but no genome-wide analysis of the CYP gene family has been conducted for a legume species. Here we performed a comprehensive database survey and identified a total of 62 CYP genes, located on 18 different chromosomes in the soybean genome (GmCYP1 to GmCYP62), of which 10 are multi- and 52 are single-domain proteins. Most of the predicted GmCYPs clustered together in pairs, reflecting the ancient genome duplication event. Analysis of gene structure revealed the presence of introns in protein-coding regions as well as in 5' and 3' untranslated regions, and that their size, abundance and distribution varied within the gene family. Expression analysis of GmCYP genes in soybean tissues displayed their differential tissue specific expression patterns. Overall, we have identified 62 CYP genes in the soybean genome, the largest CYP gene family known to date. This is the first genome-wide study of the CYP gene family of a legume species. The expansion of GmCYP genes in soybean, and their distribution pattern on the chromosomes strongly suggest genome-wide segmental and tandem duplications.
Phytochemicals – Biosynthesis, Function and Application, 2014
Soybean - Biochemistry, Chemistry and Physiology, 2011
... Dixon 2005, Lozovaya et al. 2007, Sreevidya et al. 2006, Subramanian et al. 2005) or introduc... more ... Dixon 2005, Lozovaya et al. 2007, Sreevidya et al. 2006, Subramanian et al. 2005) or introducing foreign transcription factors to up-regulate the synthesis of the metabolites (Yu & McGonigle 2005, Yu et al. 2003). One of the in ...
PLOS ONE, 2015
14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play... more 14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses.
Phytochemistry Reviews, 2015
MYB transcription factors are one of the largest transcription factor families characterized in p... more MYB transcription factors are one of the largest transcription factor families characterized in plants. They are classified into four types: R1 MYB, R2R3 MYB, R3 MYB and R4 MYB. GmMYB176 is an R1 MYB transcription factor that regulates chalcone synthase (CHS8) gene expression and isoflavonoid biosynthesis in soybean. Silencing of GmMYB176 supressed the expression of CHS8 gene and reduced the accumulation of isoflavonoids in soybean hairy roots while the overexpression of GmMYB176 did not alter both the target gene expression and metabolite level suggesting that GmMYB176 alone is not sufficient for CHS8 gene regulation and it may act cooperatively with other factor (s) for CHS8 gene activation in soybean. The current research is designed to identify and characterize the GmMYB176 interactome for CHS8 gene regulation. GmMYB176 interacting proteins have been purified by over-expressing GmMYB176–YFP fusion proteins in soybean hairy roots, followed by co-immunoprecipitation. The purified ...
PROTEOMICS, 2015
Soybean seeds are the major human dietary source of isoflavonoids, a class of plant natural produ... more Soybean seeds are the major human dietary source of isoflavonoids, a class of plant natural products almost entirely exclusive to legumes. Isoflavonoids reduce the risk of a number of chronic human illnesses. Biosynthesis and accumulation of this class of compounds is a multigenic and complex trait, with a great deal of variability among soybean cultivars and with respect to the environment. There is a wealth of genomic, transcriptomic, and metabolomics data regarding isoflavonoid biosynthesis, but the connection between multigene families and their cognate proteins is a missing link that could provide us with a great deal of functional information. The changing proteome of the developing seed can shed light on the correlative increase in isoflavonoids, while the maternal seed coat proteome can provide the link with inherited metabolic and signaling machinery. In this effort, 'seed-filling' proteomics has revealed key secondary metabolite enzymes that quantitatively vary throughout seed development. Seed coat proteomics has revealed the existence of metabolic apparatus specific to isoflavonoid biosynthesis (isoflavonoid reductase) that could potentially influence the chemical content of this organ. The future of proteomic analysis of isoflavonoid biosynthesis should be centered on the development of quantitative, tissue-specific proteomes that emphasize low-abundance metabolic proteins to extract the whole suite of factors involved.
Genetics, Genomics, and Breeding of Soybean, 2010
Seed Development: OMICS Technologies toward Improvement of Seed Quality and Crop Yield, 2012
Planta, 2014
Soybean chalcone isomerase (CHI) family contains twelve members with unique evolutionary backgrou... more Soybean chalcone isomerase (CHI) family contains twelve members with unique evolutionary background, expression patterns and is compartmentalized to specific subcellular locations. The phenylpropanoid pathway produces a diverse array of plant natural products. A key branch-point enzyme, chalcone isomerase, catalyzes the reaction producing flavanones, the backbone for many downstream metabolites such as flavonoids and isoflavonoids. We have identified twelve soybean GmCHIs that fall into four subfamilies. The study of this family in soybean in the context of various CHIs and CHI-like proteins, across divisions in the plant kingdom and beyond, shows an evolutionary journey from fatty acid-binding proteins (FAPs) to sterically restricted folds that gave rise to the chalcone-to-flavanone isomerase. There are four GmCHIs with this functionality, three of which belong to a legume-specific clade known as 'type II' CHIs. Tissue-specific expression of eight core members of the soybean CHI family showed differential temporal and spatial expression, pointing to the potential function of GmCHI1A in seed isoflavonoid production. Promoter analysis of the GmCHIs described the minutiae of sub-organ expression patterns. Subcellular localization of the family was conducted to investigate the possibility of pathway-specific compartmentalization. Subfamilies 1, 2 and 4 localized to the nucleus and cytoplasm, with nuclear localization of CHIs raising questions about alternate function. GmCHI3 isoforms localized to the chloroplast, which, in conjunction with their position on the phylogenetic tree and expression patterns, closely associates them with the FAPs. This study provides the first comprehensive look at soybean CHIs, a family of unique evolutionary background and biochemical function, with the catalytically active members producing the backbone substrate in an important plant metabolic pathway.
Plant Signaling & Behavior, 2010
We recently identified a novel R1 MYB transcription factor, GmMYB176, which regulates the CHS8 ge... more We recently identified a novel R1 MYB transcription factor, GmMYB176, which regulates the CHS8 gene expression and influences isoflavonoid biosynthesis in soybeans. GmMYB176 recognizes a unique sequence motif [TAGT(T/A)(A/T)] in CHS8 promoter and binds with it. The in planta role of GmMYB176 was established by RNAi silencing of GmMYB176 in soybean hairy roots. Silencing of GmMYB176 reduced the expression of CHS8 gene expression and isoflavonoid accumulation in hairy roots. However, the overexpression of GmMYB176 did not lead to increase in both CHS8 expression and isoflavonoid level in hairy roots suggesting that GmMYB176 is essential but not sufficient for CHS8 gene activation.
Journal of Experimental Botany, 2008
Isoflavonoids are a diverse group of biologically active natural products that accumulate in soyb... more Isoflavonoids are a diverse group of biologically active natural products that accumulate in soybean seeds during development. The majority of isoflavonoids are accumulated in the form of their glyco-and malonylconjugates in soybean seeds. The conjugation step confers stability and solubility to isoflavone aglycones enabling their compartmentalization to vacuoles or transport to the site of accumulation. A functional genomic approach was used to identify isoflavonoid specific glycosyltransferase (UGT) and malonyltransferase (MT) from soybean (Glycine max) seeds. An expressed sequence tag database for soybean was searched by key words to make a list of candidate genes. The full-length cDNAs for candidate UGTs and MTs were obtained and cloned into an expression vector for the production of recombinant enzymes. The in vitro enzymatic activity assays were conducted for recombinant UGTs and MTs using uridine diphosphate glucose and malonyl CoA, respectively, as donors with isoflavone substrates. Among several recombinant enzymes, UGT73F2 showed glycosylation activity towards all three soybean isoflavone aglycones and GmMT7 exhibited malonylation activity towards isoflavone glycosides. The subcellular localization study revealed both UGT73F2 and GmMT7 to be in the cytoplasm. The transcripts and protein accumulation patterns for UGT73F2 and GmMT7 genes have provided further support for their in planta function.
Journal of Experimental Botany, 2012
In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylati... more In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short-(6 h) and long-(2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.
The Plant journal : for cell and molecular biology, 2012
Isoflavonoids are legume-specific natural plant compounds that play important functions in nitrog... more Isoflavonoids are legume-specific natural plant compounds that play important functions in nitrogen fixation as well as biotic and abiotic stress responses. Many clinical studies have suggested a role for isoflavonoids in human health and nutrition. We have recently identified an R1 MYB transcription factor GmMYB176 that regulates CHS8 gene expression and isoflavonoid biosynthesis. Here we demonstrate that binding of 14-3-3 proteins to GmMYB176 modulates this function. GmMYB176 interacts with all 16 14-3-3 proteins (SGF14s) in soybean (Glycine max) with varying activity. The detailed analysis of 14-3-3-binding sites within GmMYB176 identified a critical motif (D2) where Ser29 is potentially phosphorylated. Deletion of the D2 motif from GmMYB176 or substitution of Ser29 with an alanine abolished binding with SGF14 proteins, which altered the subcellular localization of GmMYB176. Overexpression of SGF14l in soybean hairy roots did not affect the transcript level of GmMYB176 but it red...
The Plant Journal, 2010
Here we demonstrate that GmMYB176 regulates CHS8 expression and affects isoflavonoid synthesis in... more Here we demonstrate that GmMYB176 regulates CHS8 expression and affects isoflavonoid synthesis in soybean. We previously established that CHS8 expression determines the isoflavonoid level in soybean seeds by comparing the transcript profiles of cultivars with different isoflavonoid contents. In the present study, a functional genomic approach was used to identify the factor that regulates CHS8 expression and isoflavonoid synthesis. Candidate genes were cloned, and co-transfection assays were performed in Arabidopsis leaf protoplasts. The results showed that GmMYB176 can trans-activate the CHS8 promoter with maximum activity. Transient expression of GmMYB176 in soybean embryo protoplasts increased endogenous CHS8 transcript levels up to 169-fold after 48 h. GmMYB176 encodes an R1 MYB protein, and is expressed in soybean seed during maturation. Furthermore, GmMYB176 recognizes a 23 bp motif containing a TAGT(T/A)(A/T) sequence within the CHS8 promoter. A subcellular localization study confirmed nuclear localization of GmMYB176. A predicted pST binding site for 14-3-3 protein is required for subcellular localization of GmMYB176. RNAi silencing of GmMYB176 in hairy roots resulted in reduced levels of isoflavonoids, showing that GmMYB176 is necessary for isoflavonoid biosynthesis. However, over-expression of GmMYB176 was not sufficient to increase CHS8 transcript and isoflavonoid levels in hairy roots. We conclude that an R1 MYB transcription factor, GmMYB176, regulates CHS8 expression and isoflavonoid synthesis in soybean.
We have used cDNA microarray analysis to examine changes in gene expression during embryo develop... more We have used cDNA microarray analysis to examine changes in gene expression during embryo development in soybean and to compare gene expression profiles of two soybean cultivars that differ in seed isoflavonoid content. The analysis identified 5,910 genes that were differentially expressed in both soybean cultivars grown at two different locations for two consecutive years in one of the five different stages of embryo development. An ANOVA analysis with P value < 0.05 and < 0.01 indicated that gene expression changes due to environmental factors are greater than those due to cultivar differences. Most changes in gene expression occurred at the stages when the embryos were at 30 or 70 days after pollination (DAP). A significantly larger fraction of genes (48.5%) was expressed throughout the development and showed little or no change in expression. Transcript accumulation for genes related to the biosynthesis of storage components in soybean embryos showed several unique temporal expressions. Expression patterns of several genes involved in isoflavonoid biosynthesis, such as PAL, CHS7, CHS8 and IFS2, were higher at 70 DAP in both the cultivars. Thus, expression of these genes coincides with the onset of accumulation of isoflavonoids in the embryos. A comparative analysis of genes involved in isoflavonoid biosynthesis in RCAT Angora (high seed isoflavonoid cultivar) and Harovinton (low seed isoflavonoid cultivar) revealed that CHS7 and CHS8 were expressed at significantly greater level in RCAT Angora than in Harovinton. Our study provides a detailed transcriptome profiling of soybean embryos during development and indicates that differences in the level of seed isoflavonoids between these two cultivars could be as a result of differential expression of CHS7 and CHS8 during late stages of seed development.
Brassinosteroids are plant growth-promoting compounds that exhibit structural similarities to ani... more Brassinosteroids are plant growth-promoting compounds that exhibit structural similarities to animal steroid hormones. Recent studies have indicated that brassinosteroids are essential for proper plant development. In addition to a role in development, several lines of evidence suggest that brassinosteroids exert anti-stress effects on plants. However, the mechanism by which they modulate plant stress responses is not understood. We show here that Brassica napus and tomato seedlings grown in the presence of 24-epibrassinolide (EBR) are significantly more tolerant to a lethal heat treatment than are control seedlings grown in the absence of the compound. Since a preconditioning treatment of seedlings was not required to observe this effect, we conclude that EBR treatment increases the basic thermotolerance of seedlings. An analysis of heat shock proteins (HSPs) in B. napus seedlings by western blot analysis indicated that the HSPs did not preferentially accumulate in EBR-treated seedlings at the control temperature. However, after heat stress, HSP accumulation was higher in EBR-treated than in untreated seedlings. The results of the present study provide the first direct evidence for EBR-induced expression of HSPs. The higher accumulation of HSPs in EBR-treated seedlings raises the possibility that HSPs contribute, at least in part, to thermotolerance in EBR-treated seedlings. A search for factors other than HSPs, which may directly or indirectly contribute to brassinosteroid-mediated increase in thermotolerance, is underway.
Plant molecular biology, 2003
Isoflavonoids are biologically active natural products that accumulate in soybean seeds during de... more Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pod...
Plant biotechnology journal, 2013
Plants are one of the most economical platforms for large-scale production of recombinant protein... more Plants are one of the most economical platforms for large-scale production of recombinant proteins for biopharmaceutical and industrial uses. A large number of human recombinant proteins of therapeutic value have been successfully produced in plant systems. One of the main technical challenges of producing recombinant proteins in plants is to obtain sufficient level of protein. This research aims to identify the factors that control synthesis and accumulation of recombinant proteins in stable transgenic plants. A stepwise dissection of human immune-regulatory interleukin-10 (IL-10) protein production was carried out using Arabidopsis thaliana as a model system. EMS-mutagenized transgenic Arabidopsis IL-10 lines, at2762 and at3262, produced significantly higher amount of IL-10 protein than the non-mutagenized IL-10 line (WT-IL-10). The fates of trans-gene in these sets of plants were compared in detail by measuring synthesis and accumulation of IL-10 transcript, transcript stability,...
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Papers by Sangeeta Dhaubhadel