https://digital.csic.es/bitstream/10261/248575/1/emergiplant.pdf

Emerging roles for conjugated sterols in plants.

Plant secondary metabolism linked glycosyltransferases: An update on expanding knowledge and scopes

Farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development.

Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches.

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De Novo Assembly, Functional Annotation and Comparative Analysis of Withania somnifera Leaf and Root Transcriptomes to Identify Putative Genes Involved in the Withanolides Biosynthesis

Background 
Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.


Methodology 
The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.


Results 
The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.


Conclusions 
Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.

/pdf/overexpression-of-wssgtl1-gene-of-withania-somnifera-5an54tbgtw.pdf

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance, Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Withania somnifera (L.) Dunal (Family, Solanaceae), commonly known as Ashwagandha is one of the most valuable medicinal plants synthesizing large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized a gene encoding farnesyl diphosphate synthase (FPPS; EC 2.5.1.10), a key enzyme in the pathway of biosynthesis of isoprenoids, from W. somnifera. The full-length cDNA of Withania somnifera FPPS (WsFPPS) of 1,253 bps encodes a polypeptide of 343 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsFPPS has close similarity to its counterparts from tomato (SlFPPS) and capsicum (CaaFPPS). Using semi quantitative RT–PCR, the expression pattern of the WsFPPS gene was analyzed in different tissues of Withania chemotypes (NMITLI-101, NMITLI-108, NMITLI-118 and NMITLI-135) as well as in response to elicitors (salicylic acid and methyl jasmonate) and mechanical wounding. The expression analysis suggests that WsFPPS expression varies in different tissues (with maximal expression in flower and young leaf) and chemotypes (with highest level in NMITLI-101) and was significantly elevated in response to salicylic acid, methyl jasmonate and mechanical injury. This is the first report on characterization of an isoprenoid pathway gene involved in withanolide biosynthesis.

Differential expression of farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in response to elicitors

Withania somnifera (L.) Dunal is one of the most valuable medicinal plants synthesizing a large number of pharmacologically active secondary metabolites known as withanolides, the C28-steroidal lactones derived from triterpenoids. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the biosynthetic pathway and genes responsible for biosynthesis of these compounds. In this study, we have characterized the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) catalyzing the key regulatory step of the isoprenoid biosynthesis. The 1,728-bp full-length cDNA of Withania HMGR (WsHMGR) encodes a polypeptide of 575 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsHMGR has typical structural features of other known plant HMGRs. The relative expression analysis suggests that WsHMGR expression varies in different tissues as well as chemotypes and is significantly elevated in response to exposure to salicylic acid, methyl jasmonate, and mechanical injury. The functional color assay in Escherichia coli showed that WsHMGR could accelerate the biosynthesis of carotenoids, establishing that WsHMGR encoded a functional protein and may play a catalytic role by its positive influence in isoprenoid biosynthesis.

Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant.

Sterol glycosyltransferases (SGTs) catalyze the transfer of sugar molecules to diverse sterol molecules, leading to a change in their participation in cellular metabolism. Withania somnifera is a medicinal plant rich in sterols, sterol glycosides and steroidal lactones. Sterols and their modified counterparts are medicinally important and play a role in adaptation of the plant to stress conditions. We have identified 3 members of SGT gene family through RACE (Rapid Amplification of cDNA Ends) in addition to sgtl1 reported earlier. The amino acid sequence deduced from the ORF's showed homology (45-67 %) to the reported plant SGTs. The expression of the genes was differentially modulated in different organs in W. somnifera and in response to external stimuli. Salicylic acid and methyl jasmonate treatments showed up to 10 fold increase in the expression of sgt genes suggesting their role in defense. The level of expression increased in heat and cold stress indicating the role of sterol modifications in abiotic stress. One of the members, was expressed in E. coli and the enzyme assay showed that the crude enzyme glycosylated stigmasterol. W. somnifera expresses a family of sgt genes and there is a functional recruitment of these genes under stress conditions. The genes which are involved in sterol modification are important in view of medicinal value and understanding stress.

Sterol glycosyltransferases-identification of members of gene family and their role in stress in Withania somnifera.

Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-d-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon–intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.

Nehal Akhtar

Papers

Differential expression of farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in response to elicitors

Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant.

Sterol glycosyltransferases-identification of members of gene family and their role in stress in Withania somnifera.

Cloning and characterization of 2-C-methyl-d-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera