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Functional Characterization of a Glucosyltransferase Specific to Flavonoid 7-O-Glucosides from Withania somnifera

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Abstract

Flavonoids are a large class of phenylpropanoid-derived secondary metabolites, which are usually glycosylated by UDP-glycosyltransferases with one or more sugar groups. Here, we report the cloning and biochemical characterization of a flavonoid glycosyltransferase gene from Withania somnifera (WsGT), which is an important medicinal plant used in Ayurvedic formulations. Using PCR primers, designed for a highly conserved region of previously reported glycosyltransferases, we were able to isolate the corresponding fragment of the WsGT gene. Rapid amplification of cDNA ends (RACE) was then employed to isolate full-length cDNA, which had an open reading frame of 1,371 bp that encode for 456 amino acids. Phylogenetic analysis indicated that WsGT was similar to that of family 1 GT-B glycosyltransferase. Biochemical analysis revealed that WsGT interacts with UDP-glucose and was capable of regiospecifically glycosylating flavonoid-7-ols, such as apigenin, naringenin, luteolin, diadzein and genistein. Expression profiling studies showed that WsGT was highly expressed in young and mature leaves of W. somnifera. Furthermore, exposure to salicylic acid enhanced the expression of WsGT in the leaves and heat shock treatment resulted in decreased expression of WsGT after an initial increase. This may suggest the role of WsGT in response to abiotic/biotic stresses.

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Acknowledgments

RJSK is thankful to Council of Scientific and Industrial Research (CSIR) for senior research fellowship. We are thankful to Dr. Bhushan Dholakia and Dr. D. Shanmugam for comments on manuscript. We are grateful to Dr. Shrinivas Hotha for providing LC-MS facility and his student Ashif Shaikh for help in LC-MS experiment and analysis. This work was supported by grants from network project CSIR (NWP 0008), India.

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Authors and Affiliations

  1. Plant Tissue Culture Division, National Chemical Laboratory, Homi Bhabha Road, Pune, 411008, Maharashtra, India

    R. J. Santosh Kumar,  Ruby, Somesh Singh, Prashant D. Sonawane, R. K. Vishwakarma & B. M. Khan

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  1. R. J. Santosh Kumar
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  2. Ruby
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  3. Somesh Singh
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  4. Prashant D. Sonawane
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  5. R. K. Vishwakarma
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  6. B. M. Khan
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Corresponding author

Correspondence to B. M. Khan.

Electronic supplementary material

Fig. S1

Nucleotide and deduced amino acid sequences of WsGT from Withania somnifera with 5′ (18bp) and 3′ (158bp) UTRs followed by a poly-A tail. Start codon (ATG) and stop codon (TAA) highlighted in bold and poly-A signal site (AATAAA) are underlined (JPEG 1661 kb)

Fig. S2

Amino acid sequence alignment of WsGT protein with reported glycosyltransfearses. Asterisk indicates identical or conserved residues; colon indicates a conserved substitution; dot indicates a semi-conserved substitution. The conserved PSPG box found in glycosyltransferases is highlighted with an underline (JPEG 2618 kb)

Fig. S3

SDS-PAGE analysis of WsGT expressed in E. coli. Lane M- protein molecular size marker; lane 1 & 2- Ni-NTA purified WsGT protein; lane 3, 4 & 5- anion exchange purified WsGT. The position of purified protein is about 52kDa (JPEG 341 kb)

Fig. S4

The LC-MS spectrum of the substrate and product in the reaction of WsGT enzyme with (a) diadzein and (b) genistein (JPEG 617 kb)

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Kumar, R.J.S., Ruby, Singh, S. et al. Functional Characterization of a Glucosyltransferase Specific to Flavonoid 7-O-Glucosides from Withania somnifera . Plant Mol Biol Rep 31, 1100–1108 (2013). https://doi.org/10.1007/s11105-013-0573-4

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  • DOI: https://doi.org/10.1007/s11105-013-0573-4

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