Purification, Cloning, and Expression of a Pathogen Inducible UDP-glucose:Salicylic Acid Glucosyltransferase from Tobacco

Hyung-il Lee,Ilya Raskin

doi:10.1074/jbc.274.51.36637

Hyung-il Lee, Ilya Raskin

Open Access

https://doi.org/10.1074/jbc.274.51.36637

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Abstract

Salicylic acid (SA) plays an important role in plant disease resistance. Inoculation of tobacco leaves with incompatible pathogens triggers the biosynthesis of SA which accumulates primarily as the SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS). The tobacco UDP-glucose:salicylic acid glucosyltransferase (SA GTase) capable of forming both SAG and GS was purified, characterized, and partially sequenced. It has an apparent molecular mass of 48 kDa, a pH optimum of 7.0, and an isoelectric point at pH 4.4. UDP-glucose was the sole sugar donor for the enzyme. However, SA and several phenolics served as glucose acceptors. The apparent K(m) values for UDP-glucose and SA were 0.27 and 1-2 mM, respectively. Zn(2+) and UDP inhibited its activity. The corresponding cDNA clone which encoded a protein of 459 amino acids was isolated from an SA-induced tobacco cDNA library and overexpressed in Escherichia coli. The recombinant protein catalyzed the formation of SAG and GS, and exhibited a broad specificity to simple phenolics, similar to that of the purified enzyme. Northern blot analysis showed that the SA GTase mRNA was induced both by SA and incompatible pathogens. The rapid induction timing of the mRNA by SA indicates that it belongs to the early SA response genes.

Highlights

Importance of salicylic acid (SA)1 in the signal transduction pathway of plant disease resistance has been well documented in many plants [1,2,3,4]
SA GTase Activity in tobacco mosaic virus (TMV)-inoculated Tobacco after Temperature Shift—The glucosyltransferase activity leading to the formation of either SA 2-O-␤-D-glucoside (SAG) or Glucosyl salicylate (GS) was detected in tobacco plants treated with SA or inoculated with TMV or P. syringae pv. phaseolicola where induction of both enzyme activities was localized to the infected areas (10 –12)
Tobacco plants inoculated with TMV and incubated at 32 °C for 4 days showed no change in SA content or glucosyltransferase activity

Summary

Introduction

Importance of salicylic acid (SA) in the signal transduction pathway of plant disease resistance has been well documented in many plants [1,2,3,4]. The hypersensitive response is characterized by the formation of necrotic lesions which restrict the spread of pathogens and by a dramatic increase in SA levels in the inoculated leaf and, to a lesser extent, throughout the plant. Among the SA conjugates, the SA 2-O-␤-D-glucoside (SAG) has been identified as a predominant and stable metabolite in many plants, including tobacco. Glucosyl salicylate (GS), an ester form, was recently identified as a SA metabolite in virus- or bacteria-inoculated tobacco [10]. UDP-glucose:SA GTase (SAGT) catalyzed the conversion of SA to SAG, while formation of GS was catalyzed by UDP-glucose:SA carboxyl GTase Both enzyme activities were enhanced in pathogen-inoculated or SA-treated tobacco plants. We purified to near homogeneity, characterized, and partially sequenced the glucosyltransferase that catalyzes the in vitro conversion of SA to GS (an ester form) and to a lesser extent to SAG (a more stable glucoside).

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