Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Abstract

Plants emit a variety of volatiles in response to herbivore attack, and (Z)‐3‐hexenol and its glycosides have been shown to function as defence compounds. Although the ability to incorporate and convert (Z)‐3‐hexenol to glycosides is widely conserved in plants, the enzymes responsible for the glycosylation of (Z)‐3‐hexenol remained unknown until today. In this study, uridine‐diphosphate‐dependent glycosyltransferase (UGT) candidate genes were selected by correlation analysis and their response to airborne (Z)‐3‐hexenol, which has been shown to be taken up by the tea plant. The allelic proteins UGT85A53‐1 and UGT85A53‐2 showed the highest activity towards (Z)‐3‐hexenol and are distinct from UGT85A53‐3, which displayed a similar catalytic efficiency for (Z)‐3‐hexenol and nerol. A single amino acid exchange E59D enhanced the activity towards (Z)‐3‐hexenol, whereas a L445M mutation reduced the catalytic activity towards all substrates tested. Transient overexpression of CsUGT85A53‐1 in tobacco significantly increased the level of (Z)‐3‐hexenyl glucoside. The functional characterization of CsUGT85A53 as a (Z)‐3‐hexenol UGT not only provides the foundation for the biotechnological production of (Z)‐3‐hexenyl glucoside but also delivers insights for the development of novel insect pest control strategies in tea plant and might be generally applicable to other plants.

Sesquiterpene glucosylation mediated by glucosyltransferase UGT91Q2 is involved in the modulation of cold stress tolerance in tea plants

“…The apparent Michaelis constant (Km) value of UGT91Q2 for nerolidol was 19.71 ± 2.76 µM (Fig. f) measured at optimized conditions (Figs S4–S7), which was two‐fold lower than the apparent Km value of CsUGT85A53 and UGT85K11 for ( Z )‐3‐hexenol (27.77 and 40.66 μM, respectively), two previously identified enzymes in tea plants involved in the glucosylation of ( Z )‐3‐hexenol (Ohgami et al , ; Jing et al , ). The result suggested that UGT91Q2 shows a high affinity to nerolidol.…”

“…In Arabidopsis , the UGTs that displayed catalytic activity in vitro towards terpenoids were restricted to groups D, E, G, H and L of the phylogenetic tree of the GT1 multigene family (Caputi et al , ; Cui et al , ). In the tea plant, some UGTs belonging to the subgroups A (UGT94P1) and G (UGT85K11 and UGT85A53) showed broad substrate specificity towards monoterpenes (Jing et al , ; Ohgami et al , ). Thus, UGTs involved in the glucosylation of nerolidol are most likely members of the UGT subgroups A, D, E, G, H and L, which comprise 218 UGTs.…”

“…Real‐time PCR was performed according to published protocols (Song et al , ; Jing et al , ) with gene‐specific primer sequences (Supporting Information Table S1). The glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and the relative expression was calculated using the 2 –ΔΔCT method (Livak & Schmittgen, ).…”

“…The full‐length UGT s were amplified from C. sinensis young leaves cDNA using proofreading Phusion DNA polymerase according to a published protocol (Jing et al , ). The amplified full‐length sequences were digested with Bam HI and Sal I, the resulting gene fragments were cloned into pGEX‐4T1 vector, and the recombinant plasmids were subsequently transformed into Trans1‐T1‐competent cells.…”

“…After adding 1 mM isopropyl‐β‐ d ‐thio‐galactopyranoside (IPTG) the culture was incubated at 16°C and 150 rpm overnight for the expression of protein. The fusion proteins were purified by GST bind resin (Jing et al , ). Protein concentration was determined by a photometric method (Bradford, ).…”