Extracellular β‐glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves

Abstract

Abscisic acid conjugate concentrations increased in barley xylem sap under salinity, whereas it remained at a low level in the intercellular washing fluid (IWF) of barley primary leaves (Hordeum vulgare cv. Gerbel). Here it is shown that IWF contains beta-glucosidase activity which releases abscisic acid (ABA) from the physiologically inactive ABA-glucose conjugate pool in the leaf apoplast. The following data support this conclusion and give the first biochemical and physiological characterization of the extracellular glucosidase activity in barley. Free ABA was released by the incubation of ABA glucose ester with IWF. The product exhibited the retention time of authentic ABA upon separation by thin layer chromatography and was identified by ABA-ELISA. p-Nitrophenol-beta-D-glucopyranoside (pNPG) was used as the substrate for beta-glucosidases. The K(M)(pNPG) was 1.8 mmol l(-1). The activity was affected by ABA glucopyranoside in a competitive type of inhibition with a K(I) of 400 micromol l(-1). Various hormone conjugates were compared with respect to their inhibitory effect on beta-glucosidase activity. Inhibition was highest for the ABA glucopyranoside and the zeatin riboside, but insignificant for ABA methyl ester and zeatin-9-beta-D-glucoside. The specific activity of the beta-glucosidase was 16-fold greater in IWF as compared to crude leaf extracts confirming its extracellular compartmentation. The activity of beta-glucosidase was strongly increased after growth in hydroponic medium supplemented with NaCl. The data support the hypothesis that the glucose conjugate is a long-distance transport form of ABA.

A possible stress physiological role of abscisic acid conjugates in root‐to‐shoot signalling

“…1c, 1) intensifies the long-distance ABA stress signal. Competition experiments with a range of hormone conjugates show a reasonable specificity of the enzyme for ABA-GE as substrate (Dietz et al 2000).…”

“…ABA glucose esters could either be cleaved by hydrolases with high selectivity towards hormone conjugates or by β-glucosidases with rather low substrate selectivity. Indeed, Dietz et al (2000) detected an extracellular β-glucosidase activity in barley leaves that is able to release free ABA from its conjugate. This enzyme activity increased seven-fold when plants were salt stressed.…”

“…The following problems should be investigated in the future: (1) the chemical structure of all ABA conjugates of the xylem sap needs to be elucidated; (2) the transport of ABA conjugates across root and mesophyll plasma membranes should be investigated under control and stress conditions, including a possible involvement of ABC-transporters; and (3) apoplastic β-glucosidases that hydrolyse ABA-glucose esters need to be purified and characterized. (Dietz et al 2000). The protein of 300 µL IWF protein was separated by isoelectric focusing in immobilized pH gradients (Immobilin strips 3-10, Pharmacia, Uppsala, Sweden).…”

Abscisic Acid Uridine Diphosphate Glucosyltransferases Play a Crucial Role in Abscisic Acid Homeostasis in Arabidopsis

“…In plants, ABA-GE is stored in the vacuole and apoplastic space (Dietz et al, 2000), whereas AtBG1 localizes to the ER , suggesting that ABA-GE should be imported into the ER. Dehydration stress may act as a signal to transport ABA-GE to the ER via the ER membrane .…”

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

“…Abscisic acid glucosyl ester (ABA-GE) appears to be the major conjugate, which was found in various organs of different plant species (Piotrowska and Bajguz, 2011). In contrast to the oxidative pathway, the inactivation of ABA by Glc conjugation is reversible, and hydrolysis of ABA-GE catalyzed by b-glucosidases results in free ABA (Dietz et al, 2000;Lee et al, 2006;Xu et al, 2012). ABA-GE levels were shown to substantially increase during dehydration and specific seed developmental and germination stages (Boyer and Zeevaart, 1982;Hocher et al, 1991;Chiwocha et al, 2003).…”