Scopolin-hydrolyzing  -glucosidases in roots of Arabidopsis

Abstract

Three beta-glucosidases (At1g66270-BGLU21, At1g66280-BGLU22, and At3g09260-BGLU23) were purified from the roots of Arabidopsis and their cDNAs were expressed in insect cells. In addition, two beta-glucosidase binding protein cDNAs (At3g16420; PBPI and At3g16430; PBPII) were expressed in Escherichia coli and their protein products purified. These binding proteins interact with beta-glucosidases and activate them. BGLU21, 22 and 23 hydrolyzed the natural substrate scopolin specifically and also hydrolyzed to some extent substrates whose aglycone moiety is similar to scopolin (e.g. esculin and 4-MU-glucoside). In contrast, they hydrolyzed poorly DIMBOA-glucoside and did not hydrolyze pNP- and oNP-glucosides. We determined the physicochemical properties of native and recombinant BGLUs, and found no differences between them. They were stable in a narrow pH range (5-7.5) and had temperature and pH optima for activity at 35 degrees C and pH 5.5, respectively. As for thermostability, >95% of their activity was retained at 40 degrees C but dramatically decreased at >50 degrees C. The apparent K(m) of native and recombinant enzymes for scopolin was 0.73 and 0.81 mM, respectively, and it was 5.8 and 9.7 mM, respectively, for esculin. Western blot analysis showed that all three enzymes were exclusively expressed in roots of seedlings but not in any other plant part or organ under normal conditions. Furthermore, spatial expression patterns of all eight genes belonging to subfamily 3 were investigated at the transcription level by RT-PCR.

Feruloyl-CoA 6'-Hydroxylase1-Dependent Coumarins Mediate Iron Acquisition from Alkaline Substrates in Arabidopsis

“…In addition, because scopoletin was more abundant in root exudates, it is likely that scopolin undergoes deglycosylation just before or right after being exported across the root plasma membrane. This assumption is in agreement with the existence of root-expressed b-glucosidases that have the capacity to hydrolyze scopolin (Ahn et al, 2010). Interestingly, such a modification is not restricted to the pair scopolin-scopoletin but may also apply to esculin and its aglycon esculetin.…”

“…5, G-J). Similar to scopoletin, further chemical modifications in the root apoplast may be involved in the enrichment of esculetin in root exudates, because esculin can also serve as substrate for root-expressed b-glucosidases (Ahn et al, 2010).…”

Metabolic Engineering in Nicotiana benthamiana Reveals Key Enzyme Functions in Arabidopsis Indole Glucosinolate Modification

“…The PYK10 gene product has ;50% identity to PEN2 at the amino acid level, and PEN2 was recently identified as an atypical myrosinase responsible for hydrolyzing CYP81F2-dependent 4MO-I3M in plant innate immunity reactions (Bednarek et al, 2009;Clay et al, 2009). However, heterologously expressed PYK10 did not accept sinigrin, an aliphatic glucosinolate, as a substrate (Ahn et al, 2010), but this does not preclude a potential activity with indole glucosinolates.…”

Comparative proteomic analysis of plant responses to sound waves in Arabidopsis

“…Beta glucosidase 18 is induced by various stresses, such as cold and osmotic stress (Ahn et al 2009), and is the key enzyme involved in the abscisic acid signaling pathway (Lee et al 2006;Kato-Noguchi et al 2008). Induction of this enzyme under sound wave stress suggests that the particular sound wave frequency that was tested may induce the plant defense system in Arabidopsis.…”