Post-translational processing of β-d-xylanases and changes in extractability of arabinoxylans during wheat germination

Endo-1,4-β-d-xylanase (EC 3.2.1.8, β-d-xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3-4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I β-d-xylanase as established by immunodetection. This suggests that other, yet unidentified β-d-xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like β-d-xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher β-d-xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa β-d-xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa β-d-xylanase occurred and was associated with a drastic increase in β-d-xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the β-d-xylanase is processed into more active forms to achieve extensive AX breakdown.