Abstract
Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.
Highlights
Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown
The same phenotype was observed in potato (Solanum tuberosum) antisense plants with reduced expression of the plastidial b-amylase PCTBMY1, the putative ortholog of BAM3 (Scheidig et al, 2002). b-Amylases are exoamylases that release maltose from the nonreducing ends of glucans or dextrins by cleavage of a-1,4 linkages. a-1,6 linkages are hydrolyzed by debranching enzymes
Using recombinant potato GWD (StGWD) and granules of the Arabidopsis sex1-3 mutant as tools we studied if breakdown of the native starch particles by proteins extracted from Arabidopsis leaves is stimulated by glucan phosphorylation
Summary
Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized b-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. A-Amylase, which cleaves a-1,4 bonds within the polyglucan, plays an important role in the degradation of cereal endosperm starch (Smith et al, 2005) This enzyme is not essential for starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves (Yu et al, 2005). Traces of C6- and C3-P esters could be detected in starch of the sex mutant that has no detectable GWD (Ritte et al, 2006)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
