α-Amylase Is Not Required for Breakdown of Transitory Starch in Arabidopsis Leaves

Abstract

The Arabidopsis thaliana genome encodes three ␣-amylase-like proteins (AtAMY1, AtAMY2, and AtAMY3). Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization. AtAMY3 is an unusually large ␣-amylase (93.5 kDa) with the C-terminal half showing similarity to other known ␣-amylases. When expressed in Escherichia coli, both the whole AtAMY3 protein and the C-terminal half alone show ␣-amylase activity. We show that AtAMY3 is localized in chloroplasts. The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial ␣-amylase activity, is deficient in AtAMY3 protein. Unexpectedly, T-DNA knock-out mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type. These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein. Knockout mutants in the predicted non-plastidial ␣-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark as expected for extraplastidial amylases. Furthermore, all three AtAMY double knock-out mutant combinations and the triple knock-out degraded their leaf starch normally. We conclude that ␣-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.

A Putative Phosphatase, LSF1, Is Required for Normal Starch Turnover in Arabidopsis Leaves

“…The activity band attributable to AMY3 was, as expected, weak in intensity on gels of extracts of sex4 leaves ( Fig. 4A; Zeeman et al, 1998;Yu et al, 2005). However, the activity band in extracts of lsf1 leaves was comparable with or more intense than the band for wild-type plants.…”

“…This reduction in AMY3 activity is not responsible for the accumulation of starch in the sex4 mutant: amy3 mutants that lack AMY3 have completely normal rates of starch degradation (Yu et al, 2005). In fact, despite the reduction in its activity, AMY3 plays an important role in determining the sex4 phenotype: together with ISA3 it catalyzes the release from the granule surface of phosphorylated oligosaccharides that accumulate in the stroma of sex4 chloroplasts (Kö tting et al, 2009).…”

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day

“…It is relatively unlikely that such an increase would be brought about at the level of gene expression. Although transcript levels for many enzymes of starch degradation rise with time after dawn, there is evidence for enzymes including glucan, water dikinase, a-amylase 3 (AMY3), and the glucanotransferase DPE2 that the amount of protein varies very little over the day-night cycle Lu et al, 2005;Yu et al, 2005;Skeffington et al, 2014). In general terms, marked diel fluctuations in amounts of enzymes of primary metabolism are unlikely because of their high abundance and long half-lives (Gibon et al, 2004;Piques et al, 2009;Baerenfaller et al, 2012;Nelson et al, 2014).…”

“…However neither enzyme is required for diel starch metabolism in mesophyll cells in square wave light regimes in the absence of abiotic stress (Yu et al, 2005;Fulton et al, 2008;Horrer et al, 2016).…”

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

“…A prerequisite for transient starch mobilization is the phosphorylation of a portion of the glucosyl residues by glucan, water dikinase (GWD; Ritte et al, 2002) and phosphoglucan, water dikinase (PWD; Kö tting et al, 2005). AMY, PUL, PHO, and a-glucosidase are established to play minor roles (if any) in transient starch breakdown (Lloyd et al, 2005;Yu et al, 2005), while BAM is a major enzyme (Fulton et al, 2008). Four BAM isoforms of Arabidopsis are plastid localized, and three of them are involved in degradation of transient starch to maltose (Fulton et al, 2008).…”