RESUMO
ß-Amylase3 (BAM3) is an enzyme that is essential for starch degradation in plant leaves and is also transcriptionally induced under cold stress. However, we recently reported that BAM3's enzymatic activity decreased in cold-stressed Arabidopsis leaves, although the activity of BAM1, a homologous leaf ß-amylase, was largely unaffected. This decrease in BAM3 activity may relate to the accumulation of starch reported in cold-stressed plants. The aim of this study was to explore the disparity between BAM3 transcript and activity levels under cold stress, and we present evidence suggesting BAM3 is being inhibited by post-translational modification. A mechanism of enzyme inhibition was suggested by observing that BAM3 protein levels remained unchanged under cold stress. Cold stress induces nitric oxide (NO) signaling, one result being alteration of protein activity by nitrosylation or glutathionylation through agents such as S-nitrosoglutathione (GSNO). To test whether NO induction correlates with inhibition of BAM3 in vivo, plants were treated with sodium nitroprusside, which releases NO, and a decline in BAM3 but not BAM1 activity was again observed. Treatment of recombinant BAM3 and BAM1 with GSNO caused significant, dose-dependent inhibition of BAM3 activity while BAM1 was largely unaffected. Site-directed mutagenesis, anti-glutathione Western blots, and mass spectrometry were then used to determine that in vitro BAM3 inhibition was caused by glutathionylation at cysteine 433. In addition, we generated a BAM1 mutant resembling BAM3 that was sensitive to GSNO inhibition. These findings demonstrate a differential response of two BAM paralogs to the Cys-modifying reagent GSNO and provide a possible molecular basis for reduced BAM3 activity in cold-stressed plants.
