β-Amylase1 andβ-Amylase3 Are Plastidic Starch Hydrolases in Arabidopsis That Seem to Be Adapted for Different Thermal, pH, and Stress Conditions    

Abstract

Starch degradation in chloroplasts requires b-amylase (BAM) activity, which is encoded by a multigene family. Of nine Arabidopsis (Arabidopsis thaliana) BAM genes, six encode plastidic enzymes, but only four of these are catalytically active. In vegetative plants, BAM1 acts during the day in guard cells, whereas BAM3 is the dominant activity in mesophyll cells at night. Plastidic BAMs have been difficult to assay in leaf extracts, in part because of a cytosolic activity encoded by BAM5. We generated a series of double mutants lacking BAM5 and each of the active plastidic enzymes (BAM1, BAM2, BAM3, and BAM6) and found that most of the plastidic activity in 5-week-old plants was encoded by BAM1 and BAM3. Both of these activities were relatively constant during the day and the night. Analysis of leaf extracts from double mutants and purified BAM1 and BAM3 proteins revealed that these proteins have distinct properties. Using soluble starch as the substrate, BAM1 and BAM3 had optimum activity at pH 6.0 to 6.5, but at high pH, BAM1 was more active than BAM3, consistent with its known daytime role in the guard cell stroma. The optimum temperature for BAM1, which is transcriptionally induced by heat stress, was about 10°C higher than that of BAM3, which is transcriptionally induced by cold stress. The amino acid composition of BAM1 and BAM3 orthologs reflected differences that are consistent with known adaptations of proteins from heat-and coldadapted organisms, suggesting that these day-and night-active enzymes have undergone thermal adaptation.

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

“…Thus, the reducing environment and the alkaline pH of the stroma, generated by the photosynthetic electron transport chain in the light (Heldt et al, 1973;Buchanan and Balmer, 2005), would favor BAM1 and AMY3 activity. Conversely, there is no evidence for redox regulation of BAM3, and the enzyme has optimum activity at less alkaline pH (Santelia et al, 2015;Monroe et al, 2014). Furthermore, large-scale site-specific phosphorylation profiling of Arabidopsis proteins revealed the presence of one or more phosphorylation sites on both BAM1 and AMY3 proteins (de la Fuente van Bentem et al, 2008;Reiland et al, 2009;Xue et al, 2013;Heazlewood et al, 2008).…”

“…For example, both enzymes are redox regulated, whereby they can be rapidly activated through reduction via the light-driven ferredoxin-thioredoxin system (Sparla et al, 2006;Seung et al, 2013). Furthermore, both enzymes are preferentially active at a slightly alkaline pH (Seung et al, 2013;Sparla et al, 2006;Monroe et al, 2014;Santelia et al, 2015). Thus, the reducing environment and the alkaline pH of the stroma, generated by the photosynthetic electron transport chain in the light (Heldt et al, 1973;Buchanan and Balmer, 2005), would favor BAM1 and AMY3 activity.…”

“…Pairs of BAM1 and BAM3 orthologs are found in the genomes of several angiosperms, suggesting that BAM isoform subfunctionalization is a general feature of flowering plants (Monroe et al, 2014;Fulton et al, 2008). Interestingly, bioinformatics analyses of the promoters of BAM1 and BAM3 orthologs from 30 different plant species using the MEME algorithm (Bailey et al, 2009) revealed an enrichment of ABRE elements in the proximity of the ATG codon in all examined BAM1-like gene promoters, with the exception of that of the grasses, where instead CE3-like ABAresponsive elements were found (Figure 8; Supplemental Data Set 1).…”

“…The two close homologs, BAM1 and BAM3, are differentially regulated in response to abiotic stresses ( Figure 1D; Supplemental Figure 13) Kaplan and Guy, 2004;Monroe (A) and (B) Leaf starch (A) and maltose (B) content in wild-type, nced3, and aao3 plants treated with 300 mM mannitol compared with controls. Values are means 6 SE (n = 6).…”

“…Altogether, these findings indicate that BAM1 and BAM3 are active under different conditions and in a cell typespecific manner. Subfunctionalization among members of the b-amylase family has occurred that significantly contributed to the ability of the plant to adjust starch turnover to the need of the individual cells and in response to the external environmental stimuli (Monroe et al, 2014;Zanella et al, 2016;Horrer et al, 2016).…”