title: An alpha-amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress.
abstract: Leaves are reported to contain a secreted alpha-amylase that accumulates during senescence or after biotic or abiotic stress; however, a gene encoding this enzyme has not been described. Because a secreted amylase is isolated from plastidic starch, the function of this enzyme is difficult to predict, but circumstantial evidence suggests that it may degrade starch after cell death. The Arabidopsis thaliana genome contains three alpha-amylase genes, one of which, AMY1 (At4g25000), has a putative signal sequence suggesting that the protein may be secreted. Two independent T-DNA insertion mutants in AMY1 lacked an amylase band on starch zymograms, which was previously named 'A1'. Washed leaf protoplasts contained reduced A1 activity suggesting that the enzyme is secreted. Native AMY1, fused to a weakly fluorescent form of GFP, was sensitive to proteinase K infiltrated into leaf apoplastic spaces, while a cytosolic form of GFP was unaffected until cell breakage, confirming that the AMY1 protein is secreted. Amylase A1 was transcriptionally induced in senescing leaves and in leaves exposed to heat stress, treated with abscisic acid or infected with Pseudomonas syringae pv. tomato expressing avrRpm1. The A1 amylase was also extremely heat resistant and its expression was up-regulated in cpr5-2, an activated defence response mutant.
authors.affiliation: Department of Biology, MSC 7801, James Madison University, Harrisonburg, VA 22807, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Doyle
authors.forename: Ashleigh M
authors.initials: AM
authors.lastname: Lane
authors.forename: James M
authors.initials: JM
authors.lastname: Sides
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Mudgett
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Monroe
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016922:Cellular Senescence; D019286:Endopeptidase K / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000032:analysis; D007210:Indoleacetic Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011523:Protoplasts / Q000201:enzymology; D044224:Pseudomonas syringae / Q000378:metabolism / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D007210:Indoleacetic Acids; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000516:alpha-Amylases; D019286:Endopeptidase K