Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens.
A cDNA clone encoding the Prx7 peroxidase from barley (Hordeum vulgare L.) predicted a 341-amino acid protein with a molecular weight of 36,515. N- and C-terminal putative signal peptides were present, suggesting a vacuolar location of the peroxidase. Immunoblotting and reverse-transcriptase polymerase chain reaction showed that the Prx7 protein and mRNA accumulated abundantly in barley coleoptiles and in leaf epidermis inoculated with powdery mildew fungus (Blumeria graminis). Two isoperoxidases with isoelectric points of 9.3 and 7.3 (P9.3 and P7.3, respectively) were purified to homogeneity from barley coleoptiles. P9.3 and P7.3 had Reinheitszahl values of 3.31 and 2.85 and specific activities (with 2,2'-azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid], pH 5.5, as the substrate) of 11 and 79 units/mg, respectively. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry peptide analysis identified the P9. 3 peroxidase activity as due to Prx7. Tissue and subcellular accumulation of Prx7 was studied using activity-stained isoelectric focusing gels and immunoblotting. The peroxidase activity due to Prx7 accumulated in barley leaves 24 h after inoculation with powdery mildew spores or by wounding of epidermal cells. Prx7 accumulated predominantly in the epidermis, apparently in the vacuole, and appeared to be the only pathogen-induced vacuolar peroxidase expressed in barley tissues. The data presented here suggest that Prx7 is responsible for the biosynthesis of antifungal compounds known as hordatines, which accumulate abundantly in barley coleoptiles.
Kristensen
B K
BK
Plant Biology and Biogeochemistry Department, PBK-301, Riso National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark. brian.kristensen@risoe.dk
Bloch
H
H
Rasmussen
S K
SK
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000472:pathogenicity; D001483:Base Sequence; D003001:Cloning, Molecular; D018548:Cotyledon / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004790:Enzyme Induction; D015870:Gene Expression; D017343:Genes, Plant; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics* / Q000382:microbiology; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D012333:RNA, Messenger; D018749:RNA, Plant; D010544:Peroxidases