Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots

• Published in: The Plant journal : for cell and molecular biology Vol. 56; no. 2; pp. 303 - 315
• Main Authors: Avci, Utku, Earl Petzold, H, Ismail, Ihab O, Beers, Eric P, Haigler, Candace H
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-10-2008; Blackwell Publishing Ltd; Blackwell Science
• Subjects: apoptosis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis - ultrastructure; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biological and medical sciences; Botany; Cellular biology; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; cysteine protease; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; DNA, Plant - genetics; Electron microscopes; Fundamental and applied biological sciences. Psychology; Genes, Plant; Genetic Complementation Test; Genotype; Immunoblotting; immunolabeling; Microscopy, Electron, Transmission; microwave-assisted fixation; Mutagenesis, Insertional; Plant growth. Development of the storage organs; Plant physiology and development; Plant Roots - genetics; Plant Roots - metabolism; Plant Roots - ultrastructure; programmed cell death; Proteases; tracheary element autolysis; vacuole; vacuoles; Vacuoles - genetics; Vacuoles - metabolism; Vacuoles - ultrastructure; Vegetative apparatus, growth and morphogenesis. Senescence; Xylem - genetics; Xylem - metabolism; Xylem - ultrastructure; Xylem; Histogenesis; Root; Enzyme; Cysteine endopeptidases; immunolabeling; tracheary element autolysis; Vacuole; Arabidopsis thaliana; Autolysis; cysteine protease; Peptidases; Fixation; Cruciferae; Cell death; Dicotyledones; Angiospermae; Hydrolases; Spermatophyta; programmed cell death; Apoptosis; microwave- assisted fixation
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2008.03592.x

Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.