The Arabidopsis Xylem Peptidase XCP1 Is a Tracheary Element Vacuolar Protein That May Be a Papain Ortholog

Abstract

XCP1 is a xylem-specific papain-like cysteine peptidase in Arabidopsis. To determine whether XCP1 could be involved in tracheary element autolysis, promoter activity and localization of XCP1 were investigated using XCP1 promoter-␤-glucuronidase fusions and immunofluorescence confocal microscopy. A tracheary element expression pattern was detected for XCP1. Results from confocal microscopy and biochemical subcellular fractionation indicated that XCP1 was localized in the vacuole. Ectopic expression of XCP1 resulted in a reduction in plant size in some lines and early leaf senescence, as indicated by early loss of leaf chlorophyll. Reduced plant size was correlated with higher levels of XCP1, as shown by immunoblot and peptidase activity gel analyses. The XCP1 prodomain exhibits exceptionally high similarity (greater than 80%) to the prodomains of papain and other papain-like enzymes isolated from papaya (Carica papaya) laticifers when compared with all other reported papain-like enzymes. The potential for XCP1 and papain to perform common functions as catalysts of autolytic processing following cell death due to programmed suicide or to wounding is discussed.In plants, increased peptidase gene expression is associated with remobilization of nitrogen from senescing source tissues to storage or reproductive sinks and from seed protein reserves in support of germination and seedling growth (for review, see Granell et al., 1998; Beers et al., 2000). Increased peptidase activity is also linked to the carbon starvation response (Moriyasu and Ohsumi, 1996; Brouquisse et al., 1998). A detailed understanding of how peptidases function as necessary effectors of nitrogen remobilization during these processes is lacking.Genetic evidence has contributed to an expanded view of plant peptidases that includes important roles as regulators of responses to environmental cues and plant hormones (for review, see Callis and Vierstra, 2000; Estelle, 2001). The ubiquitinproteasome pathway is important in light-and auxin-mediated signaling pathways. For example, the level of Hy5, a transcription factor important in photomorphogenesis, is likely to be determined by COP1-mediated targeting to the proteasome (Osterlund et al., 2000). Turnover of IAA/AUX proteins may also be proteasome dependent (Worley et al., 2000; Estelle, 2001). Ser and Asp peptidases are important to cellular differentiation and plant-pathogen interactions. A subtilisin-like Ser peptidase gene, SDD1, is required for regulation of stomatal density and distribution (Berger and Altmann, 2000). In addition, activation tagging of a secreted Asp peptidase in Arabidopsis led to enhanced resistance to virulent strains of Pseudomonas syringae pv. tomato and P. syringae pv. Maculicola, and was correlated with constitutive expression of defense-related genes and elevated salicylic acid levels (R. Dixon and C. Lamb, personal communication).Experimental models for studying tracheary element (TE) differentiation provide opportunities to investigate the potential of peptidases to a...

Activity Profiling of Papain-Like Cysteine Proteases in Plants

“…XCP2, for example, is hypothesized to be involved in programmed cell death of tracheary elements (Funk et al, 2002). Furthermore, RD21 is present in endoplasmic reticulum (ER) bodies that originate from the ER and fuse with the vacuole upon stress (Hayashi et al, 2001).…”

A Weed for Wood? Arabidopsis as a Genetic Model for Xylem Development

“…Zhao et al (2000) generated xylem and bark specific cDNA libraries from Arabidopsis root-hypocotyl area and subsequently identified two Cys proteases, XCP1 and XCP2, and one Ser protease, XSP1, which were specifically expressed in xylem. Funk et al (2002) were able to define that the XCP1 is localized in the vacuoles of differentiating tracheary elements in Arabidopsis, as would be expected from a protease potentially involved in wood development. Recently it was shown by Ito and Fukuda (2002) that a Zn 21 -dependent DNase, ZEN1, has a major role in the degradation of nuclear DNA during programmed cell death of tracheary elements in Zinnia.…”

Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 Directly Regulates the Genes That Govern Programmed Cell Death and Secondary Wall Formation during Xylem Differentiation

“…During the differentiation of TEs, PCD is initiated by central vacuolar rupture (Obara et al, 2001). The cellular contents are degraded by hydrolytic enzymes, such as proteinases and nucleases, which are released by the vacuolar rupture (Funk et al, 2002;Ito and Fukuda, 2002). Cellular and biochemical analyses in Zinnia xylogenic culture have revealed a tight coupling of secondary cell wall formation and PCD in the TEdifferentiating process (Fukuda, 2004).…”