51:	Exploiting the secondary growth potential of arabidopsis to study proteases from vascular tissue.

Authors:	Johnson, Bobby, J.(A)Boonthida, Kositsup(A)Chengsong, Zhao(A)Thomas , Freeman, B.(A)Beers, Eric, P.(A)
Affiliations:	(A): Department of Horticulture, Virginia Polytechnic Institute and State University
Presenter:	Johnson, Bobby J., bojohns2@vt.edu

Secondary vascular tissue development in Arabidopsis is associated with high levels of proteolytic activity. Using a combination of tissue printing, SDS-PAGE activity gels and class-specific protease inhibitors we have documented tissue-specific Cys and Ser protease profiles for the xylem, phloem and nonvascular tissues of the primary root and hypocotyl. In addition, we have prepared two cDNA libraries for the study of proteolytic pathways during secondary vascular tissue development: 1) a xylem library and 2) a bark, or cambium/phloem-enriched, library. Using both degenerate and specific primers we have screened the libraries by PCR and obtained partial cDNAs for two Cys proteases, one Ser and one Asp protease. These cDNAs are represented in both libraries. Two full-length cDNAs coding for Cys proteases have also been cloned and named XCP1 and XCP2. RNA gel-blots indicate that XCP1 and XCP2 are expressed in the xylem but not in the bark of root-hypocotyl tissue. Cys protease XCP1 was identified as chromosome 4 gene, F23E12.90 (accession AL022604). Cys protease XCP2 contains EST w43225. The mature predicted protein for XCP1 shares 70% identity with XCP2 and both XCP1 and XCP2 are most similar (70% identity at amino acid level) to p48h-17 (accession U19267), Cys protease expressed in differentiating Zinnia tracheary elements (Ye & Varner 1996 Plant Mol. Biol. 30:1233). This work was supported by NRICGP.