Arabidopsis as a model for investigating gene activity and function in vascular tissues

Using the zinnia mesophyll cell system for tracheary element differentiation, members of at least three families of mechanistically distinct (serine, cysteine and threonine) proteases have been implicated in the regulation of tracheary element differentiation. We are currently using Arabidopsis to facilitate genetic analysis of the roles of proteolytic enzymes during vascular tissue differentiation. Although not typically considered as a model for secondary growth, Arabidopsis forms a true cambium and produces a relatively large amount of secondary xylem and phloem within the root and hypocotyl. This potential for secondary growth is best realized under conditions that delay senescence, i.e., inflorescence removal and growth at low population density. After growing Arabidopsis under these conditions for at least eight weeks it is possible to isolate biochemical quantities of xylem and phloem for analysis of tissue-specific protease activities. After dissecting approximately 300 root-hypocotyl segments into xylem and bark fractions we isolated RNA for the construction of the first xylem and bark cDNA libraries from Arabidopsis. Using gene-specific primers and degenerate primers we screened the libraries by PCR and amplified fragments from seven protease genes including members of serine, cysteine, and aspartic acid protease families. Expression for three of these genes (XCP1, XCP2, and XSP1) is xylem-specific. XCP1 and XCP2 are predicted to encode papain-like cysteine proteases and XSP1 is predicted to encode a subtilisin-like serine protease. To identify additional genes with potential importance to vascular tissue differentiation and physiology, we analyzed 1,000 (500 from xylem and 500 from bark) ESTs.