Xylem is the wood-forming and water-conducting tissue in vascular plants. Xylem is a complex tissue consisting of both living and nonliving cells. The most characteristic xylem cells are the water-conducting tracheary elements. Tracheary elements undergo a remarkable differentiation process that involves extensive secondary cell wall modification, cell suicide and complete autolysis. The complete autolysis that occurs in tracheary elements contrasts with the selective autolysis that occurs during the differentiation of two other vascular tissue cell types, laticifers and sieve elements. Protoplast degradation in tracheary elements is preceded by cell death and the release of vacuolar contents and is dependent on the activity of papain-like cysteine peptidases. XCP1 and XCP2 are Arabidopsis papain-like cysteine peptidases that localize to tracheary elements and are therefore likely to be required for autolysis. XCP1 and XCP2 are also the first recognized orthologs of the best-known laticifer peptidase, papain. As such, they represent the first known genetic link between a complete autolysis program and a selective autolysis program. The selective autolysis program in papaya (Carica papaya) laticifers preserves the plasmalemma and cell turgor but degrades the nucleus and all organelles, replacing them with latex-containing vesicles. For this project, transgenic plants expressing a reporter gene under the control of the XCP1 promoter have been mutatgenized and they are being screened for altered reporter gene expression. Positional cloning from resulting mutants will identify genes required for regulation of autolysis in tracheary elements and that may be relevant to other autolytic programs. XSP1 is an Arabidopsis subtilisin-like serine peptidase that is secreted from tracheary elements. XSP1 is also under investigation for this project. Post-transcriptional gene silencing and gene knockout experiments will be conducted for XCP1, XCP2 and XSP1 to provide direct evidence for their functions in tracheary elements. Electron microscopic immunolocalization of XCP1, XCP2 and XSP1 will also be conducted.
Wood formation and latex accumulation are two economically important processes that depend on developmentally programmed autolysis. Revealing the roles of XCP1, XCP2 and XSP1 in the differentiation of tracheary elements may lead to the development of tools for engineering qualitative and quantitative changes in wood. A comprehensive description of subcellular localization for XCP1 and XCP2 compared with published immunolocalization data for orthologous papain from papaya laticifers will yield valuable clues regarding the roles of subcellular trafficking and compartmentation in regulating selective and complete autolysis. The improved understanding of autolysis gained from this project may lead to strategies for induction of selective autolysis as part of an engineered subversion of plant metabolism toward production and packaging of selected polypeptides or other desired metabolites in storage vacuoles that replace all other organelles, such as occurs naturally during differentiation of laticifers in some species.
