Function and Regulation of Xylem Cysteine Protease 1 and Xylem Cysteine Protease 2 in Arabidopsis

A functional water-conducting system, the tracheary elements of the xylem, is required to sustain plant growth and development. Tracheary element formation is dependent on many biological processes terminated by programmed cell death and cellular autolysis. The final two processes are probably dependent on the activity of hydrolytic enzymes such as XCP1 and XCP2 known to be expressed in tracheary elements during these final two processes. Thus, the transcriptional regulation of XCP1 and the function of XCP2 were investigated. Qualitative and quantitative assessments of GUS activity as directed by various fragments of the XCP1 promoter showed that a 237-bp internal region was able to drive GUS expression in a tracheary element-specific manner in Arabidopsis. A 25-bp deletion at the 3' end of this region abolished GUS expression. The 237-bp region served as bait in a yeast one-hybrid analysis. Screening of yeast colonies retrieved 109 putative positive interactions, which included a potential transcriptional regulator, indole acetic acid-induced protein 8 (IAA8). An auxin responsive element that potentially binds auxin responsive transcription factors was found within the 25-bp deletion. Cis-elements were predicted by Genomatix and Athamap computer programs. The cis-elements form pyrimidine and gibberellic acid responsive elements that can potentially bind Dof and Myb transcription factors, respectively. In an independent effort, attempts to develop a mapping population to isolate upstream regulators of XCP1 expression did not succeed. Functionally, tracheary element-specific expression of XCP2 in Arabidopsis suggested a specialized role for XCP2 in final phases of tracheary element differentiation. The function of XCP2 was assessed using T-DNA insertional mutants, post-transcriptional gene silencing, and through tracheary element-specific expression of the cysteine protease inhibitor, soyacystatin N in Arabidopsis. Our findings revealed that the absence of XCP2 expression due to T-DNA insertional mutagenesis did not affect plant growth and development in the laboratory. Soyacystatin N was an effective in vitro inhibitor of cysteine proteases. Plants expressing 35S-driven cytosolic form of soyacystatin exhibited stunting and reduced apical dominance. Plants expressing pXCP1-driven cytosolic soyacystatin did not differ from wild type plants. Additionally, transgenic plants expressing pXCP1- and 35S-directed XCP2-double-stranded RNA for the silencing of XCP2 showed no unusual phenotypes compared to their wild type counterparts

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Horticulture

Department dc:contributor.department

Horticulture

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2003

Author dc:creator

• Ismail, Ihab

Chair dc:contributor.committeechair

• Beers, Eric P.

Committee members dc:contributor.committeemember

• Welbaum, Gregory E.
• Hess, John L.
• Nowak, Jerzy
• McDowell, John M.

• autolysis
• IAA8
• auxin-response element
• tracheary element
• PTGS
• auxin

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-08152004-231624

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/11243