טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentZohar Wolchinsky
SubjectLeaf Senescence - Cytokinins Regulation and Cell Wall
Proteome
DepartmentDepartment of Biology
Supervisor Professor Emeritus Gepstein Shimon
Full Thesis textFull thesis text - English Version


Abstract

 Leaf senescence is the last stage in leaf development, leading to death. During senescence, leaf cells undergo dramatic changes in cellular metabolism, and sequential degeneration of cellular structures, under the control of a highly regulated genetic program. Like many other genetically programmed developmental processes, leaf senescence is subject to regulation by many environmental and internal factors. Physiological studies have shown that in many species cytokinins can inhibit leaf senescence and that the endogenous cytokinin levels drops with the progression of leaf senescence.

The aim of the first part of this study was to analyze cytokinin distribution in transgenic Arabidopsis and tobacco containing the construct pARR5::GUS. ARR5 is a primary-response gene to cytokinins. The fusion of the ARR5 promoter to the reporter gene GUS thus enables the analysis of the spatial-temporal distribution patterns of cytokinins. Arabidopsis seedlings containing this construct demonstrated that free bioactive cytokinins are localized not only in roots, as expected, but also in the shoot. However, the transgenic seedlings of tobacco containing the pARR5::GUS construct accumulate the reporter stain rather in the shoot. These results may indicate distribution differences in the patterns of cytokinin biosynthesis between tobacco and Arabidopsis. Moreover, our results indicate that, in contrast to the accepted paradigm that roots are the cytokinin biosynthesis site; the shoot apical meristem has a significant role in cytokinin biosynthesis and or accumulation.

The last stage of leaf senescence is characterized by programmed cell death (PCD). PCD plays a crucial role also in pathogen-induced hypersensitive response (HR). The plant cell wall is a very dynamic structure with roles in cell growth and development, signaling, plant defense, and intercellular communication.

The aim of the second part of this study was to compare between cell wall proteomes of leaf senescence and pathogen response in order to find common as well as differentially regulated proteins that may be involved in PCD processes. A total of 379 non redundant proteins were identified by iTRAQ analysis of cell wall protein fractions. Bioinformatics analysis suggests that 143 of the proteins are classified as cell wall proteins. Three proteins were upregulated both in senescent and inoculated leaves: peroxidase (At3g49120), trypsin inhibitor (At1g73260) and strictosidine synthase like (At1g74010). Further analysis of these proteins will clarify their function in senescence and plant’s responses to pathogens. Moreover, each set of the proteomic data harbors additional insights regarding senescence or response to pathogen.