טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAvni Avishai
SubjectCytokinins' Role in Conferring Stress Tolerance in
Tobacco Plants
DepartmentDepartment of Biology
Supervisor Professor Emeritus Shimon Gepstein
Full Thesis textFull thesis text - English Version


Abstract

Drought, salinity and increasing high temperatures are the most serious environmental factors limiting the productivity of agricultural crops worldwide, with devastating economical and sociological impact. Thus offering solutions for the global climate changing crisis for plants are essential. 

The phytohormones cytokinins regulate plant senescence, cell divisions and other various development processes. Accelerated leaf senescence and leaf abscission are associated with abiotic stresses in nature as a means canopy size reduction. However, this strategy reduces yields of annual crops, with concomitant economical loss to farmers. Previous work showed that temporal regulation of cytokinins biosynthesis not only delayed leaf senescence but also conferred drought resistance. The autoregulatory system consists of a construct composed of isopentenyltransferase (IPT) gene, a key gene of cytokinin synthesis controlled by a senescence related promoter (SARK). 

The molecular mechanisms underlying the phenomenon of cytokinin-induced plant stress tolerance were investigated. The present work demonstrates that transgenic tobacco plants carrying the cytokinins biosynthesis autoregulatory system composed IPT bound to specific stress related promoter of the methallothionine gene  (pMET::IPT) are stress tolerant.

Transcriptional activation study of several stress-related candidate genes has been performed and the influence of cytokinins has been specifically investigated under stress conditions. Since environmental stresses and senescence increase the concentration of ROS, ROS levels and antioxidative genes expression was investigated. Although H2O2 content was higher in WT comparing to transgenic plants during drought stress, only CAT1 was significantly upregulated in the pMET::IPT plants under drought stress while APX doesn't.  Moreover, proline content that usually increases in response to osmotic stress didn’t increase in pMET::IPT plants during drought stress. These results hint for additional mechanism of stress tolerance besides antioxidative defense. In order to decipher whether stress responses are alter in the transgenic plants, real time PCR analysis of candidate stress-related genes was performed. All four candidate gens were less upregulated during stresses than WT plants. In addition, WIPK, a MAPK gene that is known to be part of stress signal transduction, was down-regulated during salt stress in the transgenic plants. These results made us to examine the possible alteration of stress signaling. For this purpose, stress-related kinases activity was measured in BY2 cell suspension. Indeed, significant reduction in the activity of   three kinases (SIPK, WIPK and NtOSAK) under salt stress was revealed.

Widen phosphoproteomics analysis indicated similar pattern of cytokinins effects on the activation of stress-related proteins.

These results were reinforced by RNA-Seq analysis that indicates for downregulation of stress-related genes.

The results obtained in this research demonstrating the role of cytokinin in decreasing stress responses and stress signal transduction, while at the same time, increasing other responses such as antioxidative and photosynthesis related genes. Naturally, stress responses are inhibitory to developmental and growth, thus decreasing stress responses and signaling is suggested to be contributing to cytokinin-related stress tolerance.