טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentParnis Anna
SubjectTkn2- an Essential Gene in Plant Development:
Molecular Analysis of Its Mechanism of Action
DepartmentDepartment of Biology
Supervisor Professor Emeritus Eliezer Lifschytz


Abstract

Developmental events in meristems define the shape and anatomy of the shoot and its derivative organs. In order to dissect plant morphogenesis, it is essential to study the mechanisms by which operation of a given meristematic gene results in morphological alterations. Such opportunity is provided by members of the meristem-specific homeobox-containing KNOX family.

To understand the role of homeotic genes in tomato plant development, four novel KNOX genes were isolated. The genes were classified according to their sequence homology and expression patterns to class I (TKn2 and TKn3) and class II (THox1 and THox2) of the Knotted family. Genetic mapping by the RFLP procedure suggested that one of the genes, TKn2, is linked to two known dominant mutations on chromosome II: Curl and Mouse ears. The mutations were shown to be associated with two aberrant modes of TKn2 transcription. Overexpression of the wild type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. The defective Me-TKn2 transcript is generated via splicing event that merges transcripts of two closely linked genes, it is comprised of most of the 5’ end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript. The translated fusion product was shown to enter the nucleus. It is suggested that subtle alterations in KNOX genes function may be the basis for the wide diversity in growth parameters among plant species.

In order to understand the molecular mechanism by which KNOX proteins exert their developmental role, the isolation of TKn2 interacting proteins was undertaken. The screen for such proteins was carried out using the yeast two-hybrid system. Two different groups of interacting proteins were discovered.

The first group contains four homeobox genes, with homology to the Bell1 gene, known to determine the fate of the cells during ovule development in arabidopsis. Those genes might define the specificity of action of the TKn2 gene and so to participate in patterning plant forms.

The second group of TKn2-associated proteins consists of one novel Tomato Kinesin Related (TKR) protein. Sequence analysis suggests that TKR belongs to a unique plant subclass of kinesin superfamily. This protein may function as motor vehicle targeting the KNOX proteins to plasmodesmata, or as regulator of signalling pathways. The association between the TKR and TKn2 proteins was further confirmed by in vitro experiments. The TKR binding specificity was analysed: the protein interacts with class I, but not class II KNOX family members. The TKR-precise TKR-recognition region of 35 amino acids was identified in the TKn2 protein; two amino acids in this region were shown to be critical for binding between the proteins by both two-hybrid and in vitro assays. Further investigation is needed to examine the biochemical properties and molecular role of the association between KNOX proteins and the TKR motor protein.