טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAri Feder
SubjectRegulation of the Flavonoid Pathway in Cucumis melo L
DepartmentDepartment of Biotechnology and Food Engineering
Supervisors Full Professor Kashi Yechezkel
Dr. Yaakov Tadmor
Full Thesis textFull thesis text - English Version


Abstract

The flavonoids are poly-phenolic metabolites derived from the amino acid phenylalanine and are ubiquitous in plants. Over 10,000 different flavonoid structures have been characterized and are classified into six major subgroups. Flavonoids play many different roles in plant physiology and they are also considered important components of human diet. Recent findings in our lab indicated that ‘yellow canary’ melon varieties accumulate naringenin chalcone (NarCh), a yellow flavonoid pigment, in the fruit rind. This work aimed to discover the genetic factor that regulates NarCh accumulation in melon and to uncover biochemical changes related to NarCh accumulation. To implement these objectives we used a combination of four approaches:


-Genetic mapping: of a segregating population, followed by additional analysis of recombinants, narrowed down the candidate locus to a 100Kb region on chromosome 10 of melon. 


-Transcriptome analysis: RNA-seq analyses of the bulked segregants, indicated differential expression of an F-box kelch repeat protein coding gene (CmFBK) between the bulks, which was localized to the 100 kb region shown to govern the trait.  CmFBK is highly expressed in the phenotypes that do not accumulate NarCh, while only traces of its transcripts were found in the phenotypes accumulating NarCh. 


-Biochemical analysis: metabolite analysis of the bulked segregants indicated that down-stream flavonoids are accumulated together with NarCh in the NarCh accumulating genotypes while different phenylpropanoids including caffeic and ferulic acids are accumulated in the rind of fruit that do not accumulate NarCh. These findings indicate that the biochemical flux alternation, which is associated with CmFBK, occurs either at 4CL or CHS step in the pathway.


-Transgenic: validation of the candidate gene function was performed using two different systems. Functional analysis in a virus induced gene expression system indicated that over expressing of CmFBK in melon leaves indeed caused a shift in the biosynthetic flux from flavonoids to phenylpropanoids. Additionally, stable transgenic tomato plants overexpressing CmFBK showed a significant reduction in NarCh accumulation in the fruit cuticles and visible anthocyanins (NarCh downstream compounds) in the vegetative tissues of T0 and T1 plants.


FBKs are known to be involved in protein ubiquitination, targeting them to the proteasome mediated degradation. Thus we suggest that CmFBK regulates flavonoid accumulation in melon through either 4CL or CHS enzymes degradation leading to metabolic flux alteration from flavonoids to phenylpropanoids. These findings could lead to a better understanding of the specificity mechanism of F-Box Kelch repeats protein-protein interactions in plants.