PhD Thesis defense by Mr. Andreas Perrakis

"Τhe role of a prolyl 4 hydroxylase in tomato fruit development"

Thesis Title "Τhe role of a prolyl 4 hydroxylase in tomato fruit development "

Friday 14 September 2018, 11:00, Hall: Κ2.Α7

Supervisor: Nikolaos Kalogerakis

Seven-membered Examination Committee:

  1. Nikolaos Kalogerakis, Professor
  2. Dr. Panagiotis Kalaitzis, Research A MAICh
  3. Nikolaos Paranychianakis, Assistant Professor
  4. Polydeykis Chatzopoulos, Professor
  5. Elia Psillakis, Professor
  6. Danae Venieri, Assistant Professor
  7. Stamatis Rigas, Assistant Professor

Abstract:

Plant prolyl 4 hydroxylases catalyze proline hydroxylation of Arabinogalactan proteins (AGPs), extensions and hydroxyproline rich proteins. Therefore, the frequency of glycosylation is dependent on the frequency of hydroxylation indicating the importance of P4Hs for their proper structure and function. However, there are few reports investigating the physiological significance of P4Hs in tomato growth and development. Towards that direction a number of transgenic lines produced by silencing and overexpression of both Ρ4H3 ( P4H3-35S::RNAi and P4H3-35S::cDNA c.v. Ailsa-Craig) driven by the constitute prompter 35S. The down regulation of mRNA:P4H3 in 9 independent transgenic lines carrying the 35S::Ρ4H3-RNAi resulting a number of phenotypes with agronomic significance: I) Fruits with smaller size (~30%) due to alteration in cell enlargement. Microscope analysis revealed a significant reduction in the final cell area caused by a significant reduction in cell enlargement. II) A reduction by 80% the number of seeds number per fruit III) Seeds with altered morphology containing shorter embryos exhibiting also differences in germination rate, IV) Alterations in the physiological parameters of fruit ripening such as softening, V) Alterations in climacteric ethylene production rate, IV) Alteration in carotenoids consecrations and more specifically lycopene and β-carotene and The higher carotenoid levels may be the result of the higher expression levels of the genes involved in the carotnoids biosynthetic pathway as the Carotene isomerase L (CrtISO L) that was determined by qRT-PCR. On top of that microarray analysis was performed to determine the expression analysis and for the investigation of the P4H regulators and their interacting proteins by LeMoNe algorithm in four fruit ripening stages that yielded the identification of 2 transcriptional factors (ERF24 and bHLH54) regulating a significant number of genes. Moreover, only one P4H was found to be differentially expressed during fruit ripening, the P4H4. Besides that, a notable phenotype was the alteration in anther and fruit abscission time. It was monitored that in all 9 indented transformed lines there was a delay in fruit abscission in over ripe fruits comparing with over ripe w.t. fruits. These alterations in programmed fruit abscission are highly associated with the tensile strength experiments in red ripe fruits, that showed that RNAi fruits required greater force (N) and more time (sec.) for the abscission zone to break due to greater elongation percentage (%) compared to the w.t. due to the greater tensile strength required, as a result of lower expression levels of cell wall hydroxylases such as polygalacturonases, cellulases and b-galactosidases. The level of those genes expression was determined by qRT-PCR. Those alterations were explained to the lower cell number of the transgenic lines RNAi #7, #6 and #1 which were analyzed. The main goal of this work was to study the potential function of P4H3 in tomato fruit, by altering the expression of P4H3 gene. The results yielded a great number of phenotypes with significant agronomic importance, as the increase in carotenoids levels, and improvement in softening that can be used in the future the improvement of tomato fruit quality.