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Title:	Functional analysis of persicaria minor F-box1 and Arabidopsis thaliana SKIP11 genes in the biosynthesis of green leaf volatiles
Authors:	Muhammad Naeem-Ul-Hassan (P64751)
Supervisor:	Ismanizan Ismail, Prof. Dr.
Keywords:	F-box1 Arabidopsis thaliana Green leaf volatiles Biosynthesis Dissertations, Academic -- Malaysia
Issue Date:	Dec-2015
Description:	Green leaf volatiles (GLV), the products of oxylipin pathway have been identified as important components in plant defence, plant-plant and plant-insect interactions. F-box is a large group of proteins, containing variable number of different protein-protein interaction C-terminal domains. Kelch-repeat containing F-box protein subfamily is thought to be plant specific. Like other F-box proteins, their major function is to bind the substrates for protein degradation via ubiquitin-26S proteasome system. In Persicaria minor, co-regulation of an F-box (PmF-box1) and alcohol dehydrogenase genes were elucidated by exogenous application of jasmonic acid. This observation suggests that the PmF-box1 is involved in the regulation of P. minor oxylipin pathway. Objective of the present study is to evaluate the role of PmF-box1 and its homolog in Arabidopsis thaliana, AtSKIP11 in the hydroperoxide lyase (HPL) branch of oxylipin pathway of A. thaliana. Different constructs, containing PmF-box1, kelch-repeat modified PmF-box1 (KMF), AtSKIP11 and AtSKIP11 antisense sequences were created by cloning these sequences to pB2GW7, a gateway vector for the expression of plant genes. These constructs were transferred into A. thaliana plants via Agrobacterium mediated transformation. Six weeks old plants from the T2 generation of the independent transgenic events were analysed by quantitative real-time PCR (RT-qPCR) for the expression levels of HPL pathway genes and by SPME-GC-MS for the analysis of GLV. Results have shown that the production of C5 volatile, 1-penten-3-ol was reduced in most of the transgenic plants. Production of C6 alcohol GLV was reduced by about 40% in KMF and 56% in AtSKIP11 over-expressing plants compared to vector control (VC) plant samples. Whereas, the production of these compounds was found to be enhanced in the range of 5 to 11% in other types of transgenic plants and the AtSKIP11 knock-out (atskip11) plants. RT-qPCR results have shown that expression of all target genes of HPL pathway was reduced in the transgenic plants overexpressing AtSKIP11, PmF-box1 and KMF sequence than the control plants, but increased in the AtSKIP11-antisense plants and the atskip11 plants. Expression of LOX2 was highest in the atskip11 plants and lowest in the PmF-box1 overexpressing plants. Highest expression of HPL was recorded in atskip11 plants to a value of 1.44-fold than VC samples and highest down-regulation of this gene was recorded in AtSKIP11 overexpressing plants to a value of 2.85-fold as compared to control plants. Changes in the expression levels of ADH1 were found as high as 1.95-fold in the AtSKIP11-antisense expressing plants and as low as 2.11-fold in the AtSKIP11 overexpressing plants compared to control samples. These results suggest that AtSKIP11 is involved in the regulation of HPL branch of oxylipin pathway in a negative manner. This may occur through degradation of the regulatory proteins, like transcription factors by their interaction with the kelch repeats of AtSKIP11 component of SCFAtSKIP11 complex. Although levels of some GLV changed in the PmF-box1 manipulating plants, but we could not find strong evidence in favour of the involvement of PmF-box1 in the Arabidopsis HPL pathway. However, considering the results of previous studies in our lab, it is suggested that this protein may be involved in the regulation of P. minor HPL pathway following a similar mode of action as we proposed for the AtSKIP11 in A. thaliana.,Certification of Master's/Doctoral Thesis" is not available
Pages:	173
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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