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Functional identification of the chloroplastic raffinose transporter from arabidopsis

Jayakumar, Rahul (2015-12)

Thesis (MScAgric)--Stellenbosch University, 2015.


ENGLISH ABSTRACT: In plants, the raffinose family oligosaccharides (RFOs) have a wide range of functions, ranging from membrane trafficking to participation in signal transduction processes. Their most well characterized role in plants, is the ability to function as compatible solutes that provides protection against abiotic stress factors. Raffinose is a member of the RFOs that have been found to be important in the protection of the photosynthetic apparatus of Arabidopsis thaliana under cold-acclimation. It has been well characterized that raffinose accumulates in chloroplasts under coldacclimation and is transported via a plastidic membrane transporter. The true identity of this elusive transporter is currently unknown. Presently, the only known eukaryotic transporter ever characterized is Mrt, which is found in the fungus Metarhizium robertsii. A protein BLAST analysis comparing the amino acid sequences of Mrt and a well characterized Arabidopsis chloroplastic glucose transporter, pGlcT1 (plastidic glucose translocator 1, TAIR accession code: AT5G16150) revealed a 24.7 % sequence homology. This showed significant functional homology between the two transporters. Our research aimed to identify the chloroplastic raffinose transporter by employing a multipronged approach. Firstly, an Arabidopsis thaliana library with full length clones in pBlueScript SK (+) was transformed in E. coli BL21 and growth was tested on M9 minimal media supplemented with raffinose to determine whether pGlcT1 was present. The Gateway® protein expression vector, pDEST17, containing the pGlcT1 gene was also transformed into E. coli BL21. The construct was used for heterologous expression of pGlcT1 in E.coli BL21 AI on M9 minimal media supplemented with raffinose to test growth. This approach involved an in planta approach which utilized chlorophyll fluorescence to measure the quantum efficiency of Photosystem II photochemistry (Fv/Fm) in pGlcT1 (SALK_066365) mutant plants. This was to determine if pGlcT1 was essential for raffinose accumulation in Arabidopsis chloroplasts under cold-acclimation (4oC) for 7 d. The results demonstrated that E. coli was able to grow on raffinose and sucrose in the presence of pGlcT1. Chlorophyll fluorescence analyses indicated an expected Fv/Fm reduction in the raffinose synthase (RafS) mutant (RS14) plants consistent with previous studies. There was also a notable decrease in Fv/Fm (P = 0.0006) within the pGlcT1 mutant plants while the Col-0 wild type plants maintained normal Fv/Fm values (~0.80) for the duration of cold-acclimation. The decrease in Fv/Fm values for the pGlcT1 mutant plants were statistically significant (P = 0.0004) based on a repeated measures one-way ANOVA test coupled with a linear trend post-test. Despite the preliminary nature of our work, it can be deduced from our results that pGlcT1 may facilitate the uptake of raffinose into E. coli cells. Raffinose that enters cells is most likely catabolised through inherent α-galactosidase activity. When compared with the Fv/Fm values of the wild type Col-0 plants, the decrease in Fv/Fm values in both the RafS and pGlcT1 mutant plants indicates that there is a possible raffinose deficiency within the chloroplasts of these plants under cold-acclimation (4oC). This hints at the probable importance of pGlcT1 in transporting raffinose into the chloroplast which safeguards the photosynthetic machinery of PSII under coldacclimation conditions.

AFRIKAANSE OPSOMMING: In plante, die raffinose familie oligosakkariede (RVO) het 'n wye verskeidenheid van funksies, wat wissel van membraan handel deelname aan seintransduksie prosesse. Hul mees gekenmerk funksie is die vermoë om as versoenbaar opgeloste stowwe wat plante bied beskerming teen abiotiese stresfaktore te funksioneer. Raffinose is 'n lid van die RVO's wat gevind is belangrik in die beskerming van die fotosintetiese apparaat van Arabidopsis thaliana onder koue akklimatisering te wees. Dit is goed dat gekenmerk raffinose ophoop in chloroplaste onder koue akklimatisering en via 'n plastidic membraan vervoerder vervoer word. Die ware identiteit van hierdie ontwykende vervoerder is tans onbekend. Tans, die enigste bekende eukariotiese vervoerder ooit gekenmerk is Mrt, wat gevind word in die swam Metarhizium robertsii. A proteïen BLAST analise vergelyk die aminosuur volgorde van Mrt en 'n goed gekenmerk Arabidopsis chloroplastic glukose vervoerder, pGlcT1 (plastidic glukose locator 1, TAIR toetreding kode: AT5G16150) het ook 'n 27% volgorde homologie. Dit het getoon beduidende funksionele homologie tussen die twee transporters. Ons navorsing is daarop gemik om die chloroplastic raffinose vervoerder identifiseer deur die gebruik van 'n multipronged benadering. Eerstens, 'n Arabidopsis thaliana biblioteek vollengte klone in pBlueScript SK (+) was 'n proteïen uitdrukking te bou met die Gateway® pDEST17 vektor wat die pGlcT1 gene geskep is. Die konstruk is wat gebruik word vir heteroloë uitdrukking van pGlcT1 in E.coli BL21 AI op M9 minimale media aangevul met raffinose toets groei. Hierdie benadering betrokke is 'n in planta benadering wat chlorofil-fluoressensie aangewend word om die doeltreffendheid van kwantum Photosisteem II fotochemie (Fv/Fm) in pGlcT1 (SALK_066365) mutant plante te meet. Dit was om te bepaal of pGlcT1 was noodsaaklik vir raffinose opeenhoping in Arabidopsis chloroplaste onder koue akklimatisering (4oC) vir 7 d. Die resultate het getoon dat E. coli was net in staat om te groei op raffinose en sukrose in die teenwoordigheid van pGlcT1. Chlorofil-fluoressensie ontledings aangedui 'n verwagte afname Fv/Fm (P = 0.0006) in die raffinose sintase (RafS) mutant (RS14) plante in ooreenstemming met vorige studies. Daar was ook 'n redelik beduidende afname in Fv / Fm (P = 0.0004) binne die pGlcT1 mutant plante terwyl die kol-0 wildetipe plante gehandhaaf normale Fv / Fm waardes vir die duur van die koue akklimatisering. Dit is duidelik uit die resultate dat pGlcT1 fasiliteer die opname van raffinose in E. coli selle. Raffinose wat selle binnegaan is waarskynlik catabolised deur inherente α- galaktosidase aktiwiteit. In vergelyking met die Fv/Fm waardes van die wilde-tipe Kol-0 plante, die afname in Fv/Fm waardes in beide die RafS en pGlcT1 mutant plante bevestig dat daar 'n raffinose tekort binne die chloroplaste van hierdie plante onder koue akklimatisering (4oC). Dit belig die belangrikheid van pGlcT1 in die vervoer van raffinose in die chloroplast wat die fotosintetiese masjinerie van PSII beskerm onder koue akklimatisering voorwaardes.

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