Browsing by Author "Jayakumar, Rahul"
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- ItemFunctional identification of the chloroplastic raffinose transporter from arabidopsis(Stellenbosch : Stellenbosch University, 2015-12) Jayakumar, Rahul; Peters, Shaun W.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.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.