Functional identification of the chloroplastic raffinose transporter from arabidopsis

[摘要] 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 compatiblesolutes that provides protection against abiotic stress factors. Raffinose is a memberof the RFOs that have been found to be important in the protection of thephotosynthetic apparatus of Arabidopsis thaliana under cold-acclimation.It has been well characterized that raffinose accumulates in chloroplasts under coldacclimationand is transported via a plastidic membrane transporter. The true identityof this elusive transporter is currently unknown. Presently, the only known eukaryotictransporter ever characterized is Mrt, which is found in the fungus Metarhiziumrobertsii. A protein BLAST analysis comparing the amino acid sequences of Mrt and awell characterized Arabidopsis chloroplastic glucose transporter, pGlcT1 (plastidicglucose translocator 1, TAIR accession code: AT5G16150) revealed a 24.7 %sequence homology. This showed significant functional homology between the twotransporters.Our research aimed to identify the chloroplastic raffinose transporter by employing amultipronged approach. Firstly, an Arabidopsis thaliana library with full length clonesin pBlueScript SK (+) was transformed in E. coli BL21 and growth was tested on M9minimal media supplemented with raffinose to determine whether pGlcT1 was present.The Gateway® protein expression vector, pDEST17, containing the pGlcT1 gene wasalso transformed into E. coli BL21. The construct was used for heterologousexpression of pGlcT1 in E.coli BL21 AI on M9 minimal media supplemented withraffinose to test growth. This approach involved an in planta approach which utilizedchlorophyll fluorescence to measure the quantum efficiency of Photosystem IIphotochemistry (Fv/Fm) in pGlcT1 (SALK_066365) mutant plants. This was todetermine if pGlcT1 was essential for raffinose accumulation in Arabidopsischloroplasts under cold-acclimation (4oC) for 7 d.The results demonstrated that E. coli was able to grow on raffinose and sucrose in thepresence of pGlcT1. Chlorophyll fluorescence analyses indicated an expected Fv/Fmreduction in the raffinose synthase (RafS) mutant (RS14) plants consistent withprevious 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 thepGlcT1 mutant plants were statistically significant (P = 0.0004) based on a repeatedmeasures 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 thatpGlcT1 may facilitate the uptake of raffinose into E. coli cells. Raffinose that enterscells is most likely catabolised through inherent α-galactosidase activity. Whencompared with the Fv/Fm values of the wild type Col-0 plants, the decrease in Fv/Fmvalues in both the RafS and pGlcT1 mutant plants indicates that there is a possibleraffinose deficiency within the chloroplasts of these plants under cold-acclimation(4oC). This hints at the probable importance of pGlcT1 in transporting raffinose into thechloroplast which safeguards the photosynthetic machinery of PSII under coldacclimationconditions.

[发布机构] Stellenbosch University