Potassium (K+) is an essential nutrient required by plants in high concentration and its uptake is mediated by a number of different K+ channels and K+ transporters including the KT/KUP/HAK transporter family. Phylogenetic analysis of the KT/KUP/HAK transporters in Arabidopsis and rice reveals that the proteins are arranged in four distinct clusters (I-IV). Members of cluster I and II are suggested to act as high- and low-affinity transporters respectively, while transporters in cluster III and IV have been less studied. Elaeis guineensis (oil palm) is a palm oil producing crop. The palm oil is a rich source of vitamins, carotenoids, iron and antioxidant activity and is now the largest source of edible oil. Despite the scientific and economic importance of the oil palm, Elaeis guineensis jacq has been relatively unstudied compared to other important crops such as rice, tomato, and barley. The aim of this study was to clone and characterise KT/KUP/HAK transporters from the oil palm. By a combination of database searching, PCR and 5’ and 3’ RACE, three genes encoding KT/KUP/HAK transporters from the oil palm were identified and named EgKUP3, EgKUP8 and EgKUP11. Phylogenetic analysis of the obtained sequences together with other KT/KUP/HAK transporters from various plant species suggested that they belong to this transporter superfamily and grouped in either cluster II (EgKUP3 and EgKUP8) or cluster III (EgKUP11). The hydrophobicity and topology analysis by TMHMM predicted a cytoplasmic N-terminus and an extracellular C-terminus, whereas EgKUP8 was predicted to have extracellular N- and C- termini. EgKUP11 was predicted to feature cytoplasmic N- and C- termini. TMHMM predicts 13, 10 and 14 transmembrane domain for EgKUP3, 8, and 11 respectively. Transcriptomic analysis was conducted on EgKUPs seedlings at 7-, 14-, and 21-day after treatment with a range of different K+-concentrations; 0.2 mM, 12.3 mM and 21.3 mM. The results suggested EgKUP8 expression is significantly upregulated in root tissues under depleted K+ conditions (0.2 mM) at 14 and 21 day. The expression of EgKUP3 and EgKUP11 on the other hand was not sensitive to changes in external K+ concentration. Functional complementation using Escherichia coli knockout strain defective in K+ uptake systems revealed that all EgKUPs complemented growth at 50 mM K+ concentration while EgKUP8 was also able to complement growth at 5 mM K+ when tested at pH 7.5. In contrast none of the EgKUPs were able to grow at pH 5.5 under all external K+ concentrations tested. Observations from gene expression and functional complementation studies may indicate the importance of EgKUP8 in K+ sufficient environment. Next, the EgKUPs were expressed as C-terminal GFP fusions in S. cerevisiae to establish the pre-crystallisation information needed for structural characterisation. The whole-cell florescent counts of each fusion proteins were used to calculate the approximate amount of protein expression. Although all the fusion proteins expressed between 1 – 1.3 mg/L (1 mg/L is a set threshold for protein expression), it proved impossible to extract the monodispersed transporters into our test detergents, including DDM and DM, as assessed by fluorescent size exclusion chromatography (FSEC). Cellular localisation studies indicated the EgKUPs do not localise to the plasma membrane plasma as expected but rather to the endoplasmic reticulum or cytoplasm, likely as a result of misfolding and faulty processing of the proteins in the S. cerevisiae system. Attemps to optimise the protein expression were performed by producing N- or/and C-terminally truncated proteins, but to no success. I also conducted experiments searching for novel solubilizing agents to improve general membrane protein stability in solution. However, this experiment has been conducted on a different protein, the xanthine/uric acid transporter (UapA) from Aspergillus nidulans, due to lack of success in obtaining a soluble and stable EgKUPs outside the lipid bilayer environment. The UapA which was, exchanged into Maltose Neopentyl Glycol 34 (MNG-34) amphiphile retained a greater amount of folded protein (53.2%) compared to protein exchanged into the conventional detergent, DDM (40%) after 130 min of heating at 40°C. This observation was confirmed by hFSEC assay in which MNG-34 was found to confer the greatest stability to UapA at 50°C.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:726916 |
| Date | January 2016 |
| Creators | Husri, Mohd Naqiuddin Bin |
| Contributors | Byrne, Bernadette |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/53929 |
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