Identification and Functional Testing of Tonoplast Transporters in Sugarcane

Karl Pioch (2010). Identification and Functional Testing of Tonoplast Transporters in Sugarcane PhD Thesis, School of Biological Sciences, The University of Queensland.

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Author Karl Pioch
Thesis Title Identification and Functional Testing of Tonoplast Transporters in Sugarcane
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2010-03
Thesis type PhD Thesis
Supervisor Emeritus Professor Christa Critchley
Dr. Anne Rae
Professor Chris Grof
Total pages 190
Total colour pages 15
Total black and white pages 175
Language eng
Subjects 060702 Plant Cell and Molecular Biology
060110 Receptors and Membrane Biology
Abstract/Summary Sugarcane is a tropical grass species that can accumulate large amounts of sucrose. The mature stem can store 12 – 16 % of its fresh weight as sucrose and increasing the sugar yield is the focus of many breeding and research programs. Therefore understanding the metabolic processes of sucrose accumulation in the sugarcane stem would be advantageous for increasing sucrose concentration in the stem. A critical step in sucrose accumulation is sucrose crossing the tonoplast membrane into the vacuole, the hypothesised storage compartment. Transporters on the tonoplast are responsible for regulating this process and may be the key control point for sucrose accumulation. The aim in this thesis was to investigate the properties of selected tonoplast transporters from sugarcane and to unravel their roles in the process of sugar accumulation. In an attempt to develop an understanding of the cellular dynamics of the vacuole relative to sucrose accumulation, a gene expression study was conducted on a range of sugarcane varieties that accumulate high or low concentrations of sucrose. Transcript levels of genes encoding transporters with potential roles in the sucrose accumulation process were analysed and compared with plant sugar content and tissue type. A set of tools was developed for detecting tonoplast proteins in sugarcane tissues based on a new membrane fractionation technique. This technique was validated with transporters that have defined locations and was then used to examine novel transporters. In this experiment, the ShSUT4 transporter was assigned to the tonoplast, matching later localisation results. Along with ShSUT4, ShPST2a (another putative monosaccharide transporter) and H+ pyrophosphatase were also detected in the tonoplast fractions. This method was also able to distinguish plasma membrane location and showed ShSUT1 localised to this membrane. In order to characterise the putative sugar transporters, each transporter was kinetically analysed in yeast mutants and localised by expression of GFP fusion proteins in onion epidermal cell layers. ShPST5a, a member of the ERD6-like family of monosaccharide transporters, was analysed and found to probably be a proton symporter with a Km for glucose of 3.3 mM consistent with low affinity kinetics. The localisation experiment established that PST5a is a tonoplast transporter. This symporter would most likely function in export of glucose from the vacuole to the cytoplasm. ShPST5a is the second ERD6-like transporter to be functionally characterised in plants and the first glucose exporter identified. viii ShSUT4 was also found to probably be a proton symporter with a Km for sucrose of 7.5 mM, consistent with low affinity kinetics. ShSUT4 is a member of the SUT4-like transporter family which contains both plasma membrane and tonoplast located members. SUT4 was also localised to the tonoplast and this is consistent with a role for ShSUT4 in export of sucrose from the vacuole. The discovery of new tonoplast localised transporters presented an opportunity to examine the sequences for motifs that control membrane localisation. A bioinformatic method was developed for predicting membrane localisation based on predictions of transporter protein structure. This method remains untested but is an important step forward in finding sugar transporter localisation signals in plants. The evidence presented in this thesis has established the potential roles of two transporters in sugarcane as vacuolar exporters, with ShPST5a the first of its kind found in plants. Understanding the dynamics of sugar transport across the tonoplast membrane is a significant step forward in our understanding of the sucrose accumulation process in sugarcane.
Keyword Saccharum
Additional Notes Colour pages: 27,37, 69, 79, 97, 98, 102, 109, 113, 127, 133, 138, 142, 147, 155 Landscape pages: 179-190

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Created: Wed, 16 May 2012, 03:11:48 EST by Mr Karl Pioch on behalf of Library - Information Access Service