- Title
- The functional characterisation of novel sucrose transporters
- Creator
- Dibley, Katherine
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2012
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Sucrose is the predominant form in which photosynthetically-fixed carbon is transported over long distances in many plant species. Sucrose moves in the plant from regions of net photosynthetic fixation of carbon or storage (source organs) to sinks, where active growth and/or storage product accumulation occurs. The phloem serves as the long-distance transport conduit. One or more symplasmic discontinuities may occur along the pathway from source to sink, invoking plasma membrane transport steps. For instance, phloem unloading of nutrients includes an apoplasmic step in a number of physiologically important sinks such as developing seeds and fleshy fruits. The uptake of sucrose into plant cells has been well described, and is mediated by sucrose/proton symporter proteins (SUTs). In contrast, little is known about the molecular identity of membrane transporters contributing to sucrose efflux in apoplasmic phloem loading and unloading. The aim of this study was to identify and characterise novel sucrose transporters involved in sucrose efflux. Seed coats of pea and bean were selected as source material, as they are functionally committed to sucrose efflux. Cloning by homology to known SUT sequences, five genes were isolated from legume seed coats - three from pea (including the previously described PsSUT1) and two from French bean. Complementation of the yeast strain, SUSY7, demonstrated that each gene encodes a functional sucrose transporter. When functionally expressed in yeast, three of the five transporters studied exhibited pH- and energy-independent sucrose transport that was shown to be bi-directional. These transport properties, together with counter transport, are consistent with a sucrose facilitator (SUF) function. In addition, and unlike H+-coupled SUTs, their transport function was insensitive to diethylpyrocarbonate and did not bind maltose. Kinetically the SUFs functioned as low-affinity, high-capacity sucrose transporters. The physiological significance of these novel SUFs in mediating release of sucrose from coats to the seed apoplasm in developing pea and bean seeds is discussed. Cellular and subcellular localisation studies were also carried out to determine whether SUFs are present at putative sites of sucrose efflux. The cellular and subcellular localisation of PsSUT1, PsSUF1 and PsSUF4, cloned from pea, was carried out to determine their potential contribution to phloem loading and unloading of sucrose in planta. Transient expression of GFP-tagged protein showed that all three transporters were plasma membrane localised. Thus, the likely function of the two facilitators is to mediate sucrose movement into or out of cells down a sucrose concentration gradient. In contrast, the sucrose symporter PsSUT1 would be expected to mediate sucrose retrieval from the source leaf or seed apoplasms. Immunolocalisation studies in seed coat tissues revealed that PsSUF1 and PsSUF4 are present in the inner layers of parenchyma, the putative site of sucrose release in developing seed coats. In contrast, PsSUT1 was restricted to the seed coat vascular bundles, suggesting a role in the retrieval of leaked sucrose along the delivery pathway. PsSUF4 was also present within the vascular bundles, and its function is less apparent, given the polarity of the sucrose gradient and the transporter functions as a facilitator. In pea, phloem loading of leaf minor veins follows an apoplasmic pathway (Wimmers and Turgeon, 1991). As such, two transport events- efflux from the mesophyll symplasm and subsequent uptake by collection phloem- occur in series. Thus, source leaf minor veins were investigated as another region of symplasmic discontinuity and hence a site of sucrose efflux. SUFs were not located in putative efflux cells (bundle sheath or phloem parenchyma cells), but were present, along with PsSUT1, on the plasma membranes of sieve elements. In addition, another sucrose transporter, of unknown identity, was established to be located on plasma membranes of companion cells using a generic SUT1 antibody. To further understand the mechanisms of sucrose efflux, the need to access and study the cytoplasmic face of the SUFs was recognised. A system was developed to enable this by utilising the sec6-4 mutation in yeast, which results in the production of inside-out plasma membrane vesicles. When the transporter gene of interest is transformed into the yeast mutant and transporter protein is incorporated into inside-out vesicles, the cytoplasmic face of the membrane protein is exposed for study. Rapid, real time evaluation of sucrose transporter activity of the membrane vesicles can be monitored using stopped-flow fluorimetry. The technology achieves this outcome by measuring changes in light scattering as membrane vesicles osmotically shrink or swell in response to sucrose transport into or from the vesicles respectively. To make this system suitable for studies of sucrose transport, the endogenous yeast invertase and maltose transporters, which also transport sucrose, needed to be removed from the yeast genome. Initial attempts to remove the invertase gene were carried out in yeast harbouring the sec6-4 mutation, relying on homologous recombination-mediated gene disruption. However, multiple attempts at disruption indicated that more than one invertase gene was present in sec6-4 yeast. A different strategy was adopted which involved incorporating the vesicle accumulation mutation into a suitable (invertase and maltose transporter free) yeast strain. The resultant yeast strain developed, s6s7, successfully accumulated inside-out membrane vesicles, and when combined with the appropriate expression plasmid, offers a new system to functionally characterise sucrose transporters from their cytoplasmic face. Overall, the work presented in this thesis has increased our knowledge of the mechanisms of sucrose efflux in plants. We have demonstrated that the SUT gene family includes novel sucrose facilitators (SUFs) in addition to the sucrose/proton symporters previously reported. These plasma membrane SUFs are localised (but not restricted) to regions supporting high sucrose fluxes, including seed coats and the minor veins of source leaves. In developing seed coats, the contribution of SUFs (relative to other, as yet unidentified, energised transporters) to sucrose efflux may vary across seed development. The engineering of the s6s7 strain of yeast for sucrose transporter characterisation provides the opportunity to investigate the kinetics of the cytoplasmic face of sucrose transporters, including SUFs, SUTs and non-SUT family transporters. This will allow sucrose efflux to be better understood in planta.
- Subject
- sucrose transporter; sucrose facilitator; SUT; SUF; transport; seed; legume
- Identifier
- http://hdl.handle.net/1959.13/937218
- Identifier
- uon:12527
- Rights
- Copyright 2012 Katherine Dibley
- Language
- eng
- Full Text
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