- Title
- Phloem loading and unloading pathways in sorghum bicolor: the role of sucrose transporters in export and storage of sucrose
- Creator
- Milne, Ricky
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Stem sucrose accumulation within C4 monocotyledonous plant species is of both commercial and biological interest. Although mechanisms underlying stem sucrose accumulation are not well understood, it is likely that sucrose transporters (SUTs) play an important role in delivery of sucrose to stem sinks. Sorghum bicolor is an ideal monocotyledonous model for investigating SUTs, as this species can store sucrose within its stem to high concentrations (exceeding 500 mM). Further, the full genomic sequence is publicly available. Sucrose produced in photosynthesising leaves (source tissues) enters the phloem and is transported to regions of growth and storage (sink tissues). This source-sink pathway of sucrose transport in sweet Sorghum was investigated using histochemical stains and tracer dyes. The involvement of SUTs in phloem loading and unloading was also investigated by expression, localisation and functional characterisation studies. Cell to cell transport of sucrose can occur via plasmodesmal connections (symplasmic) or via the apoplasm with transporters mediating sucrose passage across plasma membranes (apoplasmic). Deposition of lignin and suberin is associated with vascular bundles, especially in stems. These complex polymers deposited in the apoplasmic space can restrict apoplasmic solute movement. In the Sorghum source leaf, minimal lignification and suberisation was observed. At the predicted site of phloem loading, small vascular bundles, lignin and suberin were primarily deposited in bundle sheath cell walls interfacing with mesophyll cells. The apoplasmic tracer dye, PTS was unable to pass through these lignified and suberised bundle sheaths. The symplasmic tracer dye, CF (loaded as CFDA) was restricted in sieve elements (SEs). This indicated that sucrose may follow a symplasmic path through the bundle sheath, before it is released to the apoplasm and actively transported into sieve element-companion cell (SE-CC) complexes. This sucrose is then exported to sinks through the long distance transport pathway. Lignin was present in walls of protoxylem elements of meristematic, elongating and recently elongated internodal zones. Suberin was absent from these zones. Because of this, the apoplasmic tracer, PTS was unrestricted between SE and storage parenchyma apoplasms. In maturing and mature internodal zones, heavy lignification and suberisation of sclerenchyma sheath cell walls restricted apoplasmic tracer movement between SE and storage parenchyma apoplasms. Hence symplasmic unloading was required for sucrose to pass the lignified and suberised sclerenchyma sheath. Symplasmic continuity between SEs and storage parenchyma cells was comfirmed by PTS as a symplasmic tracer dye (loaded as PTSA). These observations were used to investigate the function of sucrose transporters in loading and unloading. Six sucrose transporters were identified in the Sorghum genomic sequence and cloned from low and high sugar accumulating cultivars – a grain cultivar (BTx623) and a sweet cultivar (Rio). The SUTs were cloned and transformed into the mutant SEY6210 strain of Saccharomyces cerevisiae (not capable of using sucrose as a carbon source) and confirmed as sucrose transport capable by complementation on solid media. Additionally, a variant of SUT5 was identified in a sweet cultivar, Rio, which possessed nine amino acid differences to the SUT5 from cv. BTx623, and was also transport functional. Transporter expression in yeast and oocytes revealed that SbSUT1 and SbSUT5 were sucrose transport capable in both, and additionally SbSUT5G (SUT5 from grain Sorghum cultivar, BTx623) was sucrose transport capable in oocytes. SbSUT4 was found to be non-functional in yeast or oocytes as it was not localised to the plasma membrane. SbSUT1 and SbSUT5 were dependent on a proton gradient as treatment by the protonophores, 2,4-DNP and CCCP resulted in a reduction in sucrose uptake in yeast. SbSUT1 and SbSUT5 were also found to be DEPC sensitive and possessed a conserved histidine residue. All three SUTs were dependent on the proton motive force (pmf) and both components (pH and membrane potential) affected sucrose transport into oocytes. All were highly selective for sucrose and affinities were similar to other monocot SUTs. Three SbSUTs were expressed strongly in source leaves (SbSUT1, SbSUT4, SbSUT6). SbSUT4 localised to the tonoplast of tobacco leaf protoplasts and was immunolocalised to Sorghum mesophyll cells. SbSUT4 is likely to buffer cytosolic sucrose levels for phloem loading. Supporting apoplasmic loading in Sorghum, sucrose transporters were immunolocalised with the generic PEP2 antiserum to SE plasma membranes in source leaf minor veins. SbSUT1, localised to the plasma membrane of tobacco leaf protoplasts, may load sucrose into sieve elements of small vascular bundles. SbSUT1 demonstrated high affinity and selectivity for sucrose, supporting a phloem loading function. SbSUT6 is predicted to have a similar function. Unloading from protophloem SEs in meristematic and elongating zones of the internode was predicted to be apoplasmic, due to symplasmic isolation of protophloem SEs in other species. SbSUTs were immunolocalised to SEs and are likely to function in sucrose retrieval. Low expression observed in developing zones may indicate low levels of retrieval by SbSUT2 and SbSUT5, hence unloading to the vein apoplasm may occur by diffusion. Sucrose may be cleaved by cell wall invertase and loaded into parenchyma cells by hexose transporters. Alternatively, SUTs localised to the plasma membrane of parenchyma cells may load sucrose into these cells. In maturing internode zones, radial sucrose transport was via a symplasmic route. SbSUT1 was highly expressed, and SbSUTs were localised by generic antiserum (PEP2) to SE-CC and storage parenchyma plasma membranes. These SUTs are likely to function in sucrose retrieval, in order to maintain turgor homeostasis to support unloading to storage parenchyma cells by bulk flow. Preferential expression of the tonoplast antiporters SbTMT1 and SbTMT2 in zones actively accumulating sucrose suggests they may function to mediate sucrose entry into vacuoles. Overall, work presented in this thesis extends the limited understanding of source to sink transport in monocotyledonous species which accumulate sucrose. Sucrose transporters were found to be directly involved in phloem loading, and they are likely to be associated with phloem unloading in different capacities across developmental stages.
- Subject
- sorghum; source to sink pathway; sucrose; phloem transport; sucrose transporter
- Identifier
- http://hdl.handle.net/1959.13/1057618
- Identifier
- uon:16223
- Rights
- Copyright 2014 Ricky Milne
- Language
- eng
- Full Text
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