Thursday, August 18, 2011

Uptake of Carbon into Amyloplasts

The form in which carbon crosses the amyloplast membrane and enters into starch biosynthesis has been the subject of considerable debate. Categorical evidence that carbon enters potato tuber, Chenopodium rubrum, maize endosperm, wheat endosperm and tobacco amyloplasts in the form of hexose monophosphates (or nucleosides), rather than triose phosphates was provided by determination of the degree of randomisation of radiolabel in glucose units isolated from starch following incubation of the various tissues with glucose labelled at the C1 or C6 positions (Keeling et al., 1988; Viola et al., 1991; Hatzfeld and Stitt, 1990; Fernie et al., 2001c). These data are in agreement with the observation that potato tubers lack plastidial fructose 1, 6-bisphosphatase activity (Entwistle and ap Rees, 1990) and the failure to find expression of plastidial FBPase in tubers (Kossmann et al., 1992).

Although it is clear that triose-phosphates are not the substrate taken up by amyloplasts to support starch synthesis there has been considerable debate as to whether glucose 1-phosphate (Naeem et al., 1997; Tetlow et al., 1994; Tyson and ap Rees, 1988) or glucose 6-phosphate (Schott et al., 1995; Wischmann et al., 1999) is the preferred substrate for uptake. More recently, particularly in cereals, the uptake of cytosolically produced ADPglucose has also been much discussed (Pozeuta-Romera et al., 1991a, 1991b; ap Rees, 1995). The necessary pathways to support starch synthesis presuming uptake of one of these three substrates are presented in Figure 25.3. The results of recent transgenic and immuno-localisation experiments have indicated that the substrate for uptake is most probably species specific with clear evidence of the predominant route of uptake in the developing tuber in the form of glucose-6-phosphate, whereas in barley, wheat, oat and possibly maize, the predominant form of uptake is as ADPglucose (Denyer et al., 1996; Thorbjornsen et al., 1996; Shannon et al., 1998).

The cloning of a hexose monophosphate transporter from potato and the finding that the cauliflower homologue is highly specific for glucose 6-phosphate provides strong support for the first theory (Kammerer et al., 1998). Furthermore, when this observation is taken together with in vivo evidence that transgenic potato lines, in which the activity of the plastidial isoform of phosphoglucomutase was reduced by antisense inhibition, were characterised by a large reduction in starch content (Tauberger et al., 2000), then there are compelling grounds for asserting that glucose 6-phosphate is the major form in which tuber amyloplasts import carbon from the cytosol. Since these antisense plants were not starchless we cannot, however, exclude the possibility that glucose 1-phosphate makes some contribution to the flux to starch. Nor should we, in light of recent findings of extra-plastidial isoforms of ADPglucose pyrophosphorylase (Beckles et al., 2001), overlook the possibility of production of ADPglucose by a cytosolically localised enzyme and its subsequent transport in to the plastid to supplement starch synthesis.

However, this seems unlikely since following non-aqueous fractionation of potato tuber tissue AGPase activity always co-localised with pyrophosphatase activity which is known to be located exclusively in the plastid (Farre et al., 2001b) and expression of a bacterial AGPase in the cytosol of potato tubers did not result in an altered starch content (Stark et al., 1991). Furthermore, results from recent comprehensive studies in which the ratio of ADPglucose to UDPglucose was determined in a wide range of species suggest that the presence of a cytoplasmic AGPase isoform is limited to Graminaceous endosperms and is not a general feature of starch-storing organs (Beckles et al., 1991). This rationale behind these measurements is that the metabolite ratio is expected to be high in organs in which UDPglucose and ADPglucose are both mainly produced in the cytosol since the reactions of AGPase and UGPase will be coupled and close to equilibrium.

The results from this study are in direct contrast to earlier immunolocalisation studies using antisera against AGPase which suggested that there was an extraplastidiary isoform of AGPase in tomato fruit (Chen et al., 1998), but not in maize endosperm (Miller and Chourey, 1995; Brangeon et al., 1997). However, it is possible that the immunogold-labelling patterns seen in these studies do not accurately reflect the in vivo situation. Further studies by Beckles and Smith (2001) indicated that this is indeed probably the case since the proportion of the total activity of AGPase that was confined to the plastid was similar to that of the total activity of enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antisera raised against AGPase, most or all of the protein detected was plastidial.

The utilisation of UDPglucose as a substrate for starch synthesis has not been discussed here as it is unlikely to be a major route of starch synthesis in plants for several reasons. Firstly, unlike certain glycogen synthases that can efficiently utilise UDPglucose as a substrate, plant starch synthases are either specific for ADPglucose, or have affinities for this nucleoside that are far in excess of those for UDPglucose (Smith, 1990). Furthermore, since UGPase appears to be absent from the plastid (Entwistle and ap Rees, 1988) there is no route other than through starch synthases by which UDPglucose can support starch synthesis. Secondly, as described above the reduction of ADPglucose production by decreasing the AGPase activity in a wide range of species by approaches of mutagenesis or transgenesis reduces starch accumulation (Tsuai and Nelson 1966; Lin et al., 1988a, 1988b; Smith et al., 1989; Müller-Rober et al., 1992). Additionally, as described above genetic manipulation of SuSy which produces UDPglucose had no effect on starch synthesis.

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