Transport Of Substrates Into The Amyloplast

Starch synthesis in higher plants occurs exclusively within the plastids. In heterotrophic cells this process is carried out in specialized starch-accumulating plastids (amyloplasts) and is fuelled by carbon substrates and energy (ATP) imported from the cytosol. The nature of the substrates imported in the amyloplast of starch-storing organs has been the subject of much investigation.

4.1. Transport of carbon substrates

Early work based on analysis of intramolecular label re-distribution during the conversion of labeled glucose into starch showed import of 6-carbon molecules into potato tuber amyloplast to sustain starch synthesis (86, 87). This implied the operation of a transport system different than from that operating between the chloroplasts and cytosol in photosynthetic cells which involves the operation of a triose-P translocator. Subsequent work confirmed that the triose-P translocator present in chloroplasts is absent from potato amyloplasts and indeed from the generality of non-green plastids (88, 89). Although there is no doubt that purified amyloplasts from potato tubers have the capacity for transporting glucose-P in exchange for Pi, there is still some debate as to whether the substrate imported by the amyloplast to sustain starch synthesis is Glc6P or GlclP. Naeem et al. (80) obtained preparations of purified amyloplasts from potato tubers that were able to synthesize starch from GlclP (but not Glc 6P) in the presence of ATP. On the other hand, Schott et al. (79) showed with proteoliposomes reconstituted from potato tuber amyloplast preparations that Glc6P and not GlclP could counter exchange with Pi. However, the fate of the imported sugar phosphate was not followed. Recently, Kammerer et al. (89) purified a Glc6P/Pi translocator (GPT) from maize endosperm. The corresponding peptide sequences were then used to isolate cDNAs from maize endosperm, pea roots, cauliflower inflorescences and potato tubers. In potato, high expression of the GPT gene was found only in the tubers, suggesting a role of this translocator in amyloplast transport. The transport characteristics of the GPT protein were studied in proteoliposomes obtained from purified membrane of transformed yeast cells expressing the GPT gene. In this system GPT accepts both G6P and triose-P as counter-exchangeable substrates for Pi whilst the transport of Glc 6P is negligible. These results would imply that Glc6P and not GlclP is the sugar imported into potato tuber amyloplasts to sustain starch synthesis. Confirmation of this hypothesis will require the production of transgenic potato with modified GPT activity.

4.2. Transport of ATP

ATP import in all types of plastids (90) appears to be mediated by a specific ATP/ADP transporter. Kampfenkel et al. (91) reported the cloning of a cDNA encoding an ATP/ADP translocator (AATP1) from Arabidopsis thaliana L. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of ca 62 kDa in chloroplast inner envelope preparation (92). Functional expression of AATPl in Saccharomyces cerevisiae and Escherichia coli resulted in increased rates of transport after reconstitution of the extracted protein into proteoliposomes and indicated that the transporter counter-exchanged ATP with ADP. More recently, a cDNA with a high degree of similarity to AATPl was cloned from potato(93). Expression of this gene was observed in several heterotrophic tissues such as young leaves, embryos, petals and sepals but was highest in the tubers. Transgenic potato plants with modified ATP/ADP transporter content were created by expressing the corresponding potato cDNA in antisense orientation or the heterologous cDNA from A. thaliana in the sense orientation (ibid). Transgenic potato tubers with decreased ATP/ADP transporter showed reduced starch content whilst tubers of sense lines showed increased starch content. The changes in starch content and in the ATP/ADP transporter activity were highly correlated (r2 = 0.91). These results indicate that the rates of ATP import into the amyloplast or of ADP recycling are limiting factors with regard to starch synthesis. It would also appear that the rate of ATP generation in the cytosol is sufficient to cope with enhanced starch biosynthesis.

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