"Proof of concept" (provided by the Max-Planck-Institute of Molecular Plant Physiology, Golm)

Sucrose and starch are the main products of photosynthesis in plants. Sucrose is synthesized in the cytosol from triose-phosphates exported from the chloroplast, whereas starch is synthesized directly in the chloroplasts. Most of the sucrose is exported from the leaf, via the phloem, to sink organs such as roots, developing leaves and seeds, and provides them with most of the carbon and energy they need for growth. In contrast, starch is retained in the leaf, but is broken down at night to provide hexose sugars for leaf respiration, and for synthesis and continued export of sucrose. Transitory starch reserves in the leaves also help to maintain sugar levels during periods when photosynthesis is limited by low light or other adverse environmental conditions.

Studies with antisense plants and other mutants with altered enzyme activities have shown that two enzymes - sucrose-phosphate synthase (SPS) and ADPglucose pyrophosphorylase (AGPase) - play key roles in controlling the rates of sucrose and starch synthesis in leaves (Stitt and Sonnewald, 1995). Both of these enzymes are subject to transcriptional, post-translational and allosteric regulation. AGPase is a heterotetramer consisting of two small catalytic (APS) subunits and two large regulatory (APL) subunits. The enzyme is allosterically activated by 3PGA and inhibited by Pi. AGPase is also regulated by redox modulation involving the reversible formation of an inter-molecular disulphide bridge between the Cys-11 residues of the two small subunits. The oxidized form (APS dimer) of the enzyme has lower substrate affinities than the reduced form (APS monomer), and is less sensitive to allosteric activation by 3PGA. SPS is allosterically activated by Glc6P and inhibited by Pi, and is subject to multi-site protein phosphorylation that affects the enzyme's substrate affinities and sensitivity to its allosteric regulators. SPS is light activated/dark deactivated by reversible phosphorylation of Ser158, and is also activated in response to osmotic stress by phosphorylation of Ser424. A further phosphorylation site, Ser229, is involved in 14-3-3 protein binding, and appears to regulate proteolytic turnover of the protein. The activation state of SPS is also affected by Pi status, low temperature and sucrose, and there is some evidence that protein phosphorylation is also involved in these responses.

The APS1 (At5g48300) gene encoding the small subunit of AGPase, and the SPS5B (At5g20280) gene encoding the major isoform of SPS in leaves, have been targetted for TILLING to generate a set of mutants with altered catalytic properties (specific activity and substrate affinities) or sensitivity to allosteric and post-translational regulation. The aim is to demonstrate the potential of TILLING to discover change-of-function mutants in addition to loss-of-function mutants. The advantages of targetting these two genes for this purpose are that the activities and kinetic properties of both enzymes can be readily measured, using highly sensitive and high-throughput assays based on a robotised microtitre-plate platform (Gibon et al., 2002), and post-translational modifications can be monitored using immunoblotting and protein mass-spectrometry. The effects of any changes in the catalytic properties and regulation of the enzymes on the rates of sucrose and starch synthesis, and photoassimilate partitioning, can also be determined using isotopically labelled tracers, and will help us to better understand the regulatory networks that control photosynthetic carbon metabolism in leaves.

References

Stitt M & Sonnewald U (1995) Regulation of metabolism in transgenic plants. Annual Review of Plant Physiology and Plant Molecular Biology 46, 341-368.

Hendriks JHM, Kolbe A, Gibon Y, Stitt M & Geigenberger P (2003) ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species. Plant Physiology 133, 838-849.

Gibon Y, Vigeolas H, Tiessen T, Geigenberger P & Stitt M (2002) Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system. Plant Journal 30, 221-235.