Cytosolic cycles regulate the turnover of sucrose in heterotrophic cell-suspension cultures of Chenopodium rubrum L.

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Abstract
We have investigated whether sucrose accumulation in heterotrophic cell-suspension cultures of Chenopodium rubrum L. is regulated by a cycle in which sucrose is simultaneously synthesised and degraded. Net sucrose accumulation was measured by monitoring the sucrose content, unidirectional synthesis was monitored by supplying pulses of [14C] glucose, and unidirectional degradation was estimated from the difference between unidirectional synthesis and net accumulation. When 50 mM glucose was supplied to carbohydrate-depleted cells there was a rapid net accumulation of sucrose, which stopped after 24 h. The incorporation of 14C into sucrose was similar to the initial rate of net sucrose accumulation, but rapid 14C incorporation continued after the cells had stopped accumulating sucrose. A method was developed to rapidly separate sucrose-phosphate synthase (SPS) from uridine-diphosphate-hydrolysing activities which interfered with the assay. The cells contained enough SPS activity to catalyse the observed rate of sucrose synthesis. SPS activity increased in cells which had stopped accumulating sucrose, and the enzyme became less sensitive to inhibition by inorganic phosphate. Sucrose synthase and alkaline invertase activity were four- and twofold higher than SPS activity, and both degradative enzymes increased in cells which had stopped accumulating sucrose. Sucrose synthase is strongly modulated by the concentration of sucrose and by competitive feedback regulation by fructose in these cells. It is concluded that sucrose accumulation ceases in these cells because the rate of degradation of sucrose increases until it matches the rate of synthesis. It is discussed how this cycle is regulated, and how it may interact with the substrate cycle between triose-phosphates and hexose-phosphates (Hatzfeld and Stitt, 1990, Planta 180, 198–204). These cycles allow sucrose turnover to respond in a highly sensitive manner to small changes in the balance between the supply of sucrose and the demand for carbon for respiration and biosynthesis in the cell.