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

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 [(14)C] 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 (14)C into sucrose was similar to the initial rate of net sucrose accumulation, but rapid (14)C 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.

The Metabolic Architecture of Plant Cells

“…The high fluxes of sucrose cycling found here are consistent with those found in ripening bananas (23) and in maize root tips (20). Possible roles for this futile cycle include the following: maintaining the balance between the accumulation of sucrose and the demand for carbon for respiration and biosynthesis in the cell (45,47,48) or the regulation of the sucrose and hexose concentrations in relation to the water status of the cell (20). It is interesting to note that the rate of sucrose cycling was closely related with glucose influx (around 6-fold) but in no case with alterations in hexose or starch accumulation.…”

“…8 and Table IV). A similarly high activity of the oxidative pentose phosphate pathway was found in maize root tips (20), and in cells of C. rubrum (47) and Daucus carota (48). A considerable recycling of triose phosphate in the cytosol has also been observed in C. rubrum cells and potato tubers (44) and an active futile cycle of sucrose in sugarcane (45), corn root tips (20), maturing banana (23), and kiwifruit (49), but lower values were found in C. rubrum cells (44).…”

Sugars modulate an unusual mode of control of the cell-wall invertase gene ( Incw 1) through its 3′ untranslated region in a cell suspension culture of maize

“…However, a similar pattern of induction by the hexose sugars is also seen in C. rubrum cell culture (2) and for the Incw2 gene in developing endosperm in vitro kernel cultures (27). A ''futile cycle'' of suc synthesis and cleavage, similar to the previously proposed suc turn-over reactions in several plant systems (2,28,29), is postulated as a possible basis for the hexose induction of the two Incw genes. In sycamore suspension cells, an SS pathway is proposed to be relatively more important than an invertase pathway when suc is limiting (15).…”

Carbon Fluxes in Mature Peach Leaves

“…Changes in the specific radioactivity of soluble carbohydrates and starch in mature peach leaves measured after a pulsechase experiment. The leaves were labeled using 14CO2 for 4 min, 7 h after the beginning of the photoperiod; the chase lasted 8 h. Soluble carbohydrates and starch were extracted and specific radioactivity was determined after HPLC separation as described in "Materials and Methods." These data represent the means ± SD of four replicates.…”