Characterization of Diurnal Changes in Activities of Enzymes Involved in Sucrose Biosynthesis

Abstract

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Meff., 'Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.Sucrose is the primary transport carbohydrate in most higher plants (1 1). In leaves of C3 plants, synthesis of sucrose occurs in the cytoplasm of mesophyll cells (3,25,30). The principal precursors for the sucrose biosynthetic pathway are triose phosphates which are generated within the chloroplast and exported to the cytoplasm by way of the phosphate translocator ( 17, 30). In the light, triose phosphates originate from concurrent photosynthesis and in the dark from degradation of starch. Decreased export of assimilates and lower sucrose levels in leaf tissue (8,10,12,16)

Enzyme Activities of Starch and Sucrose Pathways and Growth of Apical and Basal Maize Kernels

“…In growing tissues, sugars accumulate into vacuoles (9) and contribute to osmotic water potential, thus facilitating water flow into expanding cells (1). We observed that apical kernels had substantially lower sucrose concentrations and slightly higher glucose concentrations than basal kernels during the cell expansion stage (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Similar differences in the sucrose concentration of endosperm from apical and middle-position kernels have been reported recently for field-grown maize (23).…”

“…2.4.1.13) activity was determined as the UDP-Glcdependent sucrose formation in the presence of fructose substrate. Supernatant enzyme preparations were incubated with 7.5 mM UDP-Glc, 7.5 mM fructose, 15 mM MgCl2 in 50 mM HepesNaOH buffer (pH 7.5) at 25°C for 15 to 30 min (17). Reactions were terminated by heating with boiling water for 10 min, then samples were incubated 1 h with 10 IU of invertase (25C, pH 4.5) and glucose was measured as described for the invertase method.…”

“…Activities of insoluble invertase per gdw in both regions (B), and insoluble starch synthase (C) activities on a dry weight basis (left) and a kernel basis (right) during development of apical (+) and basal (0) kernels. ADP-Glc pyrophosphorylase was determined by measuringglucose-l-P formation from ADP-Glc and PPi using a coupled spectrophotometric assay (14,17). The soluble and insoluble preparations from enzyme extracts were assayed for starch synthase activity by meas-from 7 to 19 DAP, when cell division and expansion occurs (5), apical kernels had substantially lower sucrose concentration and insoluble (acid) invertase activity.…”

“…ADPG-PPase (ATP: a-i-glucose-l-phosphate adenylyltransferase; E.C 2.7.7.27) activity was determined by a coupled spectrophotometric assay (14,17). Contents of the reaction mixture were 50 mM Hepes-NaOH (pH 7.5), 5 mM MgCl2, 5 mM ADPGlc, 2 mM PPi, 0.3 mM NADP+, 1.5 IU phophoglucomutase, 1 IU 6-phosphogluconate dehydrogenase, 5 IU glucose-6-P dehydrogenase, and 25 1l ofsoluble enzyme preparation.…”

Tansley Review No. 105.

“…In some varieties of soybean the activity of SPS changes with a circadian rhythm independently of light conditions, but the activation of SPS in maize leaves is lightdependent (Rufty et al, 1983 ;Kalt-Torres et al, 1987). In maize and rice (C $ ) leaves, light alone seems to give maximal activation of SPS, whereas in spinach leaves, feeding with mannose is usually required to achieve maximal activation (Stitt et al, 1988 ;Huber et al, 1995 ;J.…”