Enzyme Regulation in C 4 Photosynthesis

Experiments were conducted with several Panicum species (representing the different C4 subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C4> C3-C4 intermediate > C3. In illuminated leaves, SPS activities were similar among the three C4 subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisukatum (C3 species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C4 subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C4 subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation. sucrose-P from fructose-6-P and UDP-glucose, plays a key role in the regulation of sucrose biosynthesis (10). In C3 species, sucrose biosynthesis occurs in the cytoplasm of mesophyll cells and the biochemical mechanism of sucrose formation has been relatively well established (28). In C4 species, however, research has concentrated almost exclusively on maize which is an NADP-ME species. The biochemical mechanism involved in the regulation of photosynthetic carbohydrate formation is considered to be more complex than in C3 species since two different cell types (i.e. MC and BSC) are involved in the photosynthetic carbon assimilation.In maize leaves, the MC is considered to be the main site of sucrose biosynthesis since SPS and other enzymes involved in sucrose formation are thought to be located largely, if not exclusively, in the MC (5...

mentioning

confidence: 87%

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C₄ Species

Ohsugi¹,

Huber²

1987

“…The biphasic substrate saturation curve reported by several groups working with plants (4,19) can probably be attributed to the use of a mixture of the low-and high-Km enzymes. The respective role of the two types of enzyme in the hydrolysis of Fru-2,6-P2 in vivo has not been defined.…”

mentioning

confidence: 99%

Fructose 2,6-Bisphosphate Hydrolyzing Enzymes in Higher Plants

Larondelle¹,

Mertens²,

Schaftingen³

et al. 1989

The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases 1, II, and ll, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a Km value of 30 micromolar, to a mixture of fructose 2-phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the ft-anomeric form. Its apparent Km for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described

“…13 as C4 plants in which there is a spatial separation between photosynthetic CO2 fixation and sucrose synthesis (7). In addition, the Fru 6-P,2K and Fru 2,6-P2ase from a C4 plant have been found to differ in certain regulatory characteristics from their C3 counterparts (15 1.5% (w/v) insoluble polyvinyl polypyrrolidone. Rats were killed by decapitation, the livers excised immediately, and 20 g of the tissue was extracted as described for leaves.…”

mentioning

confidence: 99%

Ion-Exchange Chromatography Separates Activities Synthesizing and Degrading Fructose 2,6-Bisphosphate from C₃ and C₄ Leaves but Not from Rat Liver

Macdonald

Chou²,

Buchanan³

1987

Self Cite

IFructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C3 (spinach, lettuce, and pea) and C4 (sorghum and amaranthus) plants but not from rat liver-a tissue known to contain a bifunctional enzyme with both activities. 12-32PjFructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.Fru 2,6-P2,2 a regulatory metabolite localized in the cytosol of leaf cells, plays a central role in the coordination of starch synthesis taking place in the chloroplast and sucrose synthesis and breakdown taking place in the cytosol (1, 3, 9). The concentration of Fru 2,6-P2 is defined by the activities responsible for its synthesis and hydrolysis, namely fructose-6-phosphate,2-kinase (Fru 6-P,2K; ATP:D-fructose-6-phosphate-2-phosphotransferase, EC 2.7.1.105) (Eq. 1) and fructose-2,6-bisphosphatase (Fru 2,6-P2ase; u-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 3.1.3.46) (Eq. 2). We have recently reported (12) that these two activities from certain leaves can be separated from one another on the basis of both charge and mol wt. The separated activities showed no detectable cross-contamination indicating that they are not present on a single bifunctional protein of the type found in liver and certain other mammalian tissues (6,8,17,18,20).The earlier experiments were with leaves of spinach and lettuce, both of which are C3 plants. The (25 g), lettuce (50 g), spinach (20 g), amaranthus (40 g), and sorghum (25 g) were washed and then homogenized in two volumes (w/v) of a preparative solution containing 20 mM Tris-HCl (pH 7.8), 14 mm 2-mercaptoethanol, 10% (v/v) glycerol (buffer A), and supplemented with (a) protease inhibitors, 0.5 mm phenylmethylsulfonyl fluoride, 2 mm e-amino n-caproic acid, 2 mM benzamidine hydrochloride; and (b) protectants against oxidative degradation, 20 mm sodium diethyldithiocarbamate, 1.5% (w/v) insoluble polyvinyl polypyrrolidone. Rats were killed by decapitation, the livers excised immediately, and 20 g of the tissue was extracted as described for leaves. Subsequent filtration and centrifugation of the crude extracts was as described previously for spinach leaves (12). The resulting supernatant fractions were loaded onto DEAE-cellulose columns (1.5 x 15 cm for spinach and rat liver, 2.1 x 15 cm for amaranthus, sorghum, lettuce, and pea), which were developed as described previously (12)