Carbon Assimilation and Leaf Water Status in Sugar Beet Leaves during a Simulated Natural Light Regimen

Carbon assimilation and leaf water status were studied in sugar beet (Beta vulgaris L., Klein E-type multigerm) leaves during a light period in which illumination either increased rapidly to full irradiance or changed gradually in a sinusoidal manner as generally occurs during a natural day. A light regimen that simulated the light of a natural day was produced by adjusting irradiance with a neutral-density filter under the control of a computer. Under this light regimen, photosynthesis, transpiration, and stomatal conductance followed the irradiance pattern very closely and ribulose bisphosphate carboxylase was nearly fully activated. When illumination was increased rapidly at the beginning of a light period, transpiration also increased quickly, causing leaves to wilt to some extent. The activation state of ribulose bisphosphate carboxylase increased to only 52%, but ribulose bisphosphate level was nearly twice as high as during the simulated natural day. In spite of the differences in activation state and ribulose bisphosphate levels, photosynthesis rates were very similar under both regimens. Nevertheless, differences in parameters between leaves under the two irradiance regimens can affect how a plant responds to internal or external factors, and therefore, the rate at which irradiance increases at the beginning of a light period is an important consideration when interpreting data.     

pH Dependence of Photosynthesis and Photorespiration in Soybean Leaf Cells

The effect of pH on the kinetics of photosynthesis, O(2) inhibition of photosynthesis, and photorespiration was examined with mesophyll cells isolated from soybean (Glycine max [L.] Merr.) leaves. At constant, subsaturating bicarbonate concentration (0.5 mm), O(2) inhibition of photosynthesis increased with increasing pH because high pH shifts the CO(2)-bicarbonate equilibrium toward bicarbonate, thereby reducing the CO(2) concentration. At constant, substrating CO(2) concentrations, cell photorespiration decreased with increasing pH. This was indicated by decreases in the CO(2) compensation concentration, O(2) inhibition of photosynthesis, and glycine synthesis. Km(CO(2)) values for isolated cell photosynthesis and in vitro ribulose-1, 5-diphosphate carboxylase activity decreased with increasing pH, while the Ki(O(2)) for both systems was similar at all pH values. The responses to pH of the corresponding kinetic constants of cell photosynthesis and in vitro RuDP carboxylase with respect to CO(2) and O(2) were identical. This provides additional evidence that the relative rates of photosynthesis and photorespiration in C(3) plants are determined by the kinetic properties of RuDP carboxylase.     

Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells

The occurrence of photorespiration in soybean (Glycine max [L.] Merr.) leaf cells was demonstrated by the presence of an O₂-dependent CO₂ compensation concentration, a nonlinear time course for photosynthetic ¹⁴CO₂ uptake at low CO₂ and high O₂ concentrations, and an O₂ stimulation of glycine and serine synthesis which was reversed by high CO₂ concentration. The compensation concentration was a linear function of O₂ concentration and increased as temperature increased. At atmospheric CO₂ concentration, 21% O₂ inhibited photosynthesis at 25 C by 27%. Oxygen inhibition of photosynthesis was competitive with respect to CO₂ and increased with increasing temperature. The Km (CO₂) of photosynthesis was also temperature-dependent, increasing from 12 μm CO₂ at 15 C to 38 μm at 35 C. In contrast, the Ki (O₂) was similar at all temperatures. Oxygen inhibition of photosynthesis was independent of irradiance except at 10 mm bicarbonate and 100% O₂, where inhibition decreased with increasing irradiance up to the point of light saturation of photosynthesis. Concomitant with increasing O₂ inhibition of photosynthesis was an increased incorporation of carbon into glycine and serine, intermediates of the photorespiratory pathway, and a decreased incorporation into starch. The effects of CO₂ and O₂ concentration and temperature on soybean cell photosynthesis and photorespiration provide further evidence that these processes are regulated by the kinetic properties of ribulose-1,5-diphosphate carboxylase with respect to CO₂ and O₂.     

A dye binding method for measurement of total protein in microalgae

Protein is a large component of the standing biomass of algae. The total protein content of algae is difficult to measure because of the problems encountered in extracting all of the protein from the cells. Here we modified an existing protein assay to measure total protein in microalgae cells that involves little or no extraction of protein from the cells. Aliquots of fresh or pretreated cells were spotted onto filter paper strips. After drying, the strips were stained in a 0.1% (w/v) solution of the protein stain Coomassie Brilliant Blue R-250 for 16 to 24 h and then destained. The stained protein spots were cut out from the paper, and dye was eluted in 1% (w/v) sodium dodecyl sulfate (SDS). Absorbance at 600 nm was directly proportional to protein concentration. Cells that were recalcitrant to taking up the dye could be either heated at 80°C for 10 min in 1% SDS or briefly sonicated for 3 min to facilitate penetration of the dye into the cells. Total protein measured in Chlorella vulgaris using this method compared closely with that measured using the total N method. Total protein concentrations were measured successfully in 12 algal species using this dye binding method.     

Binding of a Phosphorylated Inhibitor to Ribulose Bisphosphate Carboxylase/Oxygenase during the Night

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured at various times during the purification of the enzyme from leaves of Nicotiana tabacum which were collected either 1 hour before the start of the photoperiod (predawn) or in the middle of the photoperiod (midday). The activity of the enzyme in extracts of the predawn leaves (0.8 units/mg enzyme) was consistently about 2-fold lower than that measured in extracts of midday leaves (1.7 units/mg enzyme). The activity of the predawn enzyme was increased to that of the midday enzyme following removal of CO₂ and Mg²⁺ (deactivation), (NH₄)₂SO₄ precipitation, or incubation in SO₄²⁻ (18 millimolar required for one-half maximal increase). Following purification to >95% homogeneity, the predawn enzyme was found to have ~0.5 moles of bound organic phosphate per mole of enzyme active sites, while the midday enzyme had only ~0.08 moles of bound organic phosphate per mole of enzyme active sites. Deactivation of the predawn enzyme or treatment with 0.2 molar SO₄²⁻ resulted in the removal of most of the bound organic phosphate. These findings support the hypothesis that following the night period about 50% of the enzyme is catalytically inactive because of the tight-binding of a small molecular weight, phosphorylated inhibitor at the active site.     

Activation state of ribulose bisphosphate carboxylase in soybean leaves

Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg²⁺ and CO₂ (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO₃ (5 millimolar saturating) and Mg²⁺ (5 millimolar optimal) for 5 minutes at 25°C or 30 minutes at 0°C before assay. Initial activity was about 70% of total activity. K_act (Mg²⁺) and K_act (CO₂) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg²⁺ in the leaf extract was sufficient to support considerable catalytic activity. While Mg²⁺ was essential for both activation and catalysis, Mg²⁺ levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg²⁺ was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0°C. Initial activity quickly deactivated at 25°C in the absence of high CO₂. Total activity slowly declined irreversibly upon storage of leaf homogenate at 25°C.     

Low root temperature effects on soybean nitrogen metabolism and photosynthesis

The influences of low root temperature on soybeans (Glycine max [L.] Merr. cv. Wells) were studied by germinating and maintaining plants at root temperatures of 13 and 20 C through maturity. At 42 days from the beginning of imbibition, 13 and 20 C plants were switched to 20 and 13 C, respectively. Plants were harvested after 63 days. Control plants (13 C) did not nodulate, whereas those switched to 20 C did and at harvest had C₂H₂ reduction rates of 0.2 micromoles per minute per plant. Rates of C₂H₂ reduction decreased rapidly in plants switched from 20 to 13 C; however, after 2 days, rates recovered to original levels (0.8 micromoles per minute per plant) and then began a slow decline until harvest. Arrhenius plots of C₂H₂ reduction by whole plants indicated a large increase in the energy of activation below the inflection at 15 C. Highest C₂H₂ reduction rates (1.6 micromoles per minute per plant) were at 58 days for the 20 C control. Root respiration rates followed much the same pattern as C₂H₂ reduction in the 20 C control and transferred plants. At harvest, roots from 13 C-treated plants had the highest activities for malate dehydrogenase, glutamate oxaloacetate transaminase, and phosphoenolpyruvate carboxylase. Roots from transferred plants had intermediate activities and those from the 20 C treatment the lowest activities. Newly formed nodules from plants switched from 13 to 20 C had much higher glutamate dehydrogenase than glutamine synthetase activity.     

Effect of N-(Phosphonomethyl)glycine on Carbon Assimilation and Metabolism during a Simulated Natural Day

The effects of N-(phosphonomethyl)glycine (glyphosate) on the regulation of carbon assimilation, metabolism, and translocation were studied in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under a light regimen that began with gradually increasing irradiance as generally occurs on a natural day. Soon after application, glyphosate caused a marked increase in ribulose bisphosphate and a decrease in phosphoglyceric acid. The response is most simply explained by direct inhibition of ribulose bisphosphate carboxylase activity. The extent of inhibition was small, and the carbon assimilation rate did not decrease. As predicted, photosynthesis declined within an hour after glyphosate was applied to leaves under gradually increasing light. Inhibition resulted from a decrease in ribulose bisphosphate due to depletion of carbon from the photosynthetic carbon reduction cycle. Photoinhibition, a light-dependent limitation of photosynthetic capacity, appeared to be necessary for marked glyphosate-induced inhibition of photosynthesis. As a result, photosynthesis rate increased with irradiance until it exceeded 400 micromoles per square meter per second but then declined as the light level increased beyond 500 micromoles per square meter per second. Glyphosate changed the allocation of newly fixed carbon between starch and sucrose for export. Changes in the levels of ribulose bisphosphate and phosphoglyceric acid produced important effects on the regulation of carbon assimilation and metabolism.     

Regulation of photosynthetic carbon reduction cycle by ribulose bisphosphate and phosphoglyceric Acid

The activation states of a number of chloroplastic enzymes of the photosynthetic carbon reduction cycle and levels of related metabolites were measured in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under slowly changing irradiance during a day. The activation states of both phosphoribulokinase and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase increased early in the light period and remained constant during the middle of the day. Initial ribulose 1,5-bisphosphate carboxylase activity was already about one third of the midday level, did not change for the first 2 hours, but then increased in parallel with the rate of carbon fixation. Because the activation states increased by turns, first phosphoribulokinase and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase and later ribulose 1,5-bisphosphate carboxylase, the ratios of the activation states changed remarkably. Levels of ribulose bisphosphate and phosphoglycerate, which were high enough to affect enzyme reaction rates and changed in concert with activation state, indicate that these metabolites are involved in feedback/feedforward regulation of enzymes of carbon assimilation. This regulatory sequence is able to explain how the reaction rates for the enzymes of carbon assimilation are adjusted to maintain their activities in balance with each other and with the flux of carbon fixation.     

Sources of Carbon for Export from Spinach Leaves throughout the Day

Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity.