Effects of Pod Removal on Metabolism and Senescence of Nodulating and Nonnodulating Soybean Isolines: II. Enzymes and Chlorophyll

The objectives of this work were to determine the effect of sink strength (presence or absence of pods) and nitrogen source (nodulating versus nonnodulating plants) on enzymic activities, chlorophyll concentration, and senescence of soybean (Glycine max [L.] Merr. cv Harosoy) isolines. A 2-year (1981-1982) field study was conducted.

For both nodulated and nonnodulated plants, ribulose bisphosphate carboxylase (RuBPCase) activity of upper-canopy leaves was decreased by pod removal in both years, while chlorophyll concentration was decreased in 1981 only. Nonnodulated plants had lower RuBPCase activity in 1981 and lower chlorophyll concentration in both years compared with nodulated plants. In both years, and for all treatments, RuBPCase activity and chlorophyll began to decline at about the same time, but the rate of decline was less for depodded than for podded plants. Leaves in the middle and lower parts of the canopy had similar RuBPCase activity and chlorophyll concentration trends as upper-canopy leaves for all treatments.

Profiles of nitrate reductase activity (NRA) were similar for all treatments in both 1981 and 1982. Acetylene reduction profiles were similar for nodulated-podded and nodulated-depodded plants. The peak and decline in NRA profiles preceded the peak and decline in acetylene reduction profiles. The rate of decline in acetylene reduction activity was less for depodded plants, especially in 1982, but activities reached zero by the final sampling time. Thus, nodule senescence was not prevented by pod removal.

Based on seasonal profiles of RuBPCase activity, chlorophyll, NRA, and acetylene reduction activity, the initiation of senescence appeared to occur at the same approximate time for all treatments and, thus, did not depend on the presence or absence of pods or nodules. The hypothesis that nodules act as a nitrogen source and carbohydrate sink to delay senescence in the absence of pods was not correct.

     

Protein degradation in lemna with particular reference to ribulose bisphosphate carboxylase: I. The effect of light and dark

Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modification of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.     

Studies on Genetic Male-Sterile Soybeans: I. Distribution of Plant Carbohydrate and Nitrogen during Development

Soybeans (Glycine max [L.] Merr. cv. NC 69-2774) were used to study the nonstructural carbohydrate and nitrogen content of plant tissues, and nitrogenase activity throughout the development of male-sterile and male-fertile plants. Male-sterile plants set approximately 85% fewer pods plus seed than the male-fertile siblings and retained green leaves until a killing frost at 145 days after emergence. Reduced pod set caused increased carbohydrate accumulation in the leaf and root systems of male-sterile plants. Total carbohydrate in roots of male-sterile plants increased from 1.7 to 7.6 times that in the male-fertile roots. A high proportion (60 to 70%) of the male-sterile root carbohydrate was starch. Apparently, root starch was not metabolized by the male-sterile plants. Late in plant development per cent nitrogen was higher in the male-sterile soybean tissues. However, no difference was found in the ability of the nodulated root systems from either genotype to fix nitrogen.     

Effect of pod removal on leaf photosynthesis and soluble protein composition of field-grown soybeans

Well nodulated, field-grown soybeans (Glycine max [L.] Merr. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings.     

Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum

In free-living Rhizobium japonicum cultures, the stimulatory effect of CO₂ on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO₂ fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The CO₂-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain.     

Correlations among leaf CO2-exchange rates,areas and enzyme activities among soybean cultivars

Soybean (Glycine max (L.) Merr.) genotypes varying in area per nodal unit (usually a trifoliolate) and maturity class were grown in plots at the University of Illinois experimental farm. Leaf CO₂-exchange rates per unit area (CER) were measured under sunlight on intact plants. In addition to previously reported correlations with specific leaf weight and chlorophyll, CER was positively correlated with ribulose bisphosphate carboxylase (RuBPcase) activity, specific activity, and soluble protein, and was negatively correlated with area per leaf unit. The CER: chlorophyll correlation was destroyed by high CER values in 2 chlorophyll-deficient lines. CER values for 27 of the 35 lines tested fell within the range of those for isolines of cultivar Clark varying in leaf characteristics. The CER values were highest for fully expanded leaves during rapid pod fill. These results suggested that photoperiod (maturity) genes and genes for leaf area growth interact with genes controlling photosynthetic CO₂-exchange to produce the major differences in CER values among soybean genotypes.     

Effect of multiple factor source-sink manipulation on nitrogen and carbon assimilation by soybean

The objectives of this study were to determine the effect of light enhancement and hastened reproductive development on nitrogen and dry matter accumulation by field-grown soybean (Glycine max [L.] Merr.). The impacts of photosynthate supply and reproductive development on change in the season-long profiles of in vivo leaf nitrate reductase (NR) activity and root nodule acetylene reduction (AR) activity were evaluated.

Light enhancement resulted in significant increases in dry matter accumulation, root nodule fresh weight and AR activity. Seed yield was increased in both light enhanced treatments in 1978 and in one in 1979.

Hastened flowering and seed development was accomplished through photoperiod manipulation within a single genotype. Seasonal decline in leaf NR activity was most rapid in plants entering reproductive development early. An early increase in root nodule fresh weight and AR activity was also observed in response to this treatment and was followed similarly by early decline.

The addition of high levels of soil-applied nitrogen increased leaf NR activity and delayed late season decline in NR activity for both control and early reproductive plants. Nitrate supply was therefore implicated as limiting to leaf NR activity during the decline associated with flowering and early seed development. A limited additional increase in leaf NR activity was observed in response to light enhancement plus soil-applied nitrogen. As no significant increase in leaf NR activity was observed in response to light enhancement alone, leaf nitrate supply was further implicated as more limiting to leaf NR activity than was photosynthate supply during flowering and early seed development.

     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Occurrence of alternative respiratory capacity in soybean and pea

Capacity for the alternative respiratory pathway was assessed in leaf and root tissue of male-sterile and fertile soybean (Glycine max [L.] Merr.) plants and in leaf, embryonic axis, and epicotyl tissue as well as isolated mitochondria of pea (Pisum sativum L.) by measurement of oxygen uptake in the presence and absence of KCN and salicylhydroxamic acid. Male-sterile and fertile soybean tissues showed similar responses to the inhibitors, and both possessed a capacity for alternative respiration. We also found that tissue and isolated mitochondria from `Progress No. 9' pea possessed alternative respiratory capacity similar to that of `Alaska' pea.     

Effect of pod removal on leaf senescence in soybeans

Depodding soybean (Glycine max [L] Merr. cv Wye) plants results in an apparent inhibition of senescence as indicated by leaf chlorophyll and soluble protein retention. However, leaf photosynthesis and ribulose bisphosphate carboxylase (Rubisco) levels begin to decline earlier in depodded than in control, podded plants. The initial decline in photosynthesis is correlated with a decrease in leaf transpiration, while the latter decline is associated with the loss of Rubisco. Total soluble protein remains high in depodded plants because several polypeptides, three in particular, increase in amounts sufficient to offset the loss of Rubisco. Thus, depodding appears to change the function of the leaf rather than simply delaying or preventing the decline in leaf function. Changes in specific leaf weight and starch content following depodding suggest that the leaf may be changing to a storage organ.     

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels

Values of δ¹³C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The δ¹³C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or δ¹³C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, δ¹³C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on δ¹³C should occur through its effect on the extent of leakage of CO₂ from the bundle sheath cells. In this case, δ¹³C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in δ¹³C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.     

Inhibition of the formation of photosynthetic enzymes by inhibitors of photosynthesis

It has been shown previously that an increase in ribulose diphosphate carboxylase activity occurs upon brief illumination of leaves of dark-grown Zea mays plants; an increase in ribose 5-phosphate isomerase occurs after prolonged illumination. Both of these responses to illumination are inhibited by chloramphenicol.

The administration of p-chlorophenyldimethylurea, an inhibitor of photosynthesis, to etiolated maize does not affect the normal early rise in ribulose diphosphate carboxylase activity when the leaves are illuminated but does block the increase in ribose 5-phosphate isomerase. This pattern of response suggests that photosynthetic activity is required for the increase in isomerase—perhaps products of photosynthesis induce isomerase synthesis—but that the level of ribulose diphosphate carboxylase is controlled by other processes. Chlorophyll formation (as has been shown by others) is slightly suppressed by the inhibitor; levels of total soluble leaf protein appear to be unaffected.

Salicylaldoxime, which is a more general inhibitor of metabolism than p-chlorophenyldimethylurea, arrests the normally observed increases of ribulose diphosphate carboxylase, ribose 5-phosphate isomerase, and chlorophyll during illumination of dark-grown maize. The level of soluble leaf protein is also lower in leaves treated with this compound.

     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

An investigation into the interaction between nitrogen nutrition,photosynthesis and photorespiration

Photosynthetic CO₂ assimilation, photorespiration and levels of glycollate oxidase and ribulose bisphosphate (RuBP) carboxylase were measured in barley, wheat and maize plants grown on media containing nitrate or ammonium or in plants transferred from nitrate to ammonium. The CO₂ compensation point and photorespiratory CO₂ release were not altered by the nitrogen growth regime nor by transfer from nitrate to ammonium. In barley and wheat plants grown on ammonium the levels of glycollate oxidase and RuBP carboxylase per unit leaf area were higher than in nitrate grown material. These differences were not evident when the results were expressed on a protein or chlorophyll basis. The ratio of glycollate oxidase activity to RuBP carboxylase activity was not altered by the nitrogen regime.     

Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves

Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Dinitrogen fixation in male-sterile soybeans

Partial male-sterile (ms₄/ms₄) soybeans (Glycine max L. Merr.) and their fertile isoline (Ms₄/Ms₄) were grown in adjoining field plots. From 62 until 92 days after emergence, the nitrogenase activity, assayed by acetylene reduction, of the average male-sterile plant was approximately twice that of the average fertile plant. At approximately 100 days after emergence, the assayable nitrogenase activity of the fertile plants fell to zero, whereas the nitrogenase of the partial male-sterile plants continued to be active for two additional weeks. Thus, this male-sterile plant seems to fix dinitrogen both at a higher rate and over a longer duration than does its fertile isoline.     

中国春小麦株高、育性近等基因系的建立及应用

以矮败小麦和中国春小麦为材料,经过杂交和连续回交,得到了中国春小麦遗传背景的分别表现矮秆不育、矮秆可育、高秆不育、高秆可育的近等基因系。根据近等基因系各成员系的株高表现,计算出矮秆基因Rht10的降秆强度是69.8%。借助于赤霉酸处理,在幼芽期就可分出矮败中国春小麦后代的不育株与可育株。 Abstract：　Use Dwarfing Male-sterile Wheat and cv. Chinese Spring as parents, after cross and continuously back cross, the isogenic lines with Chinese Spring background were developed. These lines include dwarfing male-sterile line, dwarfing fertile line, tall male-sterile line and tall fertile line. The dwarfing intensity of gene Rht10 was calculated to be 69.8% according the differences between the isogenic lines. Treated with GA3solution, the male-sterile and fertile plants in Chinese Spring Dwarfing Male-sterile Wheat can be identified clearly when they are seedlings.     

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.