Effects of CO(2) Concentration on Rubisco Activity, Amount, and Photosynthesis in Soybean Leaves

Growth at an elevated CO₂ concentration resulted in an enhanced capacity for soybean (Glycine max L. Merr. cv Bragg) leaflet photosynthesis. Plants were grown from seed in outdoor controlled-environment chambers under natural solar irradiance. Photosynthetic rates, measured during the seed filling stage, were up to 150% greater with leaflets grown at 660 compared to 330 microliters of CO₂ per liter when measured across a range of intercellular CO₂ concentrations and irradiance. Soybean plants grown at elevated CO₂ concentrations had heavier pod weights per plant, 44% heavier with 660 compared to 330 microliters of CO₂ per liter grown plants, and also greater specific leaf weights. Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activity showed no response (mean activity of 96 micromoles of CO₂ per square meter per second expressed on a leaflet area basis) to short-term (~1 hour) exposures to a range of CO₂ concentrations (110-880 microliters per liter), nor was a response of activity (mean activity of 1.01 micromoles of CO₂ per minute per milligram of protein) to growth CO₂ concentration (160-990 microliters per liter) observed. The amount of rubisco protein was constant, as growth CO₂ concentration was varied, and averaged 55% of the total leaflet soluble protein. Although CO₂ is required for activation of rubisco, results indicated that within the range of CO₂ concentrations used (110-990 microliters per liter), rubisco activity in soybean leaflets, in the light, was not regulated by CO₂.     

Leaf Development in Lolium temulentum: Photosynthesis and Photosynthetic Proteins in Leaves Senescing under Different Irradiances

Changes in carbon fixation rate and the levels of photosyntheticproteins were measured in fourth leaves of Lolium temulentumgrown until full expansion at 360 µmol quanta m^–2s^–1 and subsequently at the same irradiance or shadedto 90 µmol m^–2 s^–1. Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco), light-harvesting chlorophylla/b protein of photosystem II (LHCII), 65 kDa protein of photosystemI (PSI), cytochrome f (Cytf) and coupling factor 1 (CF₁) declinedsteadily in amount throughout senescence in unshaded leaves.In shaded leaves, however, the decrease in LHCII and the 65kDa protein was delayed until later in senescence whereas theamount of Cyt f protein decreased rapidly following transferto shade and was lower than that of unshaded leaves at the earlyand middle stages of senescence. Decreases in the Rubisco andCF₁ of shaded leaves occurred at slightly reduced rates comparedwith unshaded leaves. These results indicate that chloroplastproteins in fully-expanded leaves are controlled individually,in a direction appropriate to acclimate photosynthesis to agiven irradiance during senescence. (Received August 20, 1992; Accepted January 5, 1993)     

Regulation of Nitrate Reductase Activity in Leaves of Soybean Bragg and Its Nodulated Mutants

The extracts from leaves of nodulated soybean (Glycine max (L.) Merr. ) cv. Bragg and its nodulated mutants i. e. non-nodulated Nod 49, supernodulated nts 382 and nts 246 contained inhibitors of activities iNR, c1NR and c2NR in vitro. Both white light illumination of 300 μE · m-2 · s-1 and inoculation with strain USDAll0 were essential conditions for ac- cumulating these inhibitors in leaves. Comparing inhibiting activities of the extracts from different varieties indicated that Nod 49 extract showed stronger inhibition than Bragg extract did, but nts 382 extract had only weakest inhibitory effect. The inoculated Bragg root extract possessed the same inhibitory activity as its leaf extract. The inoculated nts 382 root extract, like its leaf extract, showed only a little inhibitory activity. However inoculated Nod 49 root extract lead to an inhibition of leaf c2NR activity, which was different from its leaf extract that inhibited three kinds of NR activities. The above results suggested that both leaf and root extracts contained common inhibitory factor which was accumulated after inoculation.     

Developmental and Biochemical Regulation of 'Constitutive' Nitrate Reductase Activity in Leaves of Nodulating Soybean

The developmental profile of ‘constitutive’ nitratereductase activity (cNRA) in leaves of soybean (Glycine max(L.) cv. Bragg) plants at different ages is described. The youngestleaves had most cNRA and the activity dropped off as a newerleaf developed above it. Each leaf had its distinct active periodof in vivo cNRA. This pattern was different in urea-grown andsymbiotically-grown plants (inoculated with Bradyrhizobium japonicumstrain USDA 110), where the latter had no detectable in vivocNRA in older leaves. Urea-grown plants maintained considerablein vivo NRA in such older leaves. When symbiotically-grown plantshad their nodules removed, in vivo cNRA reappeared in olderleaves within 1 d of removal, nearly reaching levels of youngleaves at 3 d after nodule excision. Allantoic acid (ALL), oneof the known transport ureides of soybeans, was implicated asa possible signal molecule from nodules to leaves. Allantoicacid (100 µM) inhibited in vitro c₁ NRA significantly,with 400 µM ALL resulting in complete inhibition. In contrast,allantoin (ALN) had no inhibitive effect on NRA. Inhibitionof c₁NRA by ALL was by a competitive process, judging from Lineweaver-Burkeplots against nitrate. Kinetics showed a constant Vmax of around105 nmol NO₂ mg^–1 protein h^–1 and a K_m for nitrateof 15 mM, which increased to 60 mM in the presence of 200 µMallantoic acid. Non-specific (ionic and pH-related) influenceswere eliminated. Allantoic acid also had a slight stimulatingeffect of in vitro NRA (up about 25% at 400 µM). Thesefindings suggest that c1NRA may be involved in ureide metabolism,rather than in vivo nitrate metabolism. Key words: Root-shoot interaction, nitrogen metabolism, nodulation, symbiosis     

Ribonuclease and Chlorophyllase Activities in Senescing Leaves

The activities of two enzymes, ribonuclease and chlorophyllase were investigated during the senescence of leaves. Ribonuclease activities were measured in primary leaves of Phaseolus vulgaris, and related to the levels of nucleic acid, protein and chlorophyll. Similarly, changes in chlorophyllase activity during senescence of leaves of Raphanus sativus were measured and related to chlorophyll. During senescence the levels of each enzyme as well as its respective substrate declined. Retardation of senescence, by excision of young tissue from intact plants or by treatment of detached leaves with cytokinins resulted in a maintainace of both the substrate and enzyme levels. It was concluded that high levels of ribonuclease and chlorophyllase activity are not linked directly with the degradation of RNA and chlorophyll during leaf senescence.     

Studies on Protein Synthesis by Senescing and Kinetin-treated Barley Leaves

Using sterile conditions, changes in total protein synthesis were followed. over an 8 day incubation period, in detached first seedling leaves of barley from 8 day old plants during senescence and after kinetin treatment. In senescing leaves, total ¹⁴C-alanine incorporation was enhanced by nearly 20% within 6 h of leaf detachment and by about 30 % after 24 h. Kinetin treatment stimulated protein synthesis even more, for total incorporation was promoted ca. 50 % after 6 h and by ca. 60 % after 24 h incubation. The leaf supernatant (30,000 ×g for 30 min) proteins were separated on DEAE-Sephadex (A-50) columns into approximately 14 fractions and changes in ¹⁴C labelling of these fractions were studied following leaf detachment and on incubation on water or kinetin for 6 days. In senescing leaves, ¹⁴C-incorporation into supernatant proteins was sustained, even as protein levels declined rapidly The varied stabilities of the different leaf proteins was suggested by the characteristically changing specific activities of the different protein fractions. Although kinetin greatly promoted incorporation into all protein fractions, no evidence was surmised of specific effects on individual leaf proteins. Studies of changes in total protein synthesis in attached senescing first seedling leaves taken from plants aged 7 to 27 days revealed a relatively small increase in ¹⁴C-incorporation. However, incorporation could be greatly increased in leaves up to 15 days old by detaching and preincubating such leaves for up to 2 days on water, prior to measurement. The promotion of ¹⁴C-incorporation into protcins follwing leaf excision could result from early changes in permeability and precursor pool size.     

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.     

Endogenous Rhythms in Photosynthesis, Sucrose Phosphate Synthase Activity, and Stomatal Resistance in Leaves of Soybean (Glycine max [L.] Merr.)

Experiments were conducted with soybean (Glycine max [L.] Merr. cv `Ransom') plants to determine if diurnal rhythms in net carbon dioxide exchange rate (CER), stomatal resistance, and sucrose-phosphate synthase (SPS) activity persisted in constant environmental conditions (constant light, LL; constant dark DD) and to assess the importance of these rhythms to the production of nonstructural carbohydrates (starch, sucrose, and hexose). Rhythms in CER, stomatal resistance, and SPS activity were observed in constant environmental conditions but the rhythms differed in period length, amplitude, and phase. The results indicated that these photosynthetic parameters are not controlled in a coordinated manner. The activity of UDPG pyrophosphorylase, another enzyme involved in sucrose formation, did not fluctuate rhythmically in constant conditions but increased with time in plants in LL. In LL, the rhythm in CER was correlated positively with fluctuations in total chlorophyll (r = 0.810) and chlorophyll a (r = 0.791) concentrations which suggested that changes in pigment concentration were associated with, but not necessarily the underlying mechanism of, the rhythm in photosynthetic rate. Assimilate export rate, net starch accumulation rate, and leaf sucrose concentration also fluctuated in constant light. No single photosynthetic parameter was closely correlated with fluctuations in assimilate export during LL; thus, assimilate export may have been controlled by interactions among the endogenous rhythms in CER, SPS activity, or other metabolic factors which were not measured in the present study.     

Photosynthesis by Flag Leaves of Wheat in Relation to Protein, Ribulose Bisphosphate Carboxylase Activity and Nitrogen Supply

The effects of nitrate supply on the composition (cell numbers,protein and chlorophyll contents) of flag leaves of winter wheatgrown with two amounts of N fertilizer and of spring wheat grownin the glasshouse under controlled nitrate supply are describedand related to photosynthesis. Nitrogen deficiency decreasedthe size of leaves, mainly by reducing cell number and, to asmaller extent, by decreasing cell volume. Protein content perunit leaf area, per cell and per unit cell volume was largerwith abundant N. Total soluble protein, ribulose bisphosphatecarboxylase-oxygenase (RuBPc-o) protein and chlorophyll changedin proportion irrespective of nitrogen supply and leaf age.Photosynthesis per unit area of flag leaf and carboxylationefficiency in both winter and spring wheat were proportionalto the amount of total soluble protein up to 7.0 g m^–2and to the amount of RuBPc-o protein up to 4.0 g m^–2.However, photosynthesis did not increase in proportion to theamount of total soluble or RuBPc-o protein above these amounts.In young leaves with a high protein content the measured ratesof photosynthesis were lower than expected from the amount andactivity of RuBPc-o. Carboxylation per unit of RuBPc-o protein,measured in vitro, was slightly greater in N-deficient leavesof winter wheat but not of spring wheat. RuBPc-o activity perunit of RuBPc-o protein was similar in winter and spring wheatleaves and remained approximately constant with age, but increasedin leaves showing advanced senescence. RuBPc-o protein fromN-deficient leaves migrated faster on polyacrylamide gels thanprotein from leaves with high N content. Regulation of the rateof photosynthesis in leaves and chloroplasts with a high proteincontent is discussed. The conductance of the cell to the fluxof CO₂ from intercellular spaces to RuBPc-o active sites iscalculated, from cell surface areas and CO₂ fluxes, to decreasethe CO₂ partial pressure at the active site by less than 0.8Pa at an internal CO₂ partial pressure of 34 Pa. Thus the decreasein partial pressure of CO₂ is insufficient to account for theinefficiency of RuBPc-o in vivo at high protein contents. Otherlimitations to the rate of photosynthesis are considered. Key words: Wheat, photosynthesis, nitrogen, ribulose, bisphosphate carboxylase     

Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Variation in Net Photosynthesis, Rubisco Activity and Chloroplast Ultrastructure among Somatic Hybrids of Solanum tuberosum and S. brevidens

The effect of ploidy, parental chloroplast type and parentalnuclear genome dosage on net photosynthesis, Rubisco activityand chloroplast ultrastructure was studied among somatic hybridsof diploid S. brevidens and dihaploid S. tuberosum. An increasein nuclear ploidy resulted in an increase in net photosynthesisand specific leaf weight. There were no significant differencesin net photosynthesis or Rubisco activity between the hybridshaving different parental chloroplast type. Examination of thehexaploid hybrids indicated that Rubisco activity was affectedby nuclear-organelle genome incompatibility, the most affectedcombination being tuberosum chloroplast type with brevidensnuclear genome. Examination of chloroplast ultrastructure revealedwide variation in the size of chloroplasts, starch granules,plastoglobuli and in grana stacking among the hybrids and betweenfusion parents. Key words: Somatic hybrids, Solanum, net photosynthesis, Rubisco, chloroplast ultrastructure     

Regulation of Rubisco by inhibitors in the light

2-carboxy-D-arabinitol-1-phosphate (CA1P) bound to Rubisco either in leaf extracts or after purification can be displaced by SO₄^2? ions. Thus, treatment of leaf extracts with a buffer containing 200 mol m^?3 SO₄^2? displaces any bound CA1P and enables measurement of maximum car-boxylation potential. In tobacco leaves, the activity following treatment with SO₄^?2 ions (‘maximal activity’) is greater than the total Rubisco activity. The ratio of the two activities altered in a dynamic way with fluctuations in irradiance. Even in species which do not produce significant amounts of CA1P, the maximal activity greatly exceeded the total activity. Anion exchange separation of components in acid extracts confirmed the absence of CA1P in tobacco leaves harvested above an irradiance of 300 μmol quanta m^?2 s^?1, but the presence of another inhibitor of Rubisco. These results are consistent with the regulation of Rubisco activity by inhibitors other than CA1P which, like CA1P, can be displaced by SO₄^2? ions.     

Influence of Carbohydrates on Photosynthesis in Single, Rooted Soybean Leaves Used as a Source-Sink Model

The single rooted leaf of soybean (Glycine max L. Merr.) wasused to study source-sink relationships in photosynthesis. Whenthe leaves were kept under a regime of 10 h light (410–480µmol photons m^–2, 400–700 nm)–14 h dark,they did not expand, the increase in leaf dry weight almoststopped, and photosynthetic activity remained at a high andconstant level for 8 d while the dry weight of the roots increasedat a constant rate throughout the period. Thus, under this conditionthe leaf and the root system served as the only source and sinkorgans, respectively. When leaves grown for 7 d under this conditionwere placed under continuous light to alter the source/sinkbalance in photosynthate, the root dry weight increased at aconstant rate equal to that found under the 10 h light–14h dark condition. The leaf dry weight markedly increased andby day 5 of continuous light had increased 1.6-fold, mainlyas a consequence of accumulation of starch and sucrose, whichwere not translocated for root growth. The continuous lightcaused an abrupt decrease in the photosynthetic activity (40%of initial value by day 5). However, the activity recoveredalmost completely after a 32-h transfer to darkness. Significantnegative correlations existed between photosynthetic activityand the sucrose and starch contents in the rooted leaves placedunder continuous light. When the plants were treated with variouslight conditions, there was no significant difference (p<0.01)among the regression line slopes for photosynthetic activityon the sucrose content, but there was some deviation among thosefor the photosynthetic activity on the starch content. Thisresult suggests that sucrose accumulated in the leaf has a moredirect influence on photosynthetic activity when the source/sinkbalance was altered. (Received September 9, 1985; Accepted February 21, 1986)     

Developmental Regulation of Respiratory Activity in Pea Leaves

The developmental pattern of mitochondrial respiratory activity in pea (Pisum sativum) leaves has been investigated in an attempt to determine changes in mitochondrial function as plant cells mature. NADH and succinate dehydrogenase and cytochrome c oxidase activities remained relatively constant during cell maturation (from d 0 to d 14). Alternative oxidase and glycine decarboxylase activity, however, were low in young leaf tissue (d 0-6) but increased substantially as the tissue matured (d 7-14) and gained photorespiratory activity. Western blot analysis of the alternative oxidase protein revealed that it was primarily in an oxidized state in young leaves (d 0-6) but switched dramatically to the reduced form of the protein as the pea cells matured (d 7-14). The switch to the reduced form of the protein correlated with an increase in alternative oxidase activity. Results are discussed in terms of the changing function of plant mitochondria during leaf development.     

水稻转绿型白化突变系W25转绿过程中Rubisco、Rubisco活化酶活性与光合速率的变化

水稻 (OryzasativaL .)转绿型白化突变系W2 5在转绿过程中叶绿素、可溶性蛋白质和Rubisco含量的动态变化过程表明 ,白化突变体内叶绿素、可溶性蛋白质和Rubisco含量极低 ,随着转绿过程各组分含量迅速提高 ,转绿至第 30天时超过野生种 2 177s;Rubisco初始活力与Rubisco活化酶含量呈极显著正相关。Rubisco活化酶基因表达的研究结果表明 ,突变体的Rubisco活化酶表达高于野生种 2 177s。在转绿过程中 ,Rubisco活化酶含量的提高要先于Rubisco和光合速率     

Regulation of NO(3) Assimilation by Anion Availability in Excised Soybean Leaves

The regulation of NO₃⁻ assimilation by xylem flux of NO₃⁻ was studied in illuminated excised leaves of soybean (Glycine max L. Merr. cv Kingsoy). The supply of exogenous NO₃⁻ at various concentrations via the transpiration stream indicated that the xylem flux of NO₃⁻ was generally rate-limiting for NO₃⁻ reduction. However, NO₃⁻ assimilation rate was maintained within narrow limits as compared with the variations of the xylem flux of NO₃⁻. This was due to considerable remobilization and assimilation of previously stored endogenous NO₃⁻ at low exogenous NO₃⁻ delivery, and limitation of NO₃⁻ reduction at high xylem flux of NO₃⁻, leading to a significant accumulation of exogenous NO₃⁻. The supply of ¹⁵NO₃⁻ to the leaves via the xylem confirmed the labile nature of the NO₃⁻ storage pool, since its half-time for exchange was close to 10 hours under steady state conditions. When the xylem flux of ¹⁵NO₃⁻ increased, the proportion of the available NO₃⁻ which was reduced decreased similarly from nearly 100% to less than 50% for both endogenous ¹⁴NO₃⁻ and exogenous ¹⁵NO₃⁻. This supports the hypothesis that the assimilatory system does not distinguish between endogenous and exogenous NO₃⁻ and that the limitation of NO₃⁻ reduction affected equally the utilization of NO₃⁻ from both sources. It is proposed that, in the soybean leaf, the NO₃⁻ storage pool is particularly involved in the short-term control of NO₃⁻ reduction. The dynamics of this pool results in a buffering of NO₃⁻ reduction against the variations of the exogenous NO₃⁻ delivery.