Glycolate Pathway and Serine Synthesis in Relation to CO2 Concentration in Tomato Leaves

Isotopic trapping of the carbon flowing through the glycolatepathway by exogenous glycolate, glycine and L-serine was investigatedduring ¹⁴CO₂ photosynthesis at different CO₂ concentrationsin tomato leaves. L-Serine markedly trapped the carbon flowingfrom ¹⁴CO₂. The amounts of ¹⁴C incorporated into serine decreasedat a high CO₂ concentration, but increased with an increasein the CO₂ concentration in the presence of exogenous serineduring 10-min photosynthesis in ¹⁴CO₂. When ¹⁴CO₂ was fed for5 to 40 sec at 1300 ppm CO₂ to tomato leaves which had beengiven L-serine, an increase in the accumulation of ¹⁴C-serinebegan after 20 sec, and the ¹⁴C-serine molecules formed at 20and 40 sec were labeled uniformly. In the presence of exogenousserine during 10-min photosynthesis in 1300 ppm CO₂, isonicotinicacid hydrazide increased the incorporation of ¹⁴CO₂ into glycinewith a corresponding decrease in the accumulation of ¹⁴C-serine,but it did not inhibit serine accumulation completely; an evidencefor that some serine was formed by a pathway other than theglycolate pathway. The effect of the CO₂ concentration on theglycolate pathway is discussed in terms of serine synthesisin the presence of exogenous serine. (Received June 1, 1981; Accepted September 30, 1981)     

The Damping and Reinitiation of the Circadian Rhythm of CO2 Output in Bryophyllum Leaves in Relation to their Malate Content

The circadian rhythm of CO₂ output in leaves of Bryophyllumfedtschenkoi damps out after 3–4 d in continuous darknessand a CO₂-free air stream at 15°C. The rhythm is reinitiatedafter a single exposure to white light of 2, 4, 6 or 8 h duration,damps out again after a further 3–4 d and can be reinitiatedfor a second time by a further exposure to light. During the exposure to light there is a burst of CO₂ outputconsistent with the decarboxylation of malate, and the rhythmbegins afterwards with an initial high rate of CO₂ fixation.Malate gradually accumulates in the leaves in continuous darknessto attain a maximum value (35 mol m^–3) at the time whenthe circadian rhythm disappears, and decreases to a low value(19 mol m^–3) after a 4 h exposure to light which reinitiatesrhythmicity. These results support the hypothesis that damping of the rhythmof CO₂ output in continuous darkness is due to the accumulationof malate in the leaf cells, eventually reaching such a levelthat its removal from the cytoplasm into the vacuole cannottake place, with the result that PEPc activity, upon which therhythm of CO₂ output depends, remains allosterically inhibited. Key words: CAM, circadian rhythm, Bryophyllum, CO₂-fixation, malate metabolism     

Gas Exchange of Carrot Leaves in Response to Elevated CO2 Concentration

Short-term responses of four carrot (Daucus carota) cultivars: Cascade, Caro Choice (CC), Oranza, and Red Core Chantenay (RCC) to CO₂ concentrations (C _a) were studied in a controlled environment. Leaf net photosynthetic rate (P _N), intercellular CO₂ (C _i), stomatal conductance (g _s), and transpiration rate (E) were measured at C _a from 50 to 1 050 mol mol^–1. The cultivars responded similarly to C _a and did not differ in all the variables measured. The P _N increased with C _a until saturation at 650 mol mol^–1 (C _i= 350–400 mol mol^–1), thereafter P _N increased slightly. On average, increasing C _a from 350 to 650 and from 350 to 1 050 mol mol^–1 increased P _N by 43 and 52 %, respectively. The P _N vs. C _i curves were fitted to a non-rectangular hyperbola model. The cultivars did not differ in the parameters estimated from the model. Carboxylation efficiencies ranged from 68 to 91 mol m^–2 s^–1 and maximum P _N were 15.50, 13.52, 13.31, and 14.96 mol m^–2 s^–1 for Cascade, CC, Oranza, and RCC, respectively. Dark respiration rate varied from 2.80 mol m^–2 s^–1 for Oranza to 3.96 mol m^–2 s^–1 for Cascade and the CO₂ compensation concentration was between 42 and 46 mol mol^–1. The g _s and E increased to a peak at C _a= 350 mol mol^–1 and then decreased by 17 and 15 %, respectively when C _a was increased to 650 mol mol^–1. An increase from 350 to 1 050 mol mol^–1 reduced g _s and E by 53 and 47 %, respectively. Changes in g _s and P _N maintained the C _i:C _a ratio. The water use efficiency increased linearly with C _a due to increases in P _N in addition to the decline in E at high C _a. Hence CO₂ enrichment increases P _N and decreases g _s, and can improve carrot productivity and water conservation.     

Synthesis of Glycolate from Pyruvate via Isocitrate Lyase by Tobacco Leaves in Light

Tobacco (Nicotiana tabacum var Havana Seed) leaf discs were supplied tracer quantities of [2-¹⁴C]- and [3-¹⁴C]pyruvate for 60 minutes in steady state photosynthesis with 21% or 1% O₂, and the glycolate oxidase inhibitor α-hydroxy-2-pyridinemethanesulfonic acid was then added for 5 or 10 minutes to cause glycolate to accumulate. The [3-¹⁴C]pyruvate was converted directly to glycolate as shown by a 50% greater than equallabeled ¹⁴C in C-2 of glycolate, and the fraction of ¹⁴C in C-2 increased in 1% O₂ to 80% greater than equal-labeled. This suggests the pathway using pyruvate is less O₂-dependent than the oxygenase reaction producing glycolate from the Calvin cycle. The formation of glycolate from pyruvate in the leaf discs was time-dependent and with [2-¹⁴C]- and [3-¹⁴C]pyruvate supplied leaf discs the C-2 of glyoxylate derived from C-2 of isocitrate was labeled asymmetrically in a manner similar to the asymmetrical labeling of C-2 of glycolate under a number of conditions. Thus glycolate was probably formed by the reduction of glyoxylate. Isocitric lyase activity of tobacco leaves was associated with leaf mitochondria, though most of the activity was in the supernatant fraction after differential centrifugation of leaf homogenates. The total enzyme activity was at least 35 micromoles per gram fresh weight per hour. The relative contribution of the pathway to the glycolate pool is unknown, but the results support the existence of a sequence of reactions leading to glycolate synthesis during photosynthesis with pyruvate, isocitrate, and glyoxylate as intermediates.     

Relation of CO(2) Compensation Concentration to Apparent Photosynthesis in Maize

Significant differences in CO₂ compensation concentration measured in the field among varieties of the species Zea mays L. are reported for the first time. CO₂ compensation concentrations were significantly (P≤ 0.01) and negatively correlated with apparent photosynthesis at 300 μl CO₂/liter air. The Michaelis constant (as defined) for a leaf was significantly (P≤ 0.01) and positively correlated with apparent photosynthesis among varieties. While the first correlation is similar to behavior of CO₂ compensation among species of different photosynthetic efficiency, the latter correlation is the converse of the behavior of Km among species.     

Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration

High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2.     

Bicarbonate Fixation and Malate Compartmentation in Relation to Salt-induced Stoichiometric Synthesis of Organic Acid

The relationship of malate synthesis to K⁺ absorption from solutions of K₂SO₄ and KHCO₃ was compared in nonvacuolate barley (Hordeum vulgare) root tips and whole excised roots. The comparison has permitted separation of the process which evokes organic acid synthesis from that which leads to stoichiometry between net acid equivalents formed and excess K⁺ absorbed from K₂SO₄, on the one hand, and total K⁺ absorbed from KHCO₃, on the other. Both in tips and in roots K⁺ uptake from 20 mN salt solution exceeds malate synthesis in the first hour. In vacuolate roots the expected stoichiometry is achieved with time. When root tips are transferred to dilute CaSO₄, malate is rapidly metabolized, and K⁺ is lost to the solution. By contrast, in excised whole roots the malate level remains unchanged, the salt-induced organic acid presumably being retained in the vacuole. In excised roots malonate leads to a marked drop in malate levels in untreated roots as well as in roots which have experienced salt-induced net malate synthesis. In consequence, it is contended that malonate makes available normally sequestered vacuolar malate.     

Changes Induced by Low Oxygen Concentration in Photosynthetic and Respiratory CO2 Exchange in Phosphate-Deficient Bean Leaves

Effect of phosphorus deficiency on photosynthetic and respiratory CO₂ exchanges were analysed in primary leaves of 2-week-old bean (Phaseolus vulgaris L. cv. Golden Saxa) plants under non-photorespiratory (2 % O₂) and photorespiratory (21 % O₂) conditions. Low P decreased maximum net photosynthetic rate (P_Nmax) and increased the time necessary to reach it. In the leaves of P-deficient plants the relative decrease of P_Nmax at 2 % O₂ was larger than at 21 % O₂. The results suggested the influence of photorespiration in the cellular turnover of phosphates.     

Stimulation of the Alternative Pathway by Succinate and Malate

Stimulation of the cyanide-resistant oxidation of exogenous NADH in potato (Solanum tuberosum L. cv Bintje) tuber callus mitochondria was obtained with succinate, malate, and pyruvate. Half-maximal stimulation was observed at a succinate or malate concentration of 3 to 4 mM, which is considerably higher than that found for pyruvate (0.128 mM). No effect of succinate or malate addition was found when duroquinone was the electron acceptor. Duroquinol oxidation via the alternative pathway was poor and not stimulated by organic acids. Under stimulating conditions, no swelling or contraction of the mitochondria could be observed. Conversely, variation of the osmolarity did not affect the extent of stimulation. However, the assay temperature had a significant effect: no stimulation occurred at temperatures below 16 to 20[deg]C. Membrane fluidity measurements showed a phase transition at about 17[deg]C. Ubiquinone reduction levels were not significantly higher in the presence of succinate and malate, but the kinetics of the alternative oxidase were changed in a way comparable to that found for stimulation by pyruvate. At low temperatures the alternative oxidase displayed "activated" kinetics, and a role for membrane fluidity in the stimulation of the alternative pathway by carboxylic acids is suggested.     

The Growth Rate of Wheat Leaves in Relation to the Extension Zone Sugar Concentration Manipulated by Shading

The main objective of this study was to determine the relationshipbetween the relative rate of growth of emerging wheat leavesand the hexose sugar concentration of the extension zone. Shortperiods of intense shading (to 20 or 5% of full sun for up to14 d) were used to decrease hexose concentrations. Shading decreased hexose concentrations to a fraction of thatof controls and also resulted in thin and narrow leaves thatwere less in dry weight than control leaves of the same length.Shading did however increase the length of the zone of extendingtissue at the leaf base by 30%. The effect of hexose concentrations on the relative rate ofleaf growth was evaluated by determining the ratio between growthrates of shaded and control leaves. This ratio declined as hexoseconcentrations declined and the relationship was described bya rectangular hyperbola (r > 0.95, P < 0.01). Combineddata from many leaves on the main shoot and its tillers fromtwo irrigated wheat crops all conformed to the same relationship.The hexose concentrations where the ratio of growth rates washalf the maximum rate were 0.42 mg g^–1 fr. wt. for extensiongrowth and 1.74 mg g^–1 fr. wt. for dry weight growth.These values were significantly (P < 0.01) different. These results were compared with data from emerging leaves offield crops and it was concluded that hexose concentrationshad not limited leaf growth rates, the lowest values recordedbeing 2.5–3.0 mg g^–1 fr. wt. It was further suggestedas unlikely that leaf growth rates of wheat crops in the fieldwould be limited by hexose concentrations.     

Stimulation by Light of Rapid pH Regulation in the Chloroplast Stroma in Vivo as Indicated by CO2 Solubilization in Leaves

Leaves of Brassica oleracea, Helianthus annuus, and Nicotiana rustica were exposed for 20 s to high concentrations of CO2. CO2 uptake by the leaf, which was very fast, was measured as a transient increase in the concentration of oxygen. Rapid solubilization of CO2 in excess of that which is physically dissolved in aqueous phases is proposed to be caused by bicarbonate formation in the stroma of chloroplasts, which contain carbonic anhydrase. On this basis, pH values and bicarbonate accumulation in the chloroplast stroma were calculated. Buffer capacities were far higher than expected on the basis of known concentrations in the chloroplast stroma. Moreover, apparent buffer capacities increased with the time of exposure to high CO2, and they were higher when the measurements were performed in the light than in the dark. During prolonged exposure of leaves to 16% CO2, calculated bicarbonate concentrations in the chloroplast stroma exceeded 90 mM in the dark and 120 mM in the light. The observations are interpreted as indicating that under acid stress protons are rapidly exported from the chloroplasts in exchange for cations, which are imported. The data are discussed in terms of effective metabolic pH control by ion transport, first across the chloroplast envelope and, then, across the tonoplast of leaf mesophyll cells. The direct involvement of the vacuole in the regulation of the chloroplast pH in leaf cells is suggested.     

Photodestruction of Chlorophyll inZea mays L. Leaves Under Different CO2 Concentration

Photodestruction of chlorophyll (Chl) inZea mays leaves, after their irradiation with high photon fluence rate (5000 μmol m⁻² s{−1}), was determined in fragments of whole leaves (WL) and also in fraction of mesophyll cells (MC) and bundle sheath cells (BSC) after their mechanical separation. The lag phase and the phase of photooxidation of Chl in MC chloroplasts were shorter than in BSC. Duration of both phases was reduced when the leaves were placed in 0 % CO₂ concentration in the atmosphere, while the increase of CO₂ concentration up to 0.3 % totally protected Chl against photodestruction in BSC within the 9 h experiment. During that period of time Chl was destructed by about 30 % in MC.     

The Stimulation of Ethylene Synthesis in Nicotiana tabacum Leaves by the Phytotoxin Coronatine

Coronatine is a chlorosis-inducing toxin produced by the plant pathogen Pseudomonas syringae pv atropurpurea. This bacterium is the causal agent of chocolate spot disease, in which brown lesions with chlorotic margins develop on the leaves of Lolium multiflorum Lam. Among the many physiological changes to plants caused by coronatine is the stimulation of ethylene production from bean leaves. The ethyl-substituted side chain of coronatine is an analog of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). We have examined the question of whether part or all of the released ethylene comes from the breakdown of coronatine itself. The rate of ethylene release from leaves of Nicotiana tabacum was proportional to the concentration of coronatine applied to the leaf surface. The lowest effective concentration of coronatine, applied to leaves at 15 pmol cm⁻² of leaf area, resulted in the production of 44 pmol of ethylene cm⁻² over a period of 4 h. The maximum rate of ethylene production occurred 28 to 32 h after application of coronatine. The specific activity of ethylene produced by discs cut from coronatine-treated Nicotiana tabacum leaves floating on a solution containing 10 mm [U-¹⁴C]methionine was consistent with its exclusive origin from methionine. ACC accumulated in the coronatine-treated tissue. ACC synthase activity increased in Phaseolus aureus hypocotyls during a 6-h treatment with coronatine. Thus, coronatine induces the synthesis of ethylene from methionine.     

Effects of KCI Concentration on Changes in Starch and Malate Levels in Abaxial and Adaxial Epidermis in Relation to Stomatal Opening

The effects of KCI concentration in the incubation medium onstomatal opening and starch and malate metabolism in isolatedabaxial and adaxial epidermis of Commelina communis were investigated.Increasing KCI produced progressively wide apertures, reducedstarch hydrolysis and malate synthesis, but failed to eliminatethe normal disparity in abaxial and adaxial opening. The fusicoccin-stimulatedwide opening was accompanied by a dramatic enhancement of malateformation and starch hydrolysis at zero KCI, but not at 200mol m^–3 KCI, indicating a controlling influence of KCIconcentration on carbohydrate and malate metabolism. Accordingly,malate and Cl^– appear to compete for K⁺, and the one assumesan increasingly decisive role as the other becomes limiting.However, starch hydrolysis and malate production must play acentral role in stomatal opening in intact leaves because theyare unlikely to have such high Cl^– regimes as 200 molm^–3, though not necessarily in isolated epidermis incubatedin media enriched with inorganic ions. The restricted adaxial opening is attributed to limited K⁺ accumulation,starch hydrolysis and malate production in the guard cells,but the primary cause is not known. Key words: Stomata, Abaxial/Adaxial Epidermis     

Environmentally Induced Changes in the Cell Walls of Tomato Leaves in Relation to Cell and Protoplast Release

Factors involved in the isolation of protoplasts from the leaves of tomato plants grown over a wide range of environmental conditions have been studied. Increases in calcium pectate in summer grown (“hard”) plants are suggested as a barrier to cell wall degradation. A one-step method involving the addition of sodium citrate to pectinase plus cellulase gives high yield of protoplasts from hard plants. Attempts to convert isolated palisade cells to protoplasts have failed. The plant culture conditions are described such that protoplasts may be isolated throughout the year using low enzyme concentrations.     

Gas Exchange Responses to CO2 Concentration Instantaneously Elevated in Flag Leaves of Winter Wheat Cultivars Released in Different Years

Three winter wheat (Triticum aestivum L.) cultivars, representatives of those widely cultivated in Beijing over the past six decades, were grown in the same environmental conditions. Net photosynthetic rate (P _N) per unit leaf area and instantaneous water use efficiency (WUE) of flag leaves increased with elevated CO₂ concentration. With an increase in CO₂ concentration from 360 to 720 µmol mol^–1, P _N and WUE of Jingdong 8 (released in 1990s and having the highest yield) increased by 173 and 81 %, while those of Nongda 139 (released in 1970s) increased by 88 and 66 %, and Yanda 1817 (released in 1945, with lowest yield) by 76 and 65 %. Jingdong 8 had the highest P _N and WUE values under high CO₂ concentration, but Yanda 1817 showed the lowest P _N. Stomatal conductance (g _s) of Nongda 139 and Yanda 1817 declined with increasing CO₂ concentration, but g _s of Jingdong 8 firstly went down and then up as the CO₂ concentration further increased. Intercellular CO₂ concentration (C _i) of Jingdong 8 and Nongda 139 increased when CO₂ concentration elevated, while that of Yanda 139 increased at the first stage and then declined. Jingdong 8 had the lowest C _i of the three wheat cultivars, and Yanda 1817 had the highest C _i value under lower CO₂ concentrations. However, Jingdong 8 had the highest P _N and lowest C _i at the highest CO₂ concentration which indicates that its photosynthetic potential may be high.     

Stimulation of CO2 reduction to methane by methylcoenzyme M in extracts Methanobacterium.

Addition of methyl-coenzyme M (CH₃SCH₂CH₂SO₃^?) to undialized, anaerobic, cell-extracts of $\text{Methanobacterium thermoautotrophicum}$ under an atmosphere of H₂ and CO₂ (80:20 v/v) stimulates 30-fold the rate of CO₂ reduction to methane. For each mol of CH₃SCH₂CH₂SO₃^? added 12 mol of methane is produced. This stimulation phenomenon requires magnesium ion, ATP, H₂, and CH₃SCH₂CH₂SO₃^?. Neither the reduced form of the cofactor, HSCH₂CH₂SO₃^?, nor the oxidized, disulfide form will replace the methylated coenzyme.     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.