Influence of Etherel and Gibberellic Acid on Carbon Metabolism, Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Changes in growth parameters and ¹⁴CO₂ and [U-¹⁴C]-sucrose incorporation into the primary metabolic pools and essential oil were investigated in leaves and stems of M. spicata treated with etherel and gibberellic acid (GA). Compared to the control, GA and etherel treatments induced significant phenotypic changes and a decrease in chlorophyll content, CO₂ exchange rate, and stomatal conductance. Treatment with etherel led to increased total incorporation of ¹⁴CO₂ into the leaves wheras total incorporation from ¹⁴C sucrose was decreased. When ¹⁴CO₂ was fed, the incorporation into the ethanol soluble fraction, sugars, organic acids, and essential oil was significantly higher in etherel treated leaves than in the control. However, [U-¹⁴C]-sucrose feeding led to decreased label incorporation in the ethanol-soluble fraction, sugars, organic acids, and essential oils compared to the control. When ¹⁴CO₂ was fed to GA treated leaves, label incorporation in ethanol-insoluble fraction, sugars, and oils was significantly higher than in the control. In contrast, when [U-¹⁴C]-sucrose was fed the incorporation in the ethanol soluble fraction, sugars, organic acids, and oil was significantly lower than in the control. Hence the hormone treatment induces a differential utilization of precursors for oil biosynthesis and accumulation and differences in partitioning of label between leaf and stem. Etherel and GA influence the partitioning of primary photosynthetic metabolites and thus modify plant growth and essential oil accumulation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

Variation with age in the photosynthetic carbon fixation pattern by leaves of Amaranthus paniculatus and Oryza sativa: Change in the primary carboxylation but no shift from C4 or C3 pathway

Abstract The pattern of photosynthetic carbon fixation by leaves of Amaranthus paniculatus L. (a C₄ plant) and Oryza sativa L. (a C₃ plant) varied with age. Younger leaves of A. paniculatus incorporated ¹⁴CO₂ into malate and aspartate while senescent leaves fixed predominantly into phosphoglycerate (PGA) and sugar phosphates. Only developing leaves of O. sativa formed malate/aspartate whereas mature and senescent leaves produced PGA/sugar phosphates as the initial labelled products. Correspondingly the ratio of phosphoenolpyruvate/ribulose bisphosphate (RuBP) carboxylase activities was higher in younger leaves of A. paniculatus and developing leaves of O. sativa than in older leaves. However, pulse chase experiments revealed that the main donors of carbon to end products, irrespective of leaf stage, were C₄ acids and PGA in A. paniculatus and O. sativa respectively. The results suggest that although an apparent change from initial β-carboxylation to RuBP carboxylation occurs during leaf ontogeny in both the plants, the overall leaf photosynthesis remains C₄ or C₃. The high rate of ¹⁴CO₂ incorporation into PGA/sugar phosphates by senescent leaves of A. paniculatus is suggested to be partly due to the increased intercellular spaces in their mesophyll, allowing greater access of CO₂ directly to RuBP carboxylase in the bundle sheath.     

Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea L.

Summary The photosynthetic products of Portulaca oleracea differ greatly depending on leaf age and length of exposure to ¹⁴CO₂. Mature leaves of P. oleracea fix ¹⁴CO₂ primarily into organic and amino acids during a 10-s exposure period. Less than 2% of the ¹⁴CO₂ fixed appears in phosphorylated compounds. In contrast, incorporation into amino acids can account for over 60% of the total ¹⁴CO₂ fixed by young leaves in an equal time period, and incorporation into alanine alone can account for up to one half of this amount. Senescent leaves display a quantitative shift of primary products toward phosphorylated compounds with a concomitant reduction of the label residing in malate and asparate. About 8 times more phosphoglyceric acid is produced in senescent leaves than in mature leaves. The aspartate/ malate ratio is not constant and depends on the length of time the leaves are exposed to ¹⁴CO₂ and the age of the leaves under study. It appears as if the stage of leaf development is one of the most important factors determining the operation of a particular enzyme system in C₄ plants.     

Photosynthesis in c(4) plant tissue cultures: significance of kranz anatomy to c(4) Acid metabolism in c(4) plants

The pattern of photosynthetic carbon metabolism was determined in tissue cultures of Portulaca oleracea. Four-carbon acids are the most heavily labeled photosynthetic products during short term exposure to ¹⁴CO₂, containing greater than 40% of the total radioactivity incorporated. Phosphoglyceric acid and sugars account for only 10% of the label after equal exposure times. Other features of the CO₂ assimilation pattern in Portulaca callus tissue include a relatively large percentage of label located in various minor products throughout the time course studied, and a greater incorporation of ¹⁴C into sugars in tissue cultures than occurs in leaves. Ultrastructurally, the chloroplasts and cells of the callus are like those in the mesophyll cells of Portulaca leaves. The requirement for Kranz anatomy for operation of functional C₄ physiology is discussed.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

Lack of C4 photosynthetic metabolism in ears of C3 cereals

There is continuing controversy over whether a degree of C₄ photosynthetic metabolism exists in ears of C₃ cereals. In this context, CO₂ exchange and the initial products of photosynthesis were examined in flag leaf blades and various ear parts of two durum wheat (Triticum durum Desf.) and two six-rowed barley (Hordeum vulgare L.) cultivars. Three weeks after anthesis, the CO₂ compensation concentration at 210 mmol mol^?1 O₂ in durum wheat and barley ear parts was similar to or greater than that in flag leaves. The O₂ dependence of the CO₂ compensation concentration in durum wheat ear parts, as well as in the flag leaf blade, was linear, as expected for C₃ photosynthesis. In a complementary experiment, intact and attached ears and flag leaf blades of barley and durum wheat were radio-labelled with ¹⁴CO₂ during a 10s pulse, and the initial products of fixation were studied in various parts of the ears (awns, glumes, inner bracts and grains) and in the flag leaf blade. All tissues assimilated CO₂ mainly by the Calvin (C₃) cycle, with little fixation of ¹⁴CO₂ into the C₄ acids malate and aspartate (about 10% or less). These collective data support the conclusion that in the ear parts of these C₃ cereals C₄ photosynthetic metabolism is nil.     

Galactolipid Synthesis in Vicia faba Leaves: IV. Site(s) of Fatty Acid Incorporation into the Major Glycerolipids

The fatty acids of the major glycerolipids of Vicia faba leaves were analyzed immediately following ¹⁴CO₂ feeding. The leaves were fractionated into chloroplast and cytoplasmic fractions and the location of radioactivity in the fatty acids determined. The results indicate that the major site of incorporation of fatty acids is in the phospholipids. Phosphatidylcholine contained the highest level of radioactivity in the cytoplasmic fraction, whereas phosphatidylglycerol contained radioactivity in both the chloroplast and cytoplasmic fractions. The galactolipids contained very little radioactivity in comparison, this radioactivity being confined to high speed centrifugal fractions believed to contain the envelopes of the chloroplast. Our results suggest that phosphatidylcholine is a major site of incorporation of fatty acids (mainly in oleic acid) in the cytoplasm, whereas phosphatidylglycerol is also a site of incorporation involving both oleic and palmitic acids, inside and outside the chloroplast.     

Studies on Cassava, Manihot utilissima. II. Biosynthesis of Asparagine-14C from 14C-labelled Hydrogen Cyanide and its Relations with Cyanogenesis

Seeds and seedlings of Manihot utilissima were analysed for cyanogenic glycosides und free amino acids, with special reference to valine and isoleucine which serve as precursors of the aglycone moieties of linamarin and lotaustralin. Seeds contained traces of valine and isoleucine but no glycosides, whereas seedlings contained high concentrations of these amino acids and glycosides. Illumination of seedlings led to a steep increase in the concentration of glycosides followed by a decrease without excretion of detectable HCN. Seeds accumulated asparagine, while seedlings accumulated both asparagine and glutamine in the storage and transport of nitrogen. Seedlings incorporated 13.2 per cent of label from valine-¹⁴C(U) and 2.4 per cent of label from isoleucine-¹⁴C(U)into linamarin and lotaustralin, respectively. In both cases, appreciable amounts of label were also incorporated into asparagine. 49 per cent of label from H¹⁴CN was incorporated inio asparagine in which ca. 98 per cent of total radioactivity was located in the amide-carbon atom. The different patterns of labelling which occurred during the assimilation of H¹⁴CN and ¹⁴CO₂ showed that cyanide metabolism did not proceed via CO₂, and that M. utilissima contains an efficient enzyme-system which catalyses the conversion on high concentrations of HCN into asparagine, which subsequently enters different metabolic pools involved with respiration, protein and carbohydrate syntheses. Cyanogenesis in M. utilissima appears lo be directly influenced by available pools of valine and isoleucine, and the metabolism of HCN released from linamarin and lotaustralin by the action of linamarase may be directly related to respiratory and synthetic processes by way of the incorporation of HCN as a unit into asparagine.     

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L

Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO₂ fixation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH¹⁴CO₃, and the kinetics of ¹⁴C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO₂ fixation is similar in the different mesophyll tissues from which these cells were derived.     

The temperature acclimation of photosynthetic responses to CO2 in Zea mays and its relationship to the activities of photosynthetic enzymes and the CO2-concentrating mechanism of C4 photosynthesis

Abstract Associations between photosynthetic responses to CO₂ at rate-saturating light and photosynthetic enzyme activities were compared for leaves of maize grown under constant air temperatures of 19, 25 and 31°C. Key photosynthetic enzymes analysed were ribulose bisphosphatc (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, NADP-malic enzyme and pyruvate, P_i dikinasc. Rates of CO₂-saturated photosynthesis were similar in leaves developed at 19°C and 25°C but were decreased significantly by growth at 31°C. In contrast, carboxylation efficiency differed significantly between all three temperature regimes. Carboxylation efficiency was greatest in leaves developed at 19°C and decreased with increasing temperature during growth. The changes of carboxylation efficiency were highly correlated with changes in the activity of pyruvate, P_i dikinase (r= 0.95), but not with other photosynthetic enzyme activities. The activities of these latter enzymes, including that of RuBP carboxylase, were relatively insensitive to temperature during growth. The sensitivity of quantum yield to O₂ concentration was lower in leaves grown at 19°C than in leaves grown at 31°C. These observations support the novel hypothesis that variation in the capacity for CO₂ delivery to the bundle sheath by the C₄ cycle, relative to the capacity for net assimilation by the C₂ cycle, can be a principal determinant of C₄ photosynthetic responses to CO₂.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

14CO2 Fixation and Compartmentation of Carbon Metabolism in a Recombined Chloroplast- 'Cytoplasm' System

The Compartmentation of ¹⁴CO₂ fixation and concomitant metabolism of ^l4C-iabelled products in a recombined system, composed of isolated intact spinach (Spinacia oleracea) chloroplasls and a ‘cytoplasm’ fraction, has been studied. Addition of ‘cytoplasm’ to chloroplasts fixing ¹⁴CO₂ increased the label in hexoae monophosphates outside the chloroplasts at the expense of excreted dihydroxyacetone phosphate. The label in ammo acids was increased both inside and outside the chloroplasts. The results support the view that chloroplasls are not able to make 2-oxoacids for amino acid synthesis directly from fixed CO₂, but have to co-operate with the cytoplasm and other organelles. The results also show that recombined systems can be useful for studies on the compartmen tation of carbon metabolism in pholosynthesizing plant tissues.     

Relative Contribution of Ribulose 1,5-Diphosphate Carboxylase and Phosphoenolpyruvate Carboxylase to CO2 Fixation Activity in Roots of Dark-grown or Light-grown Lens culinaris Seedlings

The pathway of carbon assimilation in greening roots was compared to the pathway in leaves of Lens culinaris seedlings by means of labelling distribution analysis among the products of ¹⁴CO₂ fixation in vivo, and in vitro with ribulose 1,5-diphosphate as the substrate. In green leaves, CO₂ fixation via ribulose 1,5-diphosphate carboxylase predominated largely while, in green roots, this carboxylase activity and the phosphoenolpyruvate carboxylase contributed almost equally to the whole in vivo CO₂ fixation. A participation of the activities of both carboxylases according to the double carboxylation pathway in the synthesis of dicarboxylic acids (malate and aspartate) was demonstrated in vitro after 48 h of greening in roots but seemed to be absent in in vivo experiments.     

RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.     

Studies on Orobanche hederae Physiology: Pigments and CO2 Fixation

In order to investigate some aspects of Orobanche hederae physiology in relation to its parasitism, the pigment composition and the ¹⁴CO₂ incorporation, both in light and in dark, were studied. By means of various chromatographic techniques, it was shown that chlorophyll is probably quite absent and that the carotenoids are very largely distrihuted and consist almost exclusively of xanthophylls. Flavochrome, flavoxanthin, and a pigment resembling neoxanthin were found to be the major components; lutein-5,6-epoxide, taraxanthin, and traces of β-carotene and α-carotene-5,6-epoxide were also present. CO₂ incorporation is completely of heterotrophic type, being in no way stimulated by the light; the major radioactivity was detected in the fractions containing amino acids and organic acids.     

Studies on the biochemistry and fine structure of silica-shell formation in diatoms

Summary The distribution of radioactivity in ethanol-water-soluble compounds after short periods of photosynthetic incorporation of ¹⁴CO₂ is consistent with the operation of the photosynthetic carbon reduction (PCR) cycle in the fresh water diatom Navicula pelliculosa. Incorporation of ¹⁴CO₂ for extended time periods established the presence of the intermediates of the PCR and tricarboxylic acid (TCA) cycles, amino acids, and organic acids; free sugars were not observed. The main labelled soluble carbohydrate was a glucan. Hydrolysis of the radioactive insoluble material indicated the presence of carbohydrates containing several distinct sugars, and proteins with the usual amino-acid composition. During silicon starvation of exponentially growing cultures, rates of incorporation of both ³²P _i and ¹⁴CO₂ decreased. Incorporation into the lipid increased, with a corresponding decrease into protein and carbohydrate. Reintroduction of Si to staryed cells led to an increased rate of incorporation of both isotopes, and transient changes in the radioactivity in most metabolic intermediates investigated. After 30 min the radioactivity in all PCR cycle intermediates, except phosphoglyceric acid, had increased by about 300%. The radioactivity of citrate and -keto-glutarate increased, whereas that of other TCA-cycle intermediates decreased. An initial decrease in the levels of glutamate, aspartate and glutamine was apparently reversed by cleavage of glutamate-aspartate peptides, as radioactivity of other amino acids increased. Incorporation into the soluble glucan and into protein increased markedly although the rate of incorporation into insoluble carbohydrates remained constant.     

CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Rapid appearance of 13C in biogenic isoprene when 13CO2 is fed to intact leaves

Biogenic isoprene substantially affects atmospheric chemistry, but it is not known how or why many plants, especially trees, make isoprene. We fed ¹³CO₂ to leaves of Quercus rubra and monitored the incorporation of ¹³C into isoprene by mass spectrometry. After feeding ¹³CO₂ for 9 min we found all possible labelling patterns from completely unlabelled to fully labelled isoprene. By 18 min, 84% of the carbon atoms in isoprene were ¹³C. Labelling of the last 20% of the carbon atoms was much slower than labelling of the first 80%. The rate of labelling of isoprene was similar to that reported for phosphoglyceric acid indicating that there is a close linkage between the carbon source for isoprene synthesis and the photosynthetic carbon reduction pathway.     

Control of steady-state photosynthesis in sunflowers growing in enhanced CO2

Control coefficients were used to describe the degree to which ribulose bisphosphate carboxylase/oxygenase (Rubisco) limits the steady-state rate of CO₂ assimilation in sunflower leaves from plants grown at high (800 μmol mol⁻¹) and low (350 μmol mol⁻¹) CO₂. The magnitude of a control coefficient is approximately the percentage change in the flux that would result from a 1% rise in enzyme active site concentration. In plants grown at low CO₂, leaves of different ages varied considerably in their photosynthetic capacities. In a saturating light flux and an ambient CO₂ concentration of 350 μmol mol⁻¹, the Rubisco control coefficient was about 0.7 in all leaves, indicating that Rubisco activity largely limited the assimilation flux. The Rubisco control coefficient for leaves grown at 350 μmol mol⁻¹ CO₂ dropped to about zero when the ambient CO₂ concentration was raised to 800 μmol mol⁻¹. In relatively young, fully expanded leaves of plants grown at high CO₂, the Rubisco control coefficient was also about 0.7 at a saturating light flux and at the CO₂ concentration at which the plants were grown (800 μmol mol⁻¹). This apparently resulted from a decrease in the concentration of Rubisco active sites. In older leaves, however, the control coefficient was about 0.2. Because, on the whole, Rubisco activity still largely limits the assimilation flux in plants grown at high CO₂, the kinetics of this enzyme can still be used to model photosynthesis under these conditions. The relatively high Rubisco control coefficient under enhanced CO₂ indicates that the young sunflower leaves have the capacity to acclimate their photosynthetic biochemistry in a way consistent with an optimal use of protein resources.