Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source

Phosphoenolpyruvate (PEP) carboxylation was measured as dark ¹⁴CO₂ fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO₂ fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO₂-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO₂ fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM crassulacean acid metabolism - PEP Phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PP protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.     

Regulatory seryl-phosphorylation of C4 phosphoenolpyruvate carboxylase by a soluble protein kinase from maize leaves

A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (0-60% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEPCase from dark-adapted green maize leaves in an ATP-, Mg2+-, time-, and temperature-dependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PK-mediated incorporation of 32P from [gamma-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per 100-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J. A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261, 409-417), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C4 PEPCase observed in vivo during dark/light transitions of the parent leaf tissue.     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

Diurnal changes in phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase properties in the natural environment: interplay of light and temperature in a C4 thermophile

Activities of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured in leaf extracts of field grown Amaranthus paniculatus L. (C₄) during a natural diurnal irradiance and temperature pattern. Enzyme assays were run at both fixed (30°C) and the corresponding leaf temperature at the time of harvest. Light activation of PEP carboxylase (PEPCase) at fixed assay temperatures was expressed as a decrease in S_0–5 (PEP) after a threshold (> 330 μmol m^–2 s^–1) photon fluence rate was surpassed at noon. Earlier in the morning, increase in apparent enzyme affinity for PEP was observed when the assay was run at leaf temperature, indicating a physiologically meaningfull effect of temperature on S^0.5 (PEP). The 3.3-fold increase in PEPCase activity at low PEP and fixed assay temperature between the minimal and maximal irradiance and temperature hours of the day, became 12.8-, 11.5- and 7.4-fold when assays were run at the corresponding leaf temperature during three diurnal cycles with respective temperature differences (max minus min) of 9.0, 8.3 and 7.4°C. The extent of malate inhibition was the same for both day and night forms of PEPCase assayed at 35°C, but increased considerably with night enzyme at 25°C. The results indicate that light increases the apparent affinity of PEPCase for PEP and that at lower temperatures malate becomes more inhibitory. Pyruvate orthophosphate dikinase activity started to increase immediately after sunrise and the 10-fold increase at fixed temperature became 14.8-, 14.2- and 13.1-fold when assays were run at the above leaf temperatures. This indicates that the light effect predominates with pyruvate, orthophosphate dikinase, while with phosphoenolpyravate carboxylase, light and temperature co-operate to increase the day enzyme activities.     

Phosphoenolpyruvate carboxylase in mistletoe leaves: Regulation of gene expression, protein content, and covalent modification

Seasonal changes in the activity of phospho enol pyruvate carboxylase (PEPCase, EC 4.1.1.31), a key enzyme in the interaction of carbohydrate and nitrogen metabolism, were studied in leaves of the C₃ semiparasitic mistletoe, Viscum album, growing on different host trees. Maximum extractable PEPCase activities were higher in leaves of mistletoes growing on Betula pendula and Alnus glutinosa hosts compared with those on the conifers, Abies alba and Larix decidua . Independent of host, maximum extractable PEPCase activities were high in spring and autumn while low in summer. Samples with higher PEPCase activities showed higher amounts of PEPCase protein and higher PEPCase mRNA levels. A curvilinear correlation between leaf total nitrogen content and the maximum extractable PEPCase activity as well as PEPCase mRNA level suggested that nitrogen might affect the activity of PEPCase of mistletoe by up-regulating gene expression. In addition to extractable activity, seasonal changes of the PEPCase activation state, the ratio of activities resulting from limited:non-limited assays, were found, which was correlated to the variation of malate content in leaves of mistletoe. ATP-dependent activation of PEPCase was characterized by an increase in I_0.5( l -malate), indicating that PEPCase of leaves of mistletoes is probably regulated via phosphorylation.     

Induction of poplar leaf nitrate reductase: a test of extrachloroplastic control of isoprene emission rate

Several recent studies have suggested that control of isoprene emission rate is in part exerted by supply of extrachloroplastic phosphoenolpyruvate to the chloroplast. To test this hypothesis, we altered PEP supply by differential induction of cytosolic nitrate reductase (NR) and PEP carboxylase (PEPC) in plants of Populus deltoides grown with NO3- or NH4+ as the sole nitrogen source. Growth with 8 mM NH4+ produced a high leaf nitrogen concentration, compared with 8 mM NO3-, as well as slightly elevated rates of photosynthesis and significantly enhanced rates of isoprene emission and content of dimethylallyl diphosphate (DMAPP, a precursor to isoprene biosynthesis), chlorophyll (a+b) and carotenoids. Growth with 8 mM NO3- resulted in parallel reductions in both leaf isoprene emission rate and DMAPP. The differential effects of growth with NH4+ or NO3- were not observed when plants were grown with 4 mM nitrogen. The effects of reduced DMAPP availability were specific to isoprene emission and were not propagated to higher isoprenoids, as the correlations between nitrogen content and either leaf chlorophyll (a+b) or total carotenoids were unaffected by nitrogen source. Biochemical analysis revealed significantly higher levels of NR and PEPC activity in leaves of 8 mM NO3- -grown plants, consistent with their fundamental roles in nitrate assimilation. Taken together, these results support the hypothesis that foliar assimilation of NO3- reduces isoprene emission rate by competing for carbon skeletons (mediated by PEPC) within the cytosol and possibly reductant within the chloroplast. Cytosolic competition for PEP is a major regulator of chloroplast DMAPP supply, and we offer a new "safety valve" hypothesis to explain why plants emit isoprene.     

Light/dark regulation of maize leaf phosphoenolpyruvate carboxylase by in vivo phosphorylation

Phosphoenolpyruvate carboxylase (PEPCase) from light- and dark-adapted maize leaves was rapidly purified in the presence of L-malate and glycerol to apparent electrophoretic homogeneity by ammonium sulfate fractionation, hydroxylapatite chromatography, and fast-protein liquid chromatography on Mono Q. The resulting preparations were totally devoid of pyruvate, orthophosphate dikinase protein based on immunoblot analysis. Throughout the purification, both forms of PEPCase retained their different enzymatic properties. The specific activity of the light enzyme was consistently about twice that of the dark form when assayed at suboptimal (but physiological) pH (pH 7.0-7.3), and the former was also less sensitive to feedback inhibition by L-malate than that from darkened leaves under various conditions. Covalently bound phosphate and high-performance liquid chromatography-based phosphoamino acid analyses showed that both forms of purified PEPCase were phosphorylated exclusively on serine residues, but the degree of phosphorylation was about 50% greater in the light enzyme. Notably, incubation of purified PEPCase in vitro with exogenous alkaline phosphatase led to an increase in malate sensitivity and a decrease in specific activity of the light form enzyme to levels observed with the dark form, which was essentially not affected by phosphatase treatment. These results with the purified enzyme from light- and dark-adapted maize leaves indicate that the light-induced changes in activity and malate sensitivity of C4 PEPCase are related, at least in part, to the degree of covalent seryl phosphorylation of the protein in vivo.     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Salinity-induced tissue-specific diurnal changes in nitrogen assimilatory enzymes in tomato seedlings grown under high or low nitrate medium.

We studied the salt stress (100 mM NaCl) effects on the diurnal changes in N metabolism enzymes in tomato seedlings (Lycopersicon esculentum Mill. cv. Chibli F1) that were grown under high nitrogen (HN, 5 mM NO(3)(-)) or low nitrogen (LN, 0.1 mM NO(3)(-)). NaCl stress led to a decrease in plant DW production and leaf surface to higher extent in HN than in LN plants. Total leaf chlorophyll (Chl) content was decreased by salinity in HN plants, but unchanged in LN plants. Soluble protein content was decreased by salt in the leaves from HN and LN plants, but increased in the stems-petioles from LN plants. Nitrate reductase (NR, EC 1.6.1.6) showed an activity peak during first part of the light period, but no diurnal changes were observed for the nitrite reductase (NiR, EC 1.7.7.1) activity. Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) activities increased in HN plant leaves during the second part of the light period, probably when enough ammonium is produced by nitrate reduction. NR and NiR activities in the leaves were more decreased by NaCl in LN than in HN plants, whereas the opposite response was obtained for the GS activity. Fd-GOGAT activity was inhibited by NaCl in HN plant leaves, while salinity did not shift the peak of the NR and Fd-GOGAT activities during a diurnal cycle. The induction by NaCl stress occurred for the NR and GS activities in the roots of both HN and LN plants. Glutamate dehydrogenase (GDH, EC 1.4.1.2) activity shifted from the deaminating activity to the aminating activity in all tissues of HN plants. In LN plants, both aminating and deaminating activities were increased by salinity in the leaves and roots. The differences in the sensitivity to NaCl between HN and LN plants are discussed in relation to the N metabolism status brought on by salt stress.     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Marked modulation by phosphate of phosphoenolpyruvate carboxylase in leaves of Amaranthus hypochondriacus, a NAD-ME type C4 plant: decrease in malate sensitivity but no change in the phosphorylation status

The effect of Pi on the properties of phosphoenolpyruvate carboxylase (PEPC) from Amaranthus hypochondriacus, a NAD-ME type C4 plant, was studied in leaf extracts as well as with purified protein. Efforts were also made to modulate the Pi status of the leaf by feeding leaves with either Pi or mannose. Inclusion of 30 mM Pi during the assay enhanced the enzyme activity in leaf extracts or of purified protein by >2-fold. The effect of Pi on the enzyme purified from dark-adapted leaves was more pronounced than that from light-adapted ones. The Ki for malate increased >2.3-fold and >1.9-fold by Pi in the enzyme purified from dark-adapted leaves and light-adapted leaves, respectively. Pi also induced an almost 50-60% increase in Km for PEP or Ka for glucose-6-phosphate. Feeding the leaves with Pi also increased the activity of PEPC in leaf extracts, while decreasing the malate sensitivity of the enzyme. On the other hand, Pi sequestering by mannose marginally decreased the activity, while markedly suppressing the light activation, of PEPC. There was no change in phosphorylation of PEPC in leaves of A. hypochondriacus due to the feeding of 30 mM Pi. However, feeding with mannose decreased the light-enhanced phosphorylation of PEPC. The marked decrease in malate sensitivity of PEPC with no change in phosphorylation state indicates that the changes induced by Pi are independent of the phosphorylation of PEPC. It is suggested here that Pi is an important factor in regulating PEPC in vivo and could also be used as a tool to analyse the properties of PEPC.     

UV-B辐射对香蕉光合作用和不同氮源利用的影响

生长在NO3^--N、NH4^--N和NH4NO3－N的香蕉叶片有相近似的最大光合速率,UV－B辐射引起生长在不同氮源的香蕉叶片光合速率、表现量子产率和光肥利用效率的降低。UV－B辐射使生长在不同氮源的植株叶面积干重和叶氮含是降低。生长在NH4^--N的植株Vcmax和Jmax均较生长在其它氮源的高。UV－B辐射引起生长在NH4^-N的植株Vcmax和Jmax降低较相同处理的NO3^-－N和NH4NO3－N植株明显,表明生长在NH4^ －N的香蕉对UV－B辐射更加敏感。UV－B辐射改变植株的叶片的碳氢比和碳氮比。经过UV－B辐射处理的NH4^ －N生长植株的碳氮生长在NO3^--N和NH4NO3－N的低。UV－B辐射可能改变植株对不同氮源的吸收利用,从而引起碳氮代谢和酸碱调节的变化。UV－B辐射降低叶氮在Rubisco和生物力能学组分的分配系数,可能使这些组分合成减少,使叶片光调节的变化。UV－B辐射降低叶氮在Rubisco和生物力能学组分的分配系数,可能使这些组分合成减少,使叶片光合速率下降。结果表明,生长在不同氮源的香蕉植树对UV－B辐射有不同响应,NH4^ -N有利于主要光合参数增高,但其对UV－B辐射亦最为敏感。氮供应受限制或植株生长在中性盐如NH4NO3－N则对UV－B辐射不甚敏感。     

Phosphoenolpyruvate Carboxylase Kinase in Tobacco Leaves Is Activated by Light in a Similar but Not Identical Way as in Maize

We have previously reported the partial purification of a Ca2+- independent phosphoenolpyruvate carboxylase (PEPC) protein-serine/threonine kinase (PEPC-PK) from illuminated leaves of N-sufficient tobacco (Nicotiana tabacum L.) plants (Y.-H. Wang, R. Chollet [1993] FEBS Lett 328: 215-218). We now report that this C3 PEPC-kinase is reversibly light activated in vivo in a time-dependent manner. As the kinase becomes light activated, the activity and L-malate sensitivity of its target protein increases and decreases, respectively. The light activation of tobacco PEPC-PK is prevented by pretreatment of detached leaves with various photosynthesis and cytosolic protein-synthesis inhibitors. Similarly, specific inhibitors of glutamine synthetase block the light activation of tobacco leaf PEPC-kinase under both photorespiratory and nonphotorespiratory conditions. This striking effect is partially and specifically reversed by exogenous glutamine, whereas it has no apparent effect on the light activation of the maize (Zea mays L.) leaf kinase. Using an in situ "activity-gel" phosphorylation assay, we have identified two major Ca2+-independent PEPC-kinase catalytic polypeptides in illuminated tobacco leaves that have the same molecular masses (approximately 30 and 37 kD) as found in illuminated maize leaves. Collectively, these results indicate that the phosphorylation of PEPC in N-sufficient leaves of tobacco (C3) and maize (C4) is regulated through similar but not identical light-signal transduction pathways.     

In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ-³²P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation (~25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.     

Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 mol quanta · m^–2 · s^–1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO₂ exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CAM Crassulacean acid metabolism - Chl chlorophyll - 2,4D 2,4-dichlorophenoxyacetic acid - NADP-ME NADP-malic enzyme - PEPCase phosphoenolpyruvate carboxylase Methyl jasmonate was generously provided by Dr. Vincent Franceschi (Botany Department, Washington State University). The anti-maize leaf PEPCase was kindly supplied by Dr. Tatsuo Sugiyama (Department of Agricultural Chemistry, Nagoya University, Japan) and the anti-Flaveria trinervia leaf PEPCase was kindly supplied by Dr. Samuel Sun (Department of Plant Molecular Physiology, University of Hawaii, Honulu). This work was funded in part by U.S. Department of Agriculture Competitive Grant 90-37280-5706 and an equipment grant (DMB 8515521) from the National Science Foundation. Ziyu Dai was supported in part by Guangxi Agricultural College and Ministry of Agriculture of the People's Republic of China     

Effect of growth conditions on carboxylating enzymes of Zea mays plants

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and ribulose-1,5-bisphospate (RuBP) carboxylase (EC 4.1.1.39) activities in leaves of different maize hybrids grown under field conditions (high light intensity) and in a growth chamber (low light intensity) were determined. Light intensity and leaf age affected PEP carboxylase activity, whereas RuBP carboxylase was affected by leaf age only at low light intensity. PEP carboxylase/RuBP carboxylase activity ratio decreased according to light intensity and leaf age. Results demonstrate that Zea mays grown under field conditions is a typical C₄ species in all leaves independently from their position on the stem, whereas it may be a C₃ plant when it is grown in a growth chamber at low light intensityAbbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions.

Pea plants (Pisum sativum L.) grown initially in nutrient solutions with adequate nitrogen supply (4 mM NO3-) were transferred to solutions containing salt (50 or 100 mM NaCl), ammonium (4 mM) or a low nitrogen supply (0.4 mM NO3-). No changes of abscisic acid (ABA) content were found in roots of stressed pea plants 9 d after the beginning of the treatments; however, accumulation of ABA in the leaves was observed. Old leaves accumulated ABA to a higher extent than young leaves. Accumulation of ABA in leaves of ammonium-fed plants and plants grown under low nitrogen supply occurred in the absence of both increased ABA xylem loading rate and enhanced aldehyde oxidase (AO, EC 1.2.3.1) activity in roots. Enhanced leaf AO activity was observed in all treatments, with the highest increase in old leaves. Among the three AO isoforms (AO-1, AO-2 and AO-3) detected in extracts of pea leaves, the lowest one AO-3 (highest mobility in the gel) correlated with ABA production and showed the highest increment in response to the treatments. The increase of AO activity detected in leaves after 2 weeks of stress application was less prominent than after 9 d, suggesting a transient enhancement of ABA production following the onset of stress. An increase of ABA xylem loading rate as well as AO root activity 4 d and 9 d after application of the treatments was observed only in salt-treated plants followed by a decrease after 14 d in 100 mM NaCl. Decreased cytokinin (trans-zeatin riboside) delivery rate into the xylem sap was observed in all treatments. The role of abscisic acid and cytokinins as positive and negative growth signals, as well as the involvement of root-generated ABA on ABA accumulation in leaves is discussed.     

Maximal biomass production can occur in corn (Zea mays) in the absence of NO3 accumulation in either leaves or roots

In order to investigate effects of limited NO₃ availability in corn ( Zea mays L. cv. Brulouis) 17-day-old plants were grown for a further 25 days on sand in a growth chamber. The plants received frequent irrigation with a complete nutrient solution containing 0.2, 0.6, 1.5 or 3.0 mM NO₃. With 0.2 mM NO; nitrate levels in both roots and leaves diminished rapidly and were almost zero after 10 days treatment. Concurrently, as signs of nitrogen deficiency appeared, shoot growth was restricted, whereas root growth was enhanced. In addition, the concentration of reduced nitrogen and malate in the leaves declined, and in vitro nitrate reductase activity (NRA. EC 1.6.6.1), soluble protein and chlorophyll levels of leaf tissue were depressed and starch concentration was enhanced. With 0.6 mM NO₃ in the nutrient solution, the decrease in NO₃ levels in the tissues and the increase in root development were similar to those observed with 0.2 mM NO₃. However, shoot growth, reduced nitrogen concentration in leaves, and the above-mentioned biochemical characteristics were almost identical to those obtained at 1.5 and 3.0 mM NO₃. This indicates that when supplied with 0.6 mM NO₃, corn plants were able to absorb sufficient NO3 to support maximal biomass production without appreciable NO₃ accumulation in roots or shoot. It is, thus, suggested that the plants responded to low NO₃, availability in medium by enhancing root growth and by maximizing NO₃ reduction relative to NO₃ accumulation.