Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Enzymic Activities Associated with the Ability of Aerial and Submerged Forms of Littorella uniflora (L.) Aschers to perform CAM

ABSRACT: Groenhof, A. C, Smirnoff, N. and Bryant, J. A. 1988. Enzymicactivities associated with the ability of aerial and submergedforms of Littorella uniflora (L.) Aschers to perform CAM.—J.exp. Bot. 39: 353-361. The submerged form of Littorella uniflora shows a full CAM modeof photosynthesis as shown by diel acid fluctuations and elevatedactivities (in comparison to non-submerged leaves) of the enzymesphosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme.Non-submerged plants exhibit no diel fluctuations of acidityand no changes in activity of NADP-malic enzyme or PEPC. PEPCactivity is low and NADP-malic enzyme is not detectable. Furthercharacterization of PEPC extracted from submerged plants duringthe light and dark periods of a diel cycle shows that the enzymeextracted in the dark is more active. In addition, the enzymeshows a decrease in K_m (PEP) and an increase in V_max in thepresence of glucose-6-phosphate, whilst in the presence of malateK_m (PEP) is increased and V_max decreased; this response to malateis only observed in the light and at pH 7.2. Molecular weightdeterminations using a Sephacryl S-300 column show that theenzyme extracted from plants during the dark period has an apparentmol. wt. of 375 KDa and the enzyme extracted from plants duringthe light period has an apparent mol. wt. of 307 KDa. Key words: Littorella uniflora (shoreweed), Crassulacean acid metabolism, PEP carboxylase, malic enzyme     

Isoforms of NADP-Malic Enzyme from Mesembryanthemum crystallinum L. That are Involved in C3 Photosynthesis and Crassulacean Acid Metabolism

Exposure of the facultative halophyte Mesembryanthemum crystallinumL. to salt stress induces a shift from C₃ photosynthesis toCrassulacean acid metabolism (CAM). During induction of CAM,the activity of NADP-malic enzyme (EC 1.1.1.40 [EC] ) increased asmuch as 12-fold in leaves, while the enzymatic activity in rootsfell to half of the original level. These changes in the activityof the enzyme corresponded to changes in levels of the enzymeprotein. NADP-malic enzymes extracted from leaves in the C₃and CAM modes could be distinguished by differences in electrophoreticmobility during electrophoresis on a non-denaturing polyacrylamidegel. NADP-malic enzyme extracted from roots in the C₃-mode andin the CAM mode migrated as fast as the enzyme extracted fromleaves in the CAM mode on the same gel. Although the patternof peptide fragments from NADP-malic enzyme from CAM-mode leaveswas similar to that from C₃-mode leaves, as indicated by peptidemapping, both immunoprecipitation and an enzyme-linked immunosorbentassay revealed some antigenic differences between the enzymesextracted from leaves in the C₃ and the CAM modes. These resultssuggest the existence of at least two isoforms of NADPmalicenzyme that differ in their levels of expression during inductionof CAM. (Received April 21, 1994; Accepted September 5, 1994)     

NADP—苹果酸酶活性变化及其在CAM运行中的调节

ＮＡＤＰ－苹果酸酶是ＣＡＭ植物一种重要脱羧酶。实验结果表明,专一ＣＡＭ植物瓦松和兼性ＣＡＭ植物长药景天及露花其ＮＡＤＰ－苹果酸酶活性昼高夜低;５－８月,兼性ＣＡＭ植物长药景天和露花随着Ｃ３光合型向ＣＡＤ型转化,其中ＮＡＤＰ－苹果酸活活性逐渐升高。     

The Response of the C3-CAM Tree, Clusia rosea, to Light and Water Stress: I. GAS EXCHANGE CHARACTERISTICS

Gas exchange measurements were undertaken on 2-year-old plantsof Clusia rosea. The plants were shown to have the ability toswitch from C₃-photosynthesis to CAM and vice versa regardlessof leaf age and, under some conditions, CO₂ was taken up continuously,throughout the day and night. The light response was saturatedby 120 µmol m^–2 s^–1 typical of a shade plant. Gas exchange patterns in response to light, water and VPD wereexamined. All combinations of daytime and night-time CO₂ uptakewere observed, with rates of CO₂ uptake ranging from 2 to 11µmol m^–2 s^–1 depending upon water status andlight. Categorization of this plant asC₃, CAM or an intermediateis impossible. Differing VPD affected the magnitude of changesfrom CAM to C₃-photosynthesis (0 to 0.5 and 0 to 6.0 µmolm^–2 s^–1 CO₂, respectively) when plants were watered.Under well-watered conditions, but not under water stress, highPPFD elicited changes from CAM to C₃ gas exchange. This is unusualnot only for a shade plant but also for a plant with CAM. Itis of ecological importance for C. rosea, which may spend theearly years of its life as an epiphyte or in the forest understorey,to be able to maximize photosynthesis with minimal water loss. Key words: Clusia rosea, CAM, C₃, stress     

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.     

Pinitol, a Compatible Solute in Mesembryanthemum crystallinum L.?

The irrigation of Mesembryanthemum crystallinum L. plants with400 mol m^–3 NaCl to induce crassulacean acid metabolism(CAM) was accompanied by the accumulation of pinitol. Pinitolconstituted 71% of the soluble carbohydrate fraction and 9.7%dry weight in the CAM form. Pinitol in the C₃ form did not exceed5% of the soluble carbohydrate fraction. Pinitol appeared metabolicallyinert: it was not readily degraded during 96 h of darkness inthe CAM form or during CAM deinduction. Preparations of CAMM. crystallinum protoplasts, vacuoles and chloroplasts showedpinitol to be chloroplastic at a concentration of about 230mol m^–3 and cytosolic at about 100 mol m^–3. No pinitolwas detected in vacuoles. CAM leaf extracts possessed a highermyo-inositol phosphate synthesising capacity than C₃ extracts,revealing greater activity in the CAM form of glucose-6-phosphatecycloaldolase, an enzyme in the pathway of pinitol synthesis. Although pinitol accumulation and CAM induction could not beseparated and appeared to be specific responses to water stress,there may not be a causal link between them. Pinitol may functionas a compatible solute in the cytosol and especially the chloroplaststo counteract the presence of high concentrations of Na⁺ andCl^– ions in the vacuole. The accumulation of pinitol,though apparently not directly related to CAM may, like CAM,be viewed as an aspect of the adaptation of the plant to a reductionin water availability. Key words: pinitol, Mesembryanthemum crystallinum L, CAM, compatible solute     

NADP-malic enzyme: immunolocalization in different tissues34 plant maize and the C3 plant wheat

In situimmunolocalization and Western blot analysis of separatedcellular and subcellular fractions, were used to determine thelocalization of different isoforms of NADP-malic enzyme in bothwheat (C₃) and maize (C₄) plants. In both techniques, an affinitypurified anti-(maize 62 kDa NADP-ME) lgG from the maize greenleaf isoform also reacted with a 72 kDa protein in tissues ofC4 plants as well as C3 plants. The light- inducible 62 kDaisofomi is located in bundle sheath chioroplasts of maize leaves.In etiolated leaves and in roots of maize there is evidencefor the occurrence of a 72 kDa isoform which co-migrates on2-D (SDS and isoelectric focusing) PAGE. The 72 kDa isoformis also present in low levels in green leaves. This form mayoccur in multiple intracellular compartments; but in situ immunolocalizationexperiments and Western blot and activity assays on fractionatedprotoplasts indicate that a significant amount of this isoformoccurs in plastids. With regards to C³ plants such as wheat,a 72 kDa isoform in leaves is largely confined to the chloroplastsbased on in situ immunolocalization and Western blots and enzymeactivity assays with fractionated protoplasts. In maize, itappears that the constitutive expression pattern of a possibleC₃ ancestral gene for NADP-malic enzyme has been maintained,and a high level expression of a light-inducible isoform locatedin bundle sheath chloroplasts (62 kDa) has been acquired duringits evolution. Key words: NADP-malic enzyme, Triticum aestivum, Zea mays     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : II. Enzymology

Xerosicyos danguyi Humbert (Cucurbitaceae) is a leaf succulent endemic to Madagascar. Under well-watered conditions, the plant exhibited Crassulacean acid metabolism (CAM) but shifted to a dampened form of CAM, CAM-idling, when subjected to water stress. The purpose of this investigation was to examine the effects of a shift in carbon metabolism on phosphoenolpyruvate carboxylase and on NADP-malic enzyme in X. danguyi. Experiments were conducted to determine the diurnal patterns of enzyme activity and pH optima of both enzymes, as well as the approximate molecular mass, kinetic patterns, malate inhibition, and glucose-6-phosphate stimulation of phosphoenolpyruvate carboxylase. The two enzymes extracted from well-watered and water-stressed plants were similar in most parameters investigated; thus, CAM-idling appeared to be only a dampened form of CAM photosynthesis.     

NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways

NADP-malic enzyme (NADP-ME) is a widely distributed enzyme that catalyzes the oxidative decarboxylation of L-malate. Photosynthetic NADP-MEs are found in C4 bundle sheath chloroplasts and in the cytosol of CAM plants, while non-photosynthetic NADP-MEs are either plastidic or cytosolic in various plants. We propose a classification of plant NADP-MEs based on their physiological function and localization and we describe recent advances in the characterization of each isoform. Based on the alignment of amino acid sequences of plant NADP-MEs, we identify putative binding sites for the substrates and analyze the phylogenetic origin of each isoform, revealing several features of the molecular evolution of this ubiquitous enzyme.     

Induction of the Inactivation and Degradation of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Maize Leaves by Freezing and Thawing

When frozen leaves of 24-day-old maize (Zea mays L.) plant werethawed on moist filter paper at 26°C (freeze-thaw treatment)several enzymes, including phosphoenolpyruvate carboxylase (PEPC)and ribulose-1,5-bisphosphate carboxylase (RuBPC), were rapidlyinactivated and degraded. The kinetics of the inactivation anddegradation were pseudo first-order, and the halftimes for inactivationof PEPC and RuBPC were 3.2 and 2.4 min, respectively. The effectof the freeze-thaw treatment on the inactivation and degradationdiffered among various enzymes: the residual activities of RuBPC,PEPC, hydroxypyruvate reductase, Cyt c oxidase, NADP-malic enzymeand a-mannosidase 10 min after the start of the thawing treatmentwere 7, 16, 54, 64, 97 and 98% of the initial respective levels.Thirty min after the starting of thawing treatment, the amountsof total soluble protein, the large subunit of RuBPC, the smallsubunit of RuBPC, the PEPC subunit and the NADP-malic enzymesubunit had fallen to 61, 2, 16, 8, and 66% of the initial respectiveamounts. The effect of freeze-thaw treatment on PEPC was greater in oldleaves than in young leaves. There was a steady increase ofthe rate of degradation of PEPC by freeze-thaw treatment asplants aged from 6 to 24 days. These results are discussed inthe context of protein degradation in plant cells. (Received August 9, 1993; Accepted January 10, 1994)     

Blue light-induced formation of phosphoenolpyruvate carboxylase in colorless Chlorella mutant cells

The activities of NAD-malic dehydrogenase, aspartate aminotransferase,phosphoenolpyruvate carboxylase and NADP-malic enzyme in colorlessmutant cells of Chlorella vulgaris (Mutant No. 125) decreasedduring starvation (in phosphate buffer in darkness). The mostpronounced decrease was observed in phosphoenolpyruvate carboxylaseactivity. A trace of ribulose diphosphate carboxylase activitydetected in the growing cells disappeared on starvation andno activity of pyruvate carboxylase was detected in these mutantcells. Blue light (462 or 465 nm) enhanced phosphoenolpyruvatecarboxylase activity in the starved cells about 2-fold, whilethe activities of aspartate aminotransferase and NADP-malicenzyme were slightly lowered by the blue light. Red light (mainly600–650 nm) brought about a slight decrease in all theenzyme activities tested. Cycloheximide (5 µ/ml) completelyabolished the enhancing effect of blue light on phosphoenolpyruvatecarboxylase activity, indicating that the short wavelength lightspecifically increased the de novo synthesis of this enzyme. (Received March 14, 1975; )     

Rapid triggering of malate accumulation in the C3/CAM intermediate plant Sedum telephium: relationship with water status and phosphoenolpyruvate carboxylase

Sedum telephium is a C₃/CAM intermediate plant in which expressionof CAM is caused by water deficit. The timing of the C₃-CAMswitch and its relationship with water status and phosphoenolpyruvate(PEP) carboxylase activity have been investigated. Water deficitwas provided by application of polyethylene glycol (PEG) solutionsso that roots were exposed to water potentials from 0 to –2.0 MPa below that of the nutrient solution. The response ofthe plants was measured during the first dark period after PEGaddition and 7 d later. Malic acid accumulation was triggeredduring the first dark period at root water potentials of –0.3MPa or less. This corresponded with very small decreases inleaf water potential and relative water content. The capacityof PEP carboxylase was not altered at any water potential duringthe first dark period. After 7 d the capacity of PEP carboxylaseprogressively increased as water potential declined to –0.4MPa. At this, and more negative, water potentials it was 5-foldhigher than in well-watered leaves. Malic acid fluctuationsincreased with decreasing PEG water potential below a thresholdof –0.1 MPa. Malic acid levels at the end of the lightperiod were progressively lower as water potential decreased.NAD- and NADP-malic enzyme activity were not affected by lowwater potential. Leaves detached from well-watered plants in the middle of thelight period and kept hydrated did not accumulate malic acidduring the following dark period. Allowing the leaves to lose10% of their water content induced malic acid accumulation duringthe same time. Conversely, leaves detached from long-term droughtedplants (which had malate fluctuations and a PEP carboxylasecapacity 5-fold higher than well-watered plants) accumulatedmalate during the night if maintained at the same low hydrationstate (82%RWC), whereas malic acid accumulation was promptlyreduced if they were rehydrated. Malic acid accumulation couldtherefore be rapidly altered by changing the hydration stateof the leaves. The short-term rehydration treatments did notalter PEP carboxylase capacity. However, alteration of leafhydration affected the apparent K_m (PEP) of PEP carboxylaseextracted 1 h before the end of the dark period. The K_m wasincreased by rehydration and decreased by dehydration. Sensitivityto feedback inhibition by malate was not affected by hydrationstate and was high for PEP carboxylase from well-watered leavesand lower for PEP carboxylase from long-term droughted leaves. Taken together, the responses of intact plants and detachedleaves show that malic acid accumulation can be triggered veryrapidly by small water deficits in the leaves. The extent ofnight-time malic acid accumulation is independent of PEP carboxylasecapacity. However, a change in the hydration state of the leavescan rapidly alter the affinity of PEP carboxylase for PEP. Theregulation of malic acid accumulation in relation to the drought-inducedtriggering of CAM is discussed. Key words: Crassulacean acid metabolism, water stress, Sedum telephium, phosphoenolpyruvate carboxylase (PEP carboxylase), malic enzyme     

Prolonged Survival of CAM-Mode Mesembryanthemum crystallinum in Darkness and its Possible Dependence on Malate

A remarkable difference was found in the survival of leavesof Mesembryanthemum crystallinum with plants grown in the C₃versus the CAM mode. With excised leaves (petiole in solution)of C₃-mode plants subjected to 6 days of darkness, there wasa large reduction in the chlorophyll content of the leaf andleaf turgor had decreased. By day 9, the chlorophyll had disappeared,except at the major veins, and the leaf tip had dried and turnedbrown. In contrast, the leaf tissue in the CAM mode showed onlya partial loss of chlorophyll during the same period, and evenafter 17 days of darkness, the tissue at the base was stillalive. Similarly, intact plants grown in the C₃ mode deterioratedmuch faster during 20 days of darkness than did plants grownin the CAM mode. Chlorophyll content, chlorophyll a/b ratio,phosphoenolpyruvate carboxylase, NADP-malic enzyme, malate andstarch content were measured. In both C₃- and CAM-mode plants,the starch content decreased rapidly during the dark periodand was nearly depleted after two days. In the CAM-mode tissue,there was a relatively high level of malate during prolongeddarkness (up to 17 days), with a transitory rise early in thedark period. In contrast, the malate content was low and rapidlydepleted in the C₃-mode leaves kept in darkness. These findingssuggest that malate may be an important source of carbon forsustaining leaves of CAM-mode M. crystallinum during prolongeddarkness. (Received May 20, 1987; Accepted October 23, 1987)     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.     

Molecular Cloning and Sequence of a Complementary DNA Encoding 1-Aminocyclopropane-l-carboxylate Synthase Induced by Tissue Wounding

1-Aminocyclopropane-l-carboxylate (ACC) synthase [EC 4.4.1.14 [EC] ]is the key enzyme regulating ethylene biosynthesis in higherplants. A complementary DNA encoding wound-induced ACC synthasefrom mesocarp of winter squash (Cucurbita maxima Duch.) fruitswas cloned, and its complete nucleotide sequence determined.The cloned cDNA contained an open reading frame of 1479 basepairs encoding a sequence of 493 amino acids. Identificationof the cDNA was accomplished by expression of active enzymein Escherichia coli harboring the cDNA and by the presence ofa partial amino acid sequence identical to that found in thepurified enzyme. A putative pyridoxal phosphate binding siteof the enzyme is suggested. Northern blot analysis showed thatthe ACC synthase gene was activated by tissue wounding, andits expression was repressed by ethylene. Genomic Southern analysisindicates the presence of at least another sequence which weaklyhybridizes with the cDNA. (Received June 26, 1990; Accepted August 7, 1990)     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane