Photosynthetic Changes in the Inducible CAM Plant Sedum telephium L. Following the Imposition of Water Stress. 1. General Characteristics

The effects of water stress (drought) on the pattern of photosynthesisin Sedum telephium have been determined. Well-watered plantsexhibit a weak-CAM pattern, with substantial CO₂ fixation inthe day, a low level of CO₂ fixation at night, high daytimestomatal conductance with a lower conductance at night, andno diurnal fluctuation in acid content. Imposition of water-stress causes a switch from weak-CAM toa full-CAM mode of photosynthesis, as indicated by cessationof daytime CO₂ fixation, a marked increase in night-time CO₂fixation, very low daytime stomatal conductance, increased night-timeconductance and significant diurnal fluctuations in acid content. Sedum telephium, CAM, CO₂ fixation, drought, malate, photosynthesis, water stress     

Comparison of photosynthesis and antioxidative protection in Sedum album and Sedum stoloniferum (Crassulaceae) under water stress

Photosynthetic gas exchange, dry mass production, water relations and inducibility of crassulacean acid metabolism (CAM) pathway as well as antioxidative protection during the C₃-CAM shift were investigated in Sedum album and Sedum stoloniferum from Crassulaceae under water stress for 20 days. Leaf relative water content (RWC), leaf osmotic and water potential decreased with increasing water stress in both studied species. Significant reduction in dry matter production and leaf thickness was detected only in S. stoloniferum after 20-d water stress. Δtitratable acidity and phosphoenolpyruvate carboxylase (PEPC) activity in S. album responded to drought at early stages of stress treatment, continued to increase throughout the entire stress period and reached levels 15 times higher than those in well-watered plants. In S. stoloniferum, however, both parameters responded later and after a transient increase declined again. In S. stoloniferum, in spite of increase by drought stress, net night-time CO₂ assimilation was negative resembling a C₃-like pattern of gas exchange. Catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) activities increased in plants subjected to mild water stress while declined as the stress became severe. Although malondialdehyde (MDA) content was higher in drought-stressed S. stoloniferum, the increase in the concentration of hydrogen peroxide (H2O₂) that may act as a signal for C₃-CAM transition was higher in S. album compared with S. stoloniferum. In drought-stressed plants, SOD activity showed a clear diurnal fluctuation that was more steadily expressed in S. album. In addition, such pattern was observed for CAT only in S. album. We concluded that temporal and diurnal fluctuation patterns in the activity of antioxidant enzymes depended on duration of drought stress and was related to the mode of photosynthesis and degree of CAM induction. According to our results, S. stoloniferum developed a low degree of CAM activity, e.g. CAM-cycling metabolism, under drought conditions.     

Properties of Phosphoenolpyruvate Carboxylase and Carbohydrate Accumulation in the C3-CAM Intermediate Sedum telephium L. Grown Under Different Light and Watering Regimes

A comparison of the activity and properties of the enzyme phosphoenolpyruvatecarboxylase (PEPC) was made for plants of Sedum telephium L.grown under low (70 µmol m^–2 s^–1) or high(500µmol m^–2 s^–1) PPFD and subjected to varyingdegrees of water stress. Under well-watered conditions onlyplants grown under high PPFD accumulated titratable acidityovernight and the extractable activity of PEPC was almost 2-foldhigher in these plants than in plants grown under low PPFD.Increasing drought stress resulted in a substantial increasein the activity of PEPC extracted both during the light anddark periods and a decrease in the sensitivity to inhibitionby malic acid. The magnitude of these changes was determinedby the severity and duration of drought and by light intensity.A comparison of the kinetic properties of PEPC from severelydroughted plants revealed that plants droughted under high PPFDhad a lower K_m for PEP than plants under low PPFD. Additionof 2·0 mol m^–3 malate resulted in an increase inthe K_m for PEP, with plants draughted under low PPFD havinga significantly higher K_m in the presence of malic acid comparedto those under high _PPFD. Response to the activator glc-6-P,which lowered the K_m for PEP, also varied between plants grownunder the two light regimes. Under well-watered conditions PEPCextracted from plants under high PPFD was more sensitive toactivation by glc-6-P than those under low PPFD. After the severedrought treatment, however, the K_m for PEP in the presence ofglc-6-P was similar for enzyme extracted from plants grown underboth light regimes. Soluble sugars and starch were depletedovernight and were both possible sources of substrate for PEPC.With increasing drought, however, the depletion of starch relativeto soluble sugars increased under both light regimes. The propertiesof PEPC and the characteristics of carbohydrate accumulation/depletionare discussed in relation to the regulation of CAM in S. telephiumgrown under different light and watering regimes. Key words: PEP carboxylase, CAM, carbohydrates, Sedum telephium     

Modification of Maize Leaf Phosphoenolpyruvate Carboxylase with Fluorescein Isothiocyanate

Fluorescein isothiocyanate inactivates phosphoenolpyruvate carboxylasefrom maize leaves, presumably by reacting with lysyl groups.The reaction appears to involve at least two groups of lysineson the enzyme. The more rapid reaction is with groups whichare protected by the substratemagnesium phosphoenolpyruvateand thus probably are located in the active site. In addition,fluorescein isothiocyanate apparently binds more slowly at asite which desensitizes the enzyme to activation by glucose-6-phosphate. Using the fluorescence of the complex of fluorescein isothiocyanatewith phosphoenolpyruvate carboxylase it was shown that bothmagnesium phosphoenolpyruvate and glucoses-6-phosphate causechanges in the conformation of the enzyme and influence thebinding of fluorescein isothiocyanate as well. Light scattering measurements showed that fluorescein isothiocyanateinduced disaggregation of the enzyme, while glucose-6-phosphatecaused aggregation, although less when fluorescein isothiocyanatewas present. ¹Supported in part by National Science Foundation grant no.DMB 88-12484.     

Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C₃ photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte.     

Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum

In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C₃-photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte.     

Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants

Control of C₄ photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C₄ and CAM plants is both metabolite-related and posttranslational. Specificially, the regulatory properties of the enzyme vary in accord with the physiological activity of C₄ photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by l-malate under light (C₄ plants) or at night (CAM plants) than in darkness (C₄) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currently in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C₄ plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C₄ and CAM plants, respectively.     

Inducibility of crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different degrees of CAM

The biochemical basis for photosynthetic plasticity in tropical trees of the genus Clusia was investigated in three species that were from contrasting habitats and showed marked differences in their capacity for crassulacean acid metabolism (CAM). Physiological, anatomical and biochemical measurements were used to relate changes in the activities/amounts of key enzymes of C₃ and C₄ carboxylation to physiological performance under severe drought stress. On the basis of gas-exchange measurements and day/night patterns of organic acid turnover, the species were categorised as weak CAM-inducible (C.aripoensis Britt.), C₃-CAM intermediate (C. minor L.) and constitutive CAM (C.␣rosea Jacq. 9.). The categories reflect genotypic differences in physiological response to drought stress in terms of net carbon gain; in C. aripoensis net carbon gain was reduced by over 80% in drought-stressed plants whilst carbon gain was relatively unaffected after 10 d without water in C. rosea. In turn, genotypic differences in the capacity for CAM appeared to be directly related to the capacities/amounts of phosphoenolpyruvate carboxylase (PEPCase) and phosphoenolpyruvate carboxykinase (PEPCK) which increased in response to drought in both young and mature leaves. Whilst measured activities of PEPCase and PEPCK in well-watered plants of the C₃-CAM intermediate C. minor were 5–10 times in excess of that required to support the magnitude of organic acid turnover induced by drought, close correlations were observed between malate accumulation/PEPCase capacity and citrate decarboxylation/PEPCK capacity in all the species. Drought stress did not affect the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein in any of the species but Rubisco activity was reduced by 35% in the weak CAM-inducible C. aripoensis. Similar amounts of glycine decarboxylase (GDC) protein were present in all three species regardless of the magnitude of CAM expression. Thus, the constitutive CAM species C. rosea did not appear to show reduced activity of this key enzyme of the photorespiratory pathway, which, in turn, may be related to the low internal conductance to CO₂ in this succulent species. Immuno-histochemical techniques showed that PEPCase, PEPCK and Rubisco were present in cells of the palisade and spongy parenchyma in leaves of species performing CAM. However, in leaves from well-watered plants of C. aripoensis which only performed C₃ photosynthesis, PEPCK was localized around latex-producing ducts. Differences in leaf anatomy between the species suggest that the association between mesophyll succulence and the capacity for CAM in these hemi-epiphytic stranglers has been selected for in arid environments. Received: 4 July 1997 / Accepted: 27 November 1997     

Effects of rapidly imposed water deficit on photosynthetic parameters of three C<Subscript>4</Subscript> grasses

Water deficit, when rapidly imposed on three C₄ grasses of the different metabolic subtypes, Paspalum dilatatum Poiret (NADP-malic enzyme), Cynodon dactylon (L.) Pers (NAD-malic enzyme) and Zoysia japonica Steudel (phosphoenolpyruvate carboxykinase), caused decreases in photosynthetic rates, in the quantum yield of PS II and photochemical quenching, and in the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC). The results provide evidence for non-stomatal limitations of photosynthesis differing in nature between the three species.     

The appearance of a new molecular species of phosphoenolpyruvate carboxylase (PEPC) and the rapid induction of CAM in Sedum telephium L.

Abstract. When detached leaves of Sedum telephium are incubated in the absence of water, a rapid switch from C₃ photosynthesis to CAM (as indicated by the onset of day-to-night fluctuations in titratable acidity. ΔH⁺) occurs within the first dark period. The C₃-CAM switch in intact plants occurs within 3 5d. Extractable activity of phospho enol pyruvate carboxylase (PEPC) increases five-fold in intact plants during CAM induction; however, during rapid CAM induction in detached leaves, there is only a very small increase in PEPC activity. Fractionation by anion exchange chromatography of crude extracts from leaves of intact plants subjected to water deficit shows that CAM induction is associated with the appearance of a molecular species of PEPC termed PEPC I. PEPC I is barely detectable in well-watered plants which are not performing CAM. The major form in these plants is termed PEPC II. In leaves from intact plants, there is a significant positive correlation between PEPC I activity and ΔH⁺ during a period of increasing water deficit. PEPC I exhibits day to night fluctuations in malate sensitivity, being less sensitive during the dark period. In contrast, PEPC II is more sensitive to inhibition by malate and has no day to night fluctuation in sensitivity. In detached leaves deprived of water, a small increase in PEPC I capacity is detected at the end of the first dark period (20 h after the start of treatment). The results suggest that PEPC I is required for attainment of maximum nocturnal malic acid synthesis. There is a significant correlation between leaf water status (relative water content), ΔH⁺, total PEPC and PEPC I activity suggesting that the internal water status of the plant may be a trigger for CAM induction. Abscisic acid applied to detached leaves does not cause nocturnal acidification.     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Calcium-Dependent and -Independent Phosphoenolpyruvate Carboxylase Kinases in Sorghum Leaves: Further Evidence for the Involvement of the Calcium-Independent Protein Kinase in the in situ Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase

Calcium-dependent phosphoenolpyruvate carboxylase protein kinasewas copurified with C₄ phosphoenolpyruvate carboxylase (C₄ PEPC)from illuminated Sorghum leaves during purification by variousprocedures. Isolated mesophyll cell protoplasts contained bothcalcium-dependent and -independent protein kinases. The latterwas induced by light and weak bases and was found to be themajor protein kinase phosphorylating C₄ PEPC in the mesophyll. (Received July 29, 1997; Accepted November 28, 1997)     

Limitation of C3–CAM shift in the common ice plant under high irradiance

In the halophytic plant Mesembryanthemum crystallinum salinity or drought can change the mode of photosynthesis from C₃ to crassulacean acid metabolism (CAM). These two stress factors are linked to oxidative stress, however, the induction of CAM by oxidative stress per se is not straightforward. Treatment with high light (HL) did not lead to the induction of CAM, as documented by a low night/day difference in malate level and a low expression of the CAM-related form of phosphoenolcarboxylase (Ppc1), despite causing some oxidative damage (elevated MDA level, malondialdehyde). In contrast to the action of high salinity (0.4 M NaCl), HL treatment did not activate neither the cytosolic NADP-malic enzyme nor the chloroplastic form of NADP-dependent malate dehydrogenase (NADP-MDH). In plastids of HL-treated plants a huge amount of starch was accumulated. This was associated with a weak stimulation of hydrolytic and phosphorolytic starch-degrading enzymes, in contrast to their strong up-regulation under high salinity. It is concluded that HL alone is not able to activate starch degradation necessary for CAM performance. Moreover, in the absence of salinity in C₃M. crystallinum plants an age-dependent increase in energy dissipation from PSII was documented under high irradiance, as illustrated by non-photochemical quenching (NPQ). Obtained data suggest that in this halophytic species several photoprotective strategies are strictly salinity-dependent.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

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     

Induction and Repression of CAM in Sedum telephium L. in Response to Photoperiod and Water Stress

Lee, H. S. J. and Griffiths, H. 1987. Induction and repressionof CAM in Sedurn relephluni L. in response to photopcnod andwater stress.—J. exp. Bot. 38: 834–841. The introduction and repression of CAM in Sedurn telephiunmL, a temperate succulent, was investigated in watered, progressivelydrouglited and rewatered plants in growth chambers. Measurementswere made of water vapour and CO₂ exchange, titratable acidity(TA) and xylem sap tension. Effects of photoperiod were alsostudied. CAM was induced by drought under long or short days,although when watered no CAM activity was expressed. C₃-CAM intermediate plants were used for the investigation ofwater supply. Those which had received water and those drought-stressedboth displayed a similar nocturnal increase in TA, with a day-nightmaximum (H+) of 69 µmol g^–1 fr. wt. The wateredplants took up CO₂ at a maximum rate of 2?2 µmol m^–2s^–1 only in the light period, while the droughted plantsshowed a maximum nocturnal CO₂ uptake rate of 0?69 µmolm^–2 s^–1. Subsequently, as CAM was repressed, thewatered S. telephiwn displayed little variation in TA, withconstant levels at 42 µmol g^–1 fr. wt. (day 10).After 10 d of drought stress, the CAM characteristics of S.telephiurn were aLso affected, with reduced net CO₂ uptake andH+. The transition between C₃ and CAM in S. telephium can be describedas a progression in terms of the proportion of respiratory CO₂which is recycled and refixed at night as malic acid, in comparisonwith net CO₂ uptake. Recycling increased from 20% (day 1) to44% (day 10) as a result of the drought stress and was highin both the CAM-C₃ stage (no net CO2 uptake at night) and alsoin the drought-stressed CAM stage (reduced net CO₂ uptake atnight). The complete C₃-CAM transition occurred in less than8 d, and the stages could be characterized by xylem sap tensionmeasurements: CAM = 0?50 MPa C₃-CAM = 0?36 MPa C₃ = 0?29 MPa. Key words: CAM, Sedum telephium L., recycling     

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)     

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