Transformation of a CAM plant, the facultative halophyte Mesembryanthemum crystallinum by Agrobacterium tumefaciens

This study is the first to demonstrate that a foreign DNA can be delivered into cells of facultative halophyte crassulacean acid metabolism (CAM) plant, Mesembryanthemum crystallinum L. with Agrobacterium tumefaciens. This plant can be induced to change from C3 to CAM by drought and various stresses, and is a good model to study the environment stress on metabolism not only at whole plant but also at cell level. The β-glucuronidase (GUS) and kanamycin (Km) resistance genes were introduced into this plant. The average successful rate of transformation was about 54.5% with root tissue or 53.0% with hypocotyl tissue. Based on the resistance to Km, polymerase chain reaction (PCR) detection and GUS expression, transformation with Agrobacterium tumefaciens was successful for introducing foreign genes into M. crystallinum. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

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     

Responses of chlorophyll fluorescence parameters of the facultative halophyte and C3-CAM intermediate species Mesembryanthemum crystallinum to salinity and high irradiance stress

Mesembryanthemum crystallinum L. (Aizoaceae) is a facultative annual halophyte and a C(3)-photosynthesis/crassulacean acid metabolism intermediate species currently used as a model plant in stress physiology. Both salinity and high light irradiance stress are known to induce CAM in this species. The present study was performed to provide a diagnosis of alterations at the photosystem II level during salinity and irradiance stress. Plants were subjected for up to 13 days to either 0.4M NaCl salinity or high irradiance of 1000 micromol m(-2)s(-1), as well as to both stress factors combined (LLSA=low light plus salt; HLCO=high light of 1000 micromol m(-2)s(-1), no salt; HLSA=high light plus salt). A control of LLCO=low light of 200 micromol m(-2)s(-1), no salt was used. Parameters of chlorophyll a fluorescence of photosystem II (PSII) were measured with a pulse amplitude modulated fluorometer. HLCO and LLSA conditions induced a weak degree of CAM with day/night changes of malate levels (Deltamalate) of approximately 12mM in the course of the experiment, while HLSA induced stronger CAM of Deltamalate approximately 20 mM. Effective quantum yield of PSII, DeltaF/F'(m), was only slightly affected by LLSA, somewhat reduced during the course of the experiment by HLCO and clearly reduced by HLSA. Potential quantum efficiency of PSII, F(v)/F(m), at predawn times was not affected by any of the conditions, always remaining at 0.8, showing that there was no acute photoinhibition. During the course of the days HL alone (HLCO) also did not elicit photoinhibition; salt alone (LLSA) caused acute photoinhibition which was amplified by the combination of the two stresses (HLSA). Non-photochemical, NPQ, quenching remained low (<0.5) under LLCO, LLSA and HLCO and increased during the course of the experiment under HLSA to 1-2. Maximum apparent photosynthetic electron transport rates, ETR(max), declined during the daily courses and were reduced by LLSA and to a similar extent by HLSA. It is concluded that M. crystallinum expresses effective stress tolerance mechanisms but photosynthetic capacity is reduced by the synergistic effects of salinity and light irradiance stress combined.     

Thermoluminescence studies on the facultative crassulacean-acid- metabolism plant Mesembryanthemum crystallinum L.

Thermoluminescence (TL) signals were measured from leaves of the facultative CAM (crassulacean acid metabolism) plant Mesembryanthemum crystallinum L.. Following the induction of CAM by salt treatment, a TL band at 46 °C was induced, which was charged by a single-turnover flash. The intensity of the 46 °C-band depends on the number of excitation flashes and oscillates with a period of four. A similar band was induced in C₃ plants by far-red illumination. Under CAM conditions, the intensity of the 46 °C-band underlies a diurnal rhythm. The maximal intensity of the 46 °C-band is observed in the morning after onset of the light and in the evening. At around 12 a.m. it is suppressed. The intensity of the 46 °C-band relates to diurnal changes in the ratio of dihydroxy acetone phosphate/3-phosphoglycerate (DHAP/PGA) which is an indicator of the energy status of the chloroplast. During high-intensity illumination, the 46 °C-band disappears, but it is restored in the dark. We propose that the 46 °C-band is an indicator of the metabolic state of the leaf, originating from photosystem II centres initially in the S₂(S₃)Q_B oxidation state, in which the electron acceptor Q_B becomes reduced either by reverse electron flow or reduction of the plastoquinone pool via an NAD(P)H plastoquinone oxidoreductase. We present evidence that the redox state of the electron-transport chain is different under conditions of CAM compared to C₃ metabolism and that changes induced by CAM can be monitored by measuring the amplitude of the 46 °C-band after flash excitation. Received: 7 August 1997 / Accepted: 22 December 1997     

Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+) -transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum L

A strongly increased ATP/ADP ratio was found during the nocturnal phase I in crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants. Conversely, during the daytime phase III in CAM-performing plants the ATP/ADP ratio dropped to a similar level to that of C3 plants, cytochrome c oxidase activity was stimulated and mitochondrial Mn-superoxide dismutase activity was strongly increased. The findings suggest that a salinity-induced C3-CAM transition might be an efficient energy-conserving strategy for M. crystallinum plants, in which the strong nocturnal ATP production seems to be, at least partially, independent from the coupled mitochondrial electron transport.     

Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from dark-adapted leaves of the common ice plant Mesembryanthemum crystallinum L. with crassulacean-acid metabolism (CAM). Purification by (NH4)2SO4 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23-25 U/mg protein and a protein kinase activity of 255 mumol Pi.mol-1 PyrPC.s-1. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 microM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300-305] that Ser11 is the phosphorylation site of the CAM PyrPC of M. crystallinum. At pH 7.0, the Km for ATP of the protein kinase was 25 microM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone III-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M. crystallinum, similar to C4 and constitutive CAM plants.     

Stress-dependent accumulation of spermidine and spermine in the halophyte Mesembryanthemum crystallinum under salinity conditions

We studied the effects of chloride salinity (300 and 500 mM NaCl) on the content of free polyamines (PAs) from putrescine (Put) family in Mesembryanthemum crystallinum L. leaves and roots. The contents of Put and spermidine (Spd) in leaves increased temporarily, achieving the highest values on the third day of salinity treatment; thereafter (by days 7–14), they dropped sharply. The content of spermine (Spm) increased gradually, and its high level was maintained until the end of experiment. The dynamics of Spm accumulation in leaves under salinity conditions resembled that of phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the water-saving CAM pathway of photosynthesis. This indicates indirectly the involvement of Spm in the common ice plant adaptation to salinity. A decrease in the molar ratios of Spd to Spm in the leaves under salinity conditions could point to the acceleration of Spm biosynthesis (accumulation) during plant adaptation, whereas the levels of Spm precursors, Put and Spd, did not increase. This phenomenon could be explained by an accelerated conversion of Spd into Spm, an active liberation of free Spm from its conjugates, or changes in the rates of studied PA biosynthesis and degradation under salinity. At the same time, the intracellular concentration of ethylene rose under these conditions. It was supposed and then demonstrated, that the pathway of ethylene biosynthesis and that of the synthesis of Put family PAs compete under severe salinity conditions. This competition might be based on the disturbances in sulfur metabolism and a decrease in the methionine content, an immediate precursor of S-adenosyl-L-methionine.     

Antiporter NHX2 differentially induced in Mesembryanthemum crystallinum natural genetic variant under salt stress

Plants exhibit several mechanisms to survive under high salinity conditions. The uptake and compartmentalization of Na⁺ ion by the NHX antiporter is a crucial mechanism in homeostasis maintenance. Therefore, we evaluated McNHX2 gene expression and several physiological responses induced in three natural genetic variants of ice plants under salt stress. Based on morphology and growth behavior of wild type populations from an arid region of northwestern Mexico, we identified three ice plant natural genetic variants and called P0, P9, and P11. Several physiological parameters, such as water potential, relative water content, chlorophyll, and Na⁺ and K⁺ ion contents from all natural genetic variants exhibited a differential response under high salinity conditions. Specifically, the P0 variant showed lower water potential changes and least perturbation of Na⁺/K⁺ ratio than those of the P9 and P11 variants under saline conditions, suggesting that the P0 variant is the most salt tolerant. Unexpectedly, McNHX2 expression was repressed in the P11 variant while it was upregulated in the P0 and P9 variants under saline treatments. The McNHX2 gene was sequenced showing 15 introns and 16 exons; polymorphisms were found among the cDNAs sequences from the three natural genetic variants. All these data suggest that differential responses to high salinity involve different mechanisms operating in each variant for counteracting saline stress effects.

     

Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3-CAM intermediate halophyte Mesembryanthemum crystallinum L.

BSA, bovine serum albumin
CAM, Crassulacean acid metabolism
DTT, dithiothreitol
EDTA, ethylenediaminetetraacetic acid
FPLCfast protein liquid chromatography
HEPES, N-(2-hydroxyethyl)piperazine-?-(ethanesulphonic acid)
ME, β-mercaptoethanol
NBT, nitro blue tetrazolium
PAGE, polyacrylamide gel electrophoresis
SDS, sodium dodecyl sulphate
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
SOD, superoxide dismutase (EC 1.15.1.1)
TEMED, N,N,?,?-tetramethylethylenediamine
Tris, Tris (hydroxymethyl) aminomethane
Tricine, N-Tris(hydroxymethyl)methylglycine

Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m^–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn-, Fe-, and Cu/Zn-SODs, respectively. Mn-SOD was found in the mitochondrial fraction, Fe-SOD in the chloroplast fraction, and Cu/Zn-SOD is probably localized in the cytosol. The Fe-SOD found in M. crystallinum is the first iron-containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn-, Fe-, Cu/Zn-SODs, respectively. Native Mn-SOD seems to be a tetramer, while Fe-SOD and Cu/Zn-SOD are dimers. All SOD isoforms show high thermal stability. Mn-SOD is active even after short heating at 90 °C and Fe-SOD at 70 °C. Moreover, high concentrations of β-mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build-up of large stationary O₂ concentrations in the leaf air spaces is associated with the photosynthetic CO₂ reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C₃ plants to environmental stress as imposed by, for example, SO₂ and O₃.     

Characteristics of MgATP2--dependent electrogenic proton transport in tonoplast vesicles of the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Membrane vesicles were isolated from mesophyll cells of Mesembryanthemum crystallinum in the C₃ state and in the crassulacean acid metabolism (CAM) state. The distribution of ATP-hydrolysis and H⁺-transport activities, and the activities of hydroxypyruvate reductase and Antimycin-insensitive cytochrome-c-reductase on continuous sucrose gradients was studied. For isolations carried out routinely a discontinuous sucrose gradient (24%/37%/50%) was used. Nitrate-sensitive ATP-hydrolysis and H⁺-transport activities increased several-fold during the transition from C₃ photosynthesis to CAM. Nitrate-sensitive ATPase showed a substrate preference for ATP with an apparent K_m (MgATP^2-) of 0.19–0.37 mM. In both C₃ and CAM states the ATPase showed a concentration-dependent stimulation by the anions chloride and malate. However, the pH optima of the two states were different: the ATPase of C_3- M. crystallinum had an optimum of pH 7.4 and that of CAM-M. crystallinum an optimum of pH 8.4. The optical probe oxonol-VI was used to demonstrate the formation of MgATP^2--dependent electric-potential gradients in tonoplast vesicles.Abbreviations Bistris-Pronane 1,3-bis [tris(hydroxymethyl)-methylaminol propane - CAM Crassulacean acid metabolism - DIDS 4,4-dilsothiocyano-2,2-stilbene disulfonic acid: - DTT dithiothreitol - ER endoplasmic reticulum - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - HPR hydroxypyruvate reductase - IDPase inosine 5-diphosphatase - OX-VI oxonol VI - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO(2) uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24?h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24?h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant.     

A novel Mg(2+)-dependent O-methyltransferase in the phenylpropanoid metabolism of Mesembryanthemum crystallinum

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-l-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg(2+)-dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg(2+)-dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant O-methyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.