The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity.

Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.

S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase.

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

Two-minute miniprep method for plasmid DNA isolation.

An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material. Processing time, from E. coli culture to usable plasmid DNA, is two minutes or less per sample. Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number. Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.

The Interactive Effects of pH, L-Malate, and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase.

The interactive effects of pH, L-malate, and glucose-6-phosphate (Glc-6-P) on the Vmax and Km of guard-cell (GC) phosphoenolpyruvate (PEP) carboxylase (PEPC) of Vicia faba L. were determined. Leaves of three different physiological states (closed stomata, opening stomata, open stomata) were rapidly frozen and freeze dried. GC pairs dissected from the leaves were individually extracted and individually assayed for the kinetic properties of PEPC. Vmax was 6 to 9 pmol GC pair-1 h-1 and was apparently unaffected to a biologically significant extent by the investigated physiological states of the leaf, pH (7.0 or 8.5), L-malate (0, 5, or 15 mM), and Glc-6-P (0, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the Km(PEP.Mg) was about 0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a GC [PEP] of 0.27 mM. In the study reported here, we determined that the in situ GC [Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Glc-6-P was included in the GC PEPC assay mixture, the Km(PEP.Mg) decreased to about 0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Glc-6-P at endogenous concentrations would seem both to activate the enzyme and to diminish the dramatic effect of pH on Km(PEP.Mg). Under assay conditions, L-malate is an inhibitor of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8 mM. Inclusion of 5 mM L-malate increased the Km(PEP.Mg) to about 3.6 mM (pH 7.0) or 0.4 mM (pH 8.5). Glc-6-P (0.5 mM) was sufficient to relieve L-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM Glc-6-P was required to relieve L-malate inhibition at pH 7.0. No biologically significant effect of physiological state of the tissue on GC PEPC Km(PEP.Mg) (regardless of the presence of effectors) was observed. Together, these results are consistent with a model that GC PEPC is regulated by its cytosolic chemical environment and not by posttranslational modification that is detectable at physiological levels of effectors. It is important to note, however, that we did not determine the phosphorylation status of GC PEPC directly or indirectly (by comparison of the concentration of L-malate that causes a 50% inhibition of GC PEPC).

Elevated Levels of Both Sucrose-Phosphate Synthase and Sucrose Synthase in Vicia Guard Cells Indicate Cell-Specific Carbohydrate Interconversions.

A long series of reports correlate larger stomatal aperture size with elevated concentration of sucrose (Suc) in guard cells. To assess the role and autonomy of guard cells with respect to these changes, we have determined quantitatively the cellular distribution of the synthetic enzyme, Suc-phosphate synthase (SPS) and the degradative enzyme Suc synthase (SS) in Vicia leaflet. As expected for Suc-exporting cells, the photosynthetic parenchyma had a high SPS:SS ratio of approximately 45. Also as expected, in epidermal cells, which had only few and rudimentary plastids, the SPS:SS ratio was low (0.4). Of all cells and tissues measured, those that had the highest specific activity of SPS (about 4.8 [mu]mol mg-1 of protein h-1) were guard cells. Guard cells also had a very high relative specific activity of SS.

Stress protection of transgenic tobacco by production of the osmolyte mannitol.

The accumulation of sugar alcohols and other low molecular weight metabolites such as proline and glycine-betaine is a widespread response that may protect against environmental stress that occurs in a diverse range of organisms. Transgenic tobacco plants that synthesize and accumulate the sugar alcohol mannitol were engineered by introduction of a bacterial gene that encodes mannitol 1 -phosphate dehydrogenase. Growth of plants from control and mannitol-containing lines in the absence and presence of added sodium chloride was analyzed. Plants containing mannitol had an increased ability to tolerate high salinity.

Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol.

A bacterial gene encoding mannitol-1-phosphate dehydrogenase, mtlD, was engineered for expression in higher plants. Gene constructions were stably incorporated into tobacco plants. The mtlD gene was expressed and translated into a functional enzyme in tobacco, resulting in the synthesis and accumulation of mannitol, which was identified by NMR and mass spectroscopy. Mannitol concentrations exceeded 6 mumol/g (fresh weight) in the leaves and in the roots of some transformants, whereas this sugar alcohol was not detected in these organs of wild-type tobacco plants or of untransformed tobacco plants that underwent the same regeneration scheme. These experiments demonstrate that branch-points in plant carbohydrate metabolism can be generated by which novel gene products can utilize endogenous substrates to divert metabolic energy into novel compounds. Additionally, the system described here allows for physiological studies in which the responses of wild-type and transgenic tobacco to various environmental stimuli can be compared directly. Such studies will facilitate our understanding of the roles of sugar alcohols (e.g., in stress tolerance) in higher plants.

High Performance Liquid Chromatography Analysis of Carbohydrates of Cotton-Phloem Sap and of Honeydew Produced by Bemisia tabaci Feeding on Cotton.

Phloem sap from cotton (Gossypium hirsutum L.) was collected from young and mature leaves by the aphid-stylet technique. Exudate was analyzed for carbohydrates by HPLC using sensitive pulsed amperometric detection. The predominant carbohydrate present (>90%) was identified as sucrose. A second, unidentified compound that was not one of the more commonly translocated sugars was detected in mature leaves. Carbohydrates in honeydew produced by the sweet-potato whitefly (Bemisia tabaci [Genn.]) feeding on cotton were sucrose, glucose, fructose, trehalulose, and a series of oligosaccharides.

Partial characterization of guard-cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities.

Maximum velocity and Km(PEP.Mg) of phosphoenolpyruvate carboxylase (PEPC) from stomatal guard cells of Vicia faba L. were determined as a function of pH, presence of malate, and physiological state of guard cells. The biochemical rationale for these measurements is that (a) massive proton extrusion from guard cells, the primary event that drives stomatal movements, has been speculated to alkalinize the cell; (b) guard-cell malate concentration increases severalfold on stomatal opening, and malate, generally an inhibitor of PEPC's, affects the oligomeric state of some PEPC's; and (c) the apparent in vivo activity of guard-cell PEPC is greatly enhanced during stomatal opening, compared with that of other physiological states of these cells. As there are precedents for cell-specific expression of particular forms of PEPC and for labile reversible, post-translational modifications (which are manifested kinetically as distinct physiological-state isoforms), individual assays were initiated on the addition of a single stomatal complex directly to a microdroplet of assay cocktail. The stomatal complexes (each of which comprises a pair of guard cells having a mass of 6 x 10(-9) g) were dissected from lyophilized leaf tissue that had been freeze-quenched either before, during, or after a treatment to open stomata. Vmax at pH 7.0 was not significantly different from that at pH 8.5. Neither Vmax nor Km(PEP.Mg) was distinguished on the basis of the physiological state of the tissue from which the enzyme was extracted. However, Km(PEP.Mg) was greater than 4x lower at pH 8.5 than at pH 7.0. Malate inhibition was competitive at both pH's, but inhibition was greater than 3x greater at the lower pH. These data indicate that the combined effects of pH and malate over the range studied can produce changes in enzyme velocity of approximately 24-fold. Thus, the results are consistent with an interpretation that guard-cell PEPC is regulated by the cytoplasmic chemical environment and not by alternations between physiological-state isoforms.

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L.

The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.

Histochemical technique: densitometry of nanogram quantities of proteins separated in one-dimensional microslab gels.

We describe methods for densitometry of electrophoretically separated proteins in 25-millimeter microslab gels. The methods are sufficiently sensitive to use with several individually excised plant cells, for which we describe an extraction procedure. In brief, submicrogram samples are excised from freeze-dried plant tissue. Extraction takes place under oil in microliter droplets of detergent. Proteins are separated by one-dimensional microelectrophoresis (HM Poehling, V Neuhoff 1980 Electrophoresis 1: 90-102) and then stained by a sensitive Coomassie procedure (V Neuhoff, R Stamm, E Hansjorg 1985 Electrophoresis 6: 427-448). The resulting profile is scanned by a computerized densitometer based on the Leitz Diavert MPV Microphotometer. Evaluations and typical data demonstrate the high performance of this system.

Histochemical technique : a general method for quantitative enzyme assays of single cell ;extracts' with a time resolution of seconds and a reading precision of femtomoles.

Biochemists who study single cells have been constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods we describe will remove this limitation. In brief, nanogram tissue samples are dissected from frozen-dried tissue. The samples are ;extracted' in microdroplets of assay cocktail. The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. In the development of this method, we evaluated several parameters required for optimization; the most important of these evaluations, including numerous empirically derived relationships, are reported here and in supplemental material provided with reprints.With these methods, assays of pyruvate orthophosphate dikinase on samples enriched in bundlesheath cells and mesophyll cells of Flaveria brownii yielded the predictable results. Assays of this enzyme in guard cells dissected from Vicia faba leaflets gave results like those recently reported by another laboratory for protoplasts derived from these cells. The results of assays by this method and by enzymic cycling for NAD(P)triose-P dehydrogenase were comparable. Phosphoenolpyruvate carboxylase, the most extensively studied enzyme activity, was present at high levels in guard cells, which has been demonstrated previously in other reports based on diverse assay approaches.

Guard Cell Starch Biosynthesis Regulated by Effectors of ADP-Glucose Pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes the regulated step of starch bioynthesis in mesophyll chloroplasts. This enzyme is activated by a high ratio of the concentrations of 3-P-glycerate to inorganic phosphate (Pi) in light. In contrast, starch in guard cell chloroplasts is degraded when stomata open, which usually occurs in light. We have investigated the biochemical causes for this contrasting phenomenon.Vicia faba L. leaflets were sampled in darkness and after various periods of illumination. The samples were quick-frozen and freeze-dried. Guard cells and other cells were dissected out, weighed, and assayed for ADP-glucose pyrophosphorylase activity, 3-P-glycerate, and Pi. In the pyrophosphorolytic direction, ADP-glucose pyrophosphorylase specific activity in guard cells was 2.7 moles per kilogram protein per hour, which was comparable to the values obtained for palisade and spongy cells. The specific activity in epidermal cells was 4-fold lower. Under our assay conditions, the guard cell enzyme activity was 5-fold higher in the presence of 3-P-glycerate and 5-fold lower with Pi (i.e. similar to the results obtained with extracts of fresh leaflet). During three minutes of illumination, 3-P-glycerate concentration in palisade cells increased 2.5-fold to 10 millimoles per kilogram dry mass. The concentration of 3-P-glycerate in guard cells was 20-fold lower and unaffected by illumination. The concentration of Pi was approximately 17 millimoles per kilogram dry mass in palisade cells, but was 10-fold higher in guard cells. These overall cellular Pi concentrations were unaffected by illumination. We conclude that starch biosynthesis in guard cells is not activated by light because of the low and constant 3-P-glycerate concentration there. We interpret this last to be a consequence of the absence of the photosynthetic carbon reduction pathway in chloroplasts of these cells.

Histological Compartmentation of Phosphate in Vicia faba L. Leaflet : Possible Significance to Stomatal Functioning.

Guard cells, epidermal cells, palisade parenchyma cells, and spongy parenchyma cells of Vicia faba L. leaflet were analyzed for inorganic phosphate and phosphorus. On a molar basis, cells in the epidermal layer contained about 15-fold more inorganic phosphate than mesophyll cells did. Although a metabolic role for this asymmetric distribution cannot be defined unequivocally, we note that high epidermal inorganic phosphate would buffer against pH changes in the epidermis during stomatal movements.

Profile of Basic Carbon Pathways in Guard Cells and Other Leaf Cells of Vicia faba L.

Guard cells and three other cell types from Vicia faba L. ;Longpod' leaflets were assayed for enzymes that catalyze one step in each of five major carbon pathways in green plants: the photosynthetic carbon reduction pathway (ribulose-bisphosphate carboxylase, EC 4.1.1.39), the photosynthetic carbon oxidation pathway (hydroxypyruvate reductase, EC 1.1.1.81), glycolysis ([NAD] glyceraldehyde-P dehydrogenase, EC 1.2.1.12), the oxidative pentose-P pathway (6-P-gluconate dehydrogenase, EC 1.1.1.44), and the tricarboxylic acid pathway (fumarase, EC 4.2.1.2). Neither ribulose-bisphosphate carboxylase nor hydroxypyruvate reductase could be detected in guard cells or epidermal cells; high levels of these activities were present in mesophyll cells. The specific activity of fumarase (protein basis) was about 4-fold higher in guard cells than in epidermal, palisade parenchyma or spongy parenchyma cells. (NAD) glyceraldehyde-P and 6-P-gluconate dehydrogenases also were present at high protein specific activities in guard cells (2- to 4-fold that in meosphyll cells).It was concluded that the capacity for metabolite flux through the catabolic pathways is high in guard cells. In addition, other support is provided for the view that photoreduction of CO(2) by these guard cells is absent.

Taxonomic survey for the presence of ribulose-1,5-bisphosphate carboxylase activity in guard cells.

Guard cell pairs were dissected from freeze-dried leaves of plants representing 15 families, including monocots, dicots, and pteridophytes. All three major photosynthetic carbon pathways (C(2), C(4), and Crassulacean acid metabolism) were represented. These individual guard cell pairs were assayed quantitatively for ribulose-1,5-bisphosphate carboxylase specific activity. Assay sensitivity averaged 0.08 picomoles of ribulose-P(2) dependent P-glycerate formation (i.e. 100-fold more sensitive than required to detect the activity present in a single Vicia faba mesophyll cell). The calculated specific activities for guard cells and mesophyll cells averaged 4 and 472 millimoles per kilogram dry weight per hour, respectively. For all species surveyed, (a) the enzyme activity calculated for guard cells was below the detection limit of the assay, or (b) the specific activity (weight or cell basis) calculated for guard cells was less than 1% of the specific activity calculated for adjacent mesophyll cells. Based on this survey, the generalization is made that the photosynthetic carbon reduction pathway is absent, or virtually so, in guard cell chloroplasts.