Nanotubules on plant surfaces: chemical composition of epicuticular wax crystals on needles of Taxus baccata L

Needles of Taxus baccata L. were covered with tubular epicuticular wax crystals varying in diameters (100 and 250 nm) and lengths (300-500 and 500-1000 nm) on the abaxial and adaxial surfaces, respectively. Various sampling protocols were employed to study the chemical composition of the needle waxes on three different levels of spatial resolution. First, a dipping extraction of whole needles yielded the total cuticular wax mixture consisting of very long chain fatty acids (21%), alkanediols (19%), phenyl esters (15%), and secondary alcohols (9%) together with small amounts of aldehydes, primary alcohols, alkanes, alkyl esters, and tocopherols. Second, waxes from both sides of the needle were sampled separately by brushing with CHCl3-soaked fabric glass. Both sides showed very similar qualitative composition, but differed drastically in quantitative aspects, with nonacosan-10-ol (18%) and alkanediols (33%) dominating the abaxial and adaxial waxes, respectively. Third, the epi- and intracuticular wax layers were selectively sampled by a combination of mechanical wax removal and brushing extraction. This provided direct evidence that the tubular wax crystals contained high percentages of nonacosane-4,10-diol and nonacosane-5,10-diol on the abaxial surface, and nonacosan-10-ol on the adaxial surface of the needles. Together with these compounds, relatively large amounts of fatty acids and smaller percentages of aldehydes, primary alcohols, alkyl esters, and alkanes co-crystallized in the epicuticular layer. In comparison, the intracuticular wax consisted of higher portions of cyclic constituents and aliphatics with relatively high polarity. The formation of the tubular crystals is discussed as a spontaneous physico-chemical process, involving the establishment of gradients between the epi- and intracuticular wax layers and local phase separation.     

Molecular characterization of the CER1 gene of arabidopsis involved in epicuticular wax biosynthesis and pollen fertility.

The aerial parts of plants are coated with an epicuticular wax layer, which is important as a first line of defense against external influences. In Arabidopsis, the ECERIFERUM (CER) genes effect different steps of the wax biosynthesis pathway. In this article, we describe the isolation of the CER1 gene, which encodes a novel protein involved in the conversion of long chain aldehydes to alkanes, a key step in was biosynthesis. CER1 was cloned after gene tagging with the heterologous maize transposable element system Enhancer-Inhibitor, also known as Suppressor-mutator. cer1 mutants display glossy green stems and fruits and are conditionally male sterile. The similarity of the CER1 protein with a group of integral membrane enzymes, which process highly hydrophobic molecules, points to a function of the CER1 protein as a decarbonylase.     

Genomic cloning and characterization of a ricin gene from Ricinus communis.

A genomic clone that specifies a single polypeptide precursor for ricin, a toxic lectin of Ricinus communis (castor bean), was isolated, sequenced and Sl mapped. The gene encodes a 64 kDa precursor which contains, in the following order: a 24 or 35 amino acid signal peptide, the A chain, a 12 amino acid linker peptide, and the B chain. The 5'-end of the ricin mRNA maps approximately 35 bases upstream from the first methionine codon. Two putative TATA boxes and a possible CAAT box lie in the 5'-flanking region. Two possible polyadenylation signals were found in the 3' flanking region. No introns were found, which is typical of other lectin genes that have been sequenced. Southern blot analysis suggests that the castor bean genome contains approximately six ricin-like genes.     

Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production

The cuticle covering the aerial organs of land plants plays a protective role against several biotic and abiotic stresses and, in addition, participates in a variety of plant-insect interactions. Here, we describe the molecular cloning and characterization of the maize (Zea mays) GLOSSY1 (GL1) gene, a component of the pathway leading to cuticular wax biosynthesis in seedling leaves. The genomic and cDNA sequences we isolated differ significantly in length and in most of the coding region from those previously identified. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes. GL1 expression is not restricted to the juvenile developmental stage of the maize plant, pointing to a broader function of the gene product than anticipated on the basis of the mutant phenotype. Indeed, in addition to affecting cuticular wax biosynthesis, gl1 mutations have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure. Of the many wax biosynthetic genes identified so far, only a few from Arabidopsis (Arabidopsis thaliana) were found to be essential for normal cutin formation. Among these is WAX2, which shares 62% identity with GL1 at the protein level. In wax2-defective plants, cutin alterations induce postgenital organ fusion. This trait is not displayed by gl1 mutants, suggesting a different role of the maize and Arabidopsis cuticle in plant development.     

Identification of a tyrosine phosphatase involved in the response to mechanical injury in leaves of Ricinus communis

Plants defend themselves against biotic or abiotic stress by triggering intracellular signaling pathways that regulate gene expression and responses to the offending agent. Phosphorylation and dephosphorylation represent major mechanisms for the regulation of plant defense pathways. Therefore, MAP kinases and phosphatases have been the focus of many studies in this area. This study identified three phosphatase activities, namely RcPPase I, II and III. Wounding increased the activity levels of RcPPase III, while the activities of RcPPase I and II remained constant compared to the control. N-terminal partial amino acid sequence, biochemical characterization with use of specific substrates and inhibitors indicated that the RcPPase III belong to the family of tyrosine phosphatases (PTPs).     

Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production

Insertional mutagenesis of Arabidopsis ecotype C24 was used to identify a novel mutant, designated wax2, that had alterations in both cuticle membrane and cuticular waxes. Arabidopsis mutants with altered cuticle membrane have not been reported previously. Compared with the wild type, the cuticle membrane of wax2 stems weighed 20.2% less, and when viewed using electron microscopy, it was 36.4% thicker, less opaque, and structurally disorganized. The total wax amount on wax2 leaves and stems was reduced by >78% and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones, with increased acids, primary alcohols, and esters. Besides altered cuticle membranes, wax2 displayed postgenital fusion between aerial organs (especially in flower buds), reduced fertility under low humidity, increased epidermal permeability, and a reduction in stomatal index on adaxial and abaxial leaf surfaces. Thus, wax2 reveals a potential role for the cuticle as a suppressor of postgenital fusion and epidermal diffusion and as a mediator of both fertility and the development of epidermal architecture (via effects on stomatal index). The cloned WAX2 gene (verified by three independent allelic insertion mutants with identical phenotypes) codes for a predicted 632-amino acid integral membrane protein with a molecular mass of 72.3 kD and a theoretical pI of 8.78. WAX2 has six transmembrane domains, a His-rich diiron binding region at the N-terminal region, and a large soluble C-terminal domain. The N-terminal portion of WAX2 is homologous with members of the sterol desaturase family, whereas the C terminus of WAX2 is most similar to members of the short-chain dehydrogenase/reductase family. WAX2 has 32% identity to CER1, a protein required for wax production but not for cuticle membrane production. Based on these analyses, we predict that WAX2 has a metabolic function associated with both cuticle membrane and wax synthesis. These studies provide new insight into the genetics and biochemistry of plant cuticle production and elucidate new associations between the cuticle and diverse aspects of plant development.     

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.     

Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (K_m = 4.63 ± 0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.     

Enolase isozymes from Ricinus communis: Partial purification and characterization of the isozymes

The plastid and cytosolic isozymes of enolase from developing endosperm of castor oil seeds, Ricinus communis L. cv. Baker 296, were separated and partially purified. Each purified isozyme had a specific activity of approximately 200 μmol min^?1 mg protein. The isozymes have similar pH optima for the forward reaction, but different optima for the reverse reaction. The divalent metal specificity is the same for both isozymes. In addition to differences in charge, the isozymes can be distinguished by their different kinetic constants, thermostability and sensitivity to fluoride inhibition. Antibodies against yeast enolase isozyme I cross-react with Ricinus plastid enolase but not with the cytosolic isozyme.     

Cloning and characterization of a mammalian pseudouridine synthase

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.     

The 3-ketoacyl-CoA synthase WFL is involved in lateral organ development and cuticular wax synthesis in Medicago truncatula

Plant Molecular Biology - A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula....     

Effect of drought stress on the cytological status in Ricinus communis

Growing leaves of dicots are characterized by the simultaneous development of cytological structure and physiological function. Cytological development of growing leaves of castor bean (Ricinus communis L.) and the impact of drought on this process was studied. Cell division was observed when the middle lobe of the leaf was below 8 cm length. Cell densities dropped when the middle leaflet had reached 4 cm. Identical relationships between leaf size (length of the middle lobe) and (I) exposed surface area of epidermal cells (ii) height of palisade cells, (iii) cell density and stomatal density were observed. During drought, areal growth decreased, but the relationships between the cytological parameters and leaf size did not change. The impact of drought on the cellular growth processes depended on the stage of cytological development at the onset of the drought. These results are the basis for an analysis of physiological and biochemical parameters in the forthcoming studies.     

Effects of drought on nutrient and ABA transport in Ricinus communis

We studied the effects of variations of water flux through the plant, of diurnal variation of water flux, and of variation of vapour pressure deficit at the leaf on compensation pressure in the Passioura-type pressure chamber, the composition of the xylem sap and leaf conductance in Ricinus communis. The diurnal pattern of compensation pressure showed stress relaxation during the night hours, while stress increased during the day, when water limitation increased. Thus compensation pressure was a good measure of the momentary water status of the root throughout the day and during drought. The bulk soil water content at which predawn compensation pressure and abscisic acid concentration in the xylem sap increased and leaf conductance decreased, was high when the water usage of the plant was high. For all xylem sap constituents analysed, variations in concentrations during the day were larger than changes in mean concentrations with drought. Mean concentrations of phosphate and the pH of the xylem sap declined with drought, while nitrate concentration remained constant. When the measurement leaf was exposed to a different VPD from the rest of the plant, leaf conductance declined by 400mmol m^?2 s^?1 when compensation pressure increased by 1 MPa in all treatments. The compensation pressure needed to keep the shoot turgid, leaf conductance and the abscisic acid concentration in the xylem were linearly related. This was also the case when the highly dynamic development of stress was taken into account.     

Acetyl-coenzyme A carboxylase from the developing endosperm of Ricinus communis. I. Isolation and characterization

Acetyl-coenzyme A carboxylase has been purified from the plastids of developing castor oil seeds. High concentrations of the enzyme are required for stability as well as the presence of dithiothreitol, glycerol, bicarbonate, Triton X-100, and polyvinyl-pyrrolidone. It has a molecular weight of approximately 528,000 and appears to be membrane associated. Acetyl-CoA carboxylase is active over a wide pH range with an optimum at 8.0. Arrhenius plots are biphasic. The enzyme displays normal Michaelis-Menten kinetics with limiting Michaelis constants of KATP, 0.1 mM; KHCO-3, 3.0 mM; and Kacetyl-CoA, 0.05 mM. Monovalent cations, such as K+ and Cs+, exert a small activating effect on the enzyme while a divalent cation, Mn2+ or Mg2+, is essential for activity. The enzyme does not appear to be highly regulated by cellular metabolites.     

Sucrose is metabolised by sucrose synthase and glycolysis within the phloem complex of Ricinus communis L. seedlings

Metabolites and enzyme activities were measured in the phloem sap exuding from a cut hypocotyl of germinating castor-bean (Ricinus communis L.) seedlings. The sap contained considerable quantities of adenine nucleotides, uridine nucleotides, uridine diphosphoglucose (UDPGlc), all the major phosphorylated metabolites required for glycolysis, fructose-2,6-bisphosphate and pyrophosphate. Supplying 200 mM glucose instead of sucrose to the cotyledons resulted in high concentrations of glucose in the sap, but did not modify the metabolite levels. In contrast, when 200 mM fructose was supplied we found only a low level of fructose but a raised sucrose concentration in the sap, which was accompanied by a three-to fourfold decrease of UDPGlc, and an increase of pyrophosphate, UDP and UTP. The measured levels of metabolites are used to estimate the molar mass action ratios and in-vivo free-energy change associated with the various reactions of sucrose breakdown and glycolysis in the phloem. It is concluded that the reactions catalysed by ATP-dependent phosphofructokinase and pyruvate kinase are removed from equilibrium in the phloem, whereas the reactions catalysed by sucrose synthase, UDPGlc-pyrophosphorylase, phosphoglucose mutase, phosphoglucose isomerase, aldolase, triose-phosphate isomerase, phosphoglycerate mutase and enolase are close to equilibrium within the conducting elements of the phloem. Since the exuded sap contained negligible or undetectable activities of the enzymes, it is concluded, that the responsible proteins are bound, or sequesterd behind plasmodesmata, possibly in the companion cells. It is argued that sucrose mobilisation via a reversible reaction catalysed by sucrose synthase is particularily well suited to allow the rate of sucrose breakdown in the phloem to respond to changes in the metabolic requirement for ATP, and for UDPGlc during callose production. It is also calculated that the transport of nucleotides in the phloem sap implies that there must be a very considerable uptake or de-novo biosynthesis of these cofactors in the phloem.