The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi.

Alpha-1,4-galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1-->4-linked alpha-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.

Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.

Pectin is a family of complex polysaccharides present in all plant primary cell walls. The complicated structure of the pectic polysaccharides, and the retention by plants of the large number of genes required to synthesize pectin, suggests that pectins have multiple functions in plant growth and development. In this review we summarize the current level of understanding of pectin primary and tertiary structure, and describe new methods that may be useful to study localized pectin structure in the plant cell wall. We also discuss progress in our understanding of how pectin is biosynthesized and review the biological activities and possible modes of action of pectic oligosaccharides referred to as oligogalacturonides. We present our view of critical questions regarding pectin structure, biosynthesis, and function that need to be addressed in the coming decade. As the plant community works towards understanding the functions of the tens of thousands of genes expressed by plants, a large number of those genes are likely to be involved in the synthesis, turnover, biological activity, and restructuring of pectin. A combination of genetic, molecular, biochemical and chemical approaches will be necessary to fully understand the function and biosynthesis of pectin.

Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells.

The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.

Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells.

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi.

Enzymatic synthesis and purification of [(3)H]uridine diphosphate galacturonic acid for use in studying Golgi-localized transporters.

Uridine 5'-diphosphate galacturonic acid (UDP-GalA) is a substrate for the galacturonosyltransferases that synthesize the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Pectin synthesis occurs in the Golgi and it is hypothesized that UDP-GalA is transported into the lumen of the Golgi by membrane-localized transporters. To study the transport and metabolism of UDP-GalA in the Golgi, UDP-GalA labeled in the uridine moiety is required. Here we present a high-yield method for the synthesis of [(3)H]UDP-GalA from [(3)H]UTP and Glc-1-P by sequential reactions catalyzed by UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and UDP-GlcA-4-epimerase and the separation of the reaction products over a Dionex CarboPac PA1 anion-exchange column using high-performance anion-exchange chromatography (HPAEC). Approximately half of the [(3)H]UTP was converted into [(3)H]UDP-GalA and the remaining 50% was recovered as [(3)H]UDP-GlcA. Both products were purified and the identity of the [(3)H]UDP-GalA was confirmed by its conversion into [(3)H]UDP-GlcA by UDP-GlcA-4-epimerase. The enzymatic synthesis of diverse nucleotide sugars radiolabeled in the nucleotide by the use of nucleotide-converting enzymes, combined with the high-resolution separation of the nucleotide sugars and their purification by HPAEC, can provide unique substrates required for the study of diverse nucleotide sugar transporters.

Biological activity of reducing-end-derivatized oligogalacturonides in tobacco tissue cultures.

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H2O2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.

A method for biotin labeling of biologically active oligogalacturonides using a chemically stable hydrazide linkage.

Oligogalacturonides (oligomers of alpha-1,4-D-galacturonic acid) with degrees of polymerization (DP) between 8 and 16 were labeled with biotin using a rapid and simple two-reaction protocol that yields a stable oligogalacturonide derivative. In the first reaction biotin-x-hydrazide was coupled to the anomeric carbon of the reducing galacturonic acid residue by a hydrazone linkage. Carbohydrate-hydrazone linkages such as these have been widely used to label a variety of biomolecules. However, we show herein that the oligogalacturonide-hydrazone linkage is hydrolyzed in water. In the second reaction the hydrazone linkage was reduced with sodium cyanoborohydride to form a stable hydrazide. The stability of hydrazide-linked oligogalacturonides was confirmed using high-performance anion-exchange chromatography (HPAEC). The biotin and uronic acid content of the HPAEC fractions was determined using quantitative colorimetric microplate assays. Electrospray mass spectrometry and 1H NMR spectroscopy were used to confirm the structure of the HPAEC-purified biotin-derivatized oligogalacturonides. Biotin-derivatized oligogalacturonides will be useful in studies of the biological functions of oligogalacturonides.

Cell-Free Synthesis of Pectin (Identification and Partial Characterization of Polygalacturonate 4-[alpha]-Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures).

Polygalacturonate 4-[alpha]-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell-suspension cultures. Incubation of UDP-[14C]galacturonic acid with tobacco membranes results in a time-dependent incorporation of [14C]galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The optimal synthesis of product occurs at a pH of 7.8, 25 to 30[deg]C, an apparent Km for UDP-D-galacturonic acid of approximately 8.9 [mu]M, and a Vmax of approximately 150 pmol min-1 mg-1 protein. The product was characterized by scintillation counting, thin-layer chromatography, high-performance anion-exchange chromatography, and gel-filtration chromatography in combination with enzymatic and chemical treatments. The intact product has a molecular mass of approximately 105,000 D based on dextran molecular standards. The product was treated with base to hydrolyze ester linkages (e.g. methyl esters), digested with a homogeneous endopolygalacturonase (EPGase), or base and EPGase treated. Base and EPGase treatment results in cleavage of 34 to 89% of 14C-labeled product into components that co-chromatograph with mono-, di-, and trigalacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked [alpha]-D-galactosyluronic acid residues. Optimal EPGase fragmentation of the product requires base treatment prior to enzymatic digestion, suggesting that 45 to 67% of the galacturonic acid residues in the synthesized homogalacturonan are esterified. At least 40% of the base-sensitive linkages were shown to be methyl esters by comparing the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase.

Enzymatic synthesis and purification of uridine diphosphate [14C]galacturonic acid: a substrate for pectin biosynthesis.

Pectins are complex polysaccharides that contain 1,4-linked alpha-D-galactosyluronic acid residues found in the primary wall of all higher plant cells. The pectic polysaccharides play critical roles in cell wall structure and in plant growth and development. As a first step in studying pectin biosynthesis a method was developed to routinely generate and purify UDP-[U-14C]galacturonic acid (UDP-[14C]GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis. UDP-[14C]GalA was enzymatically synthesized by 4-epimerization of commercially available UDP-[U-14C]glucuronic acid (UDP-[14C]GlcA) using a particulate preparation from radish roots. The resulting mixture of UDP-[14C]GalA and UDP-[14C]GlcA was separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 anion-exchange column. The UDP-sugars were detected by their absorbance at 262 nm or by pulsed amperometric detection following postcolumn addition of NaOH. The yield of UDP-[14C]GalA obtained using this procedure was 16% of the starting UDP-[14C]GlcA. Establishment of a reliable method to synthesize and purify UDP-[14C]GalA will facilitate the identification and purification of the galacturonosyltransferase(s) involved in pectin biosynthesis.

Cell wall carbohydrates as signals in plants.

Plant and fungal cells are surrounded by a cell wall rich in diverse polysaccharides and proteins. It has become apparent in recent years that the carbohydrates in the cell wall function not only to maintain cell shape and integrity, but also may serve as signals in plants. This review summarizes the evidence that biologically-active oligosaccharides (oligosaccharins) released from plant or microbial cell walls can serve as signals to regulate plant defense and plant growth and development. The oligosaccharins discussed include the fungal-derived hepta-beta-glucoside and the plant cell wall-derived oligogalacturonides and xyloglucans. Possible mechanisms by which oligosaccharins may exert their effects on plant cells are discussed.

The control of root, vegetative shoot and flower morphogenesis in tobacco thin cell-layer explants (TCLs).

Thin cell-layer explants (TCLs) have been proposed as favorable tissues for the study of root, vegetative shoot and flower formation. We tested the effects of pH, light quality, light quantity, and IBA and kinetin concentrations on the morphogenesis of TCLs cultured individually on a liquid medium. Alterations of the amounts of exogenously supplied IBA and kinetin were sufficient to induce the formation of roots, vegetative shoots and flowers on TCLs cultured on otherwise identical media. The type and number of organs formed were sensitive to the intensity of light (55, 75, 100 and 120 muEinsteins m-2 sec-1) under which TCLs were grown. Evidence was obtained that the effects of light on TCL morphogenesis were associated with photochemical degradation of IBA in the medium. Evaluation of the organogenesis that occurred in TCLs cultured on a medium containing a range of IBA and kinetin concentrations showed that the number and type of organs formed, and overall growth, were dependent upon the initial concentrations of auxin and cytokinin. We have developed the TCL culture system into a sensitive and reproducible bioassay for the study of morphogenesis. The advantages of using the TCL morphogenesis bioassay for the identification and study of molecules (e.g. cell wall oligosaccharides) that may regulate morphogenesis are discussed.

Pectic Cell Wall Fragments Regulate Tobacco Thin-Cell-Layer Explant Morphogenesis.

Pectic fragments of cell wall polysaccharides, released from the walls of suspension-cultured sycamore cells by treatment with endopolygalacturonase, were tested for morphogenesis-regulating activity in a modified tobacco thin-cell-layer explant (TCL) bioassay (D. Mohnen, S. Eberhard, V. Marfa, N. Doubrava, P. Toubart, D. J. Gollin, T.A. Gruber, W. Nuri, P. Albersheim, and A. Darvill, manuscript submitted). The pectic fragments inhibited the formation of roots on TCLs grown on a root-inducing medium containing 15 micromolar indole-3-butyric acid and 0.5 micromolar kinetin. Addition of the pectic fragments to a root-inducing medium containing 7 micromolar indole-3-butyric acid and 0.15 micromolar kinetin caused roots to form on the basal end of TCLs. TCLs cultured on this medium in the absence of added pectic fragments formed roots along their entire length. The pectic fragments induced polar tissue enlargement and the formation of flowers on TCLs cultured on transition medium. The flower-inducing activity was stable to heat treatment and proteolytic digestion. Pectic fragments isolated from the walls of suspension-cultured tobacco cells were as effective as those from the walls of sycamore cells in inducing de novo flower formation in the TCLs. These results support the hypothesis that oligosaccharins from plant cell walls regulate morphogenesis.

Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin.

Two endochitinases (EC 3.2.1.14) of M(r) values of approximately 34,000 and approximately 32,000 have been purified from cultured tissues of Nicotiana tabacum cv. Havana 425. The chitinase content of cloned tobacco pith tissues subcultured on hormone-free medium increases by approximately 5-fold to 8% of the soluble protein over a 7-day period. This induction is inhibited >90% by addition of combinations of the plant hormones auxin and cytokinin to the culture medium. Chitinase is also developmentally regulated in the intact plant. Not detectable in leaves near the top of the plant, it is 1-4% of the soluble protein in roots and lower leaves. A cDNA clone of tobacco chitinase was isolated containing a single, large open reading frame of 310 amino acids that includes the complete amino acid sequence of the mature enzyme. Chitinase and chitinase mRNA measured by RNA blot analysis show similar patterns of regulation indicating that chitinase accumulation is controlled, at least in part, at the mRNA level. The patterns were also similar to those obtained with glucan endo-1,3-beta-glucosidase (EC 3.2.1.39) suggesting that the two enzymes are coordinately regulated.

Hormonal regulation of beta1,3-glucanase messenger RNA levels in cultured tobacco tissues.

We describe the isolation of a cDNA clone of beta1,3-glucanase mRNA from Nicotiana tabacum L. cv. ;Havana 425' and its use to measure the kinetics of mRNA accumulation in cultured tobacco tissues treated with the plant hormones auxin and cytokinin. Northern blot analysis showed that the tissues contain a single 1.6 kb-sized beta1,3-glucanase mRNA. The levels of beta1,3-glucanase and beta1,3-glucanase mRNA increase by up to seven- and 20-fold, respectively, over a 7-day period in tissues subcultured on hormone-free medium and medium containing auxin or cytokinin added separately. Over the same interval of time, the content of both the enzyme and its mRNA remains at a constant low level in tissues subcultured on medium containing both auxin and cytokinin. The results show that auxin and cytokinin block beta1,3-glucanase production at the level of the mRNA.