Debra Mohnen

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.

Pectin structure and biosynthesis.

Pectin is structurally and functionally the most complex polysaccharide in plant cell walls. Pectin has functions in plant growth, morphology, development, and plant defense and also serves as a gelling and stabilizing polymer in diverse food and specialty products and has positive effects on human health and multiple biomedical uses. Pectin is a family of galacturonic acid-rich polysaccharides including homogalacturonan, rhamnogalacturonan I, and the substituted galacturonans rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA). Pectin biosynthesis is estimated to require at least 67 transferases including glycosyl-, methyl-, and acetyltransferases. New developments in understanding pectin structure, function, and biosynthesis indicate that these polysaccharides have roles in both primary and secondary cell walls. Manipulation of pectin synthesis is expected to impact diverse plant agronomical properties including plant biomass characteristics important for biofuel production.

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides.

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up approximately 90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of alpha-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.

The Arabidopsis irregular xylem8 Mutant Is Deficient in Glucuronoxylan and Homogalacturonan, Which Are Essential for Secondary Cell Wall Integrity

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

Pectin and xylan are generally considered as separate cell wall glycan networks distinct from cell wall proteins. This work describes a cell wall proteoglycan with pectin and arabinoxylan covalently attached to an arabinogalactan protein, identifying a cross-linked matrix polysaccharide wall protein architecture with implications for wall structure, function, and synthesis. Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.

Evolving Views of Pectin Biosynthesis

Recent progress in the identification and characterization of pectin biosynthetic proteins and the discovery of pectin domain-containing proteoglycans are changing our view of how pectin, the most complex family of plant cell wall polysaccharides, is synthesized. The functional confirmation of four types of pectin biosynthetic glycosyltransferases, the identification of multiple putative pectin glycosyl- and methyltransferases, and the characteristics of the GAUT1:GAUT7 homogalacturonan biosynthetic complex with its novel mechanism for retaining catalytic subunits in the Golgi apparatus and its 12 putative interacting proteins are beginning to provide a framework for the pectin biosynthetic process. We propose two partially overlapping hypothetical and testable models for pectin synthesis: the consecutive glycosyltransferase model and the domain synthesis model.

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.

Hormonal regulation of β1,3-glucanase messenger RNA levels in cultured tobacco tissues

We describe the isolation of a cDNA clone of β1,3‐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 β1,3‐glucanase mRNA. The levels of β1,3‐glucanase and β1,3‐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 β1,3‐glucanase production at the level of the mRNA.

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex.

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

Evolution and Function of the Plant Cell Wall Synthesis-Related Glycosyltransferase Family 8

Carbohydrate-active enzyme glycosyltransferase family 8 (GT8) includes the plant galacturonosyltransferase1-related gene family of proven and putative α-galacturonosyltransferase (GAUT) and GAUT-like (GATL) genes. We computationally identified and investigated this family in 15 fully sequenced plant and green algal genomes and in the National Center for Biotechnology Information nonredundant protein database to determine the phylogenetic relatedness of the GAUTs and GATLs to other GT8 family members. The GT8 proteins fall into three well-delineated major classes. In addition to GAUTs and GATLs, known or predicted to be involved in plant cell wall biosynthesis, class I also includes a lower plant-specific GAUT and GATL-related (GATR) subfamily, two metazoan subfamilies, and proteins from other eukaryotes and cyanobacteria. Class II includes galactinol synthases and plant glycogenin-like starch initiation proteins that are not known to be directly involved in cell wall synthesis, as well as proteins from fungi, metazoans, viruses, and bacteria. Class III consists almost entirely of bacterial proteins that are lipooligo/polysaccharide α-galactosyltransferases and α-glucosyltransferases. Sequence motifs conserved across all GT8 subfamilies and those specific to plant cell wall-related GT8 subfamilies were identified and mapped onto a predicted GAUT1 protein structure. The tertiary structure prediction identified sequence motifs likely to represent key amino acids involved in catalysis, substrate binding, protein-protein interactions, and structural elements required for GAUT1 function. The results show that the GAUTs, GATLs, and GATRs have a different evolutionary origin than other plant GT8 genes, were likely acquired from an ancient cyanobacterium (Synechococcus) progenitor, and separate into unique subclades that may indicate functional specialization.