Knockout of eight hydroxyproline-O-galactosyltransferases cause multiple vegetative and reproductive growth defects.

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated hydroxyproline-rich cell wall proteins found throughout the plant kingdom. To date, eight Hydroxyproline-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to catalyze the addition of the first galactose sugar to Hyp residues in AGP protein cores. The generation and characterization of galt23456789 octuple mutants using CRISPR-Cas9 gene editing technology, provided strong reverse genetic evidence that AG glycans are essential for normal vegetative and reproductive growth, as these mutants demonstrated stunted growth, greatly delayed flowering and significant defects in floral organ development and morphogenesis. Compared to the lower seed set of galt25789 quintuple mutants being more so contributed by female gametophytic defects, dramatically low seed-set of octuple mutants was largely due to impaired male reproductive function, specifically due to shorter filaments, delayed anther dehiscence, and large decreases in pollen quantity and viability. Octuple mutant pollen had severely distorted reticulate exine, tectum patterning and intine thickness. Reduced amounts of galactose and arabinose in overall lower amounts of ß-Yariv precipitated AGPs illustrated how biological functions of AGPs are affected by abnormal glycosylation.

Type II arabinogalactans initiated by hydroxyproline-O-galactosyltransferases play important roles in pollen-pistil interactions.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions.

Hydroxyproline-O-Galactosyltransferases Synthesizing Type II Arabinogalactans Are Essential for Male Gametophytic Development in Arabidopsis.

In flowering plants, male reproductive function is determined by successful development and performance of stamens, pollen grains, and pollen tubes. Despite the crucial role of highly glycosylated arabinogalactan-proteins (AGPs) in male gamete formation, pollen grain, and pollen tube cell walls, the underlying mechanisms defining these functions of AGPs have remained elusive. Eight partially redundant Hyp-galactosyltransferases (named GALT2-GALT9) genes/enzymes are known to initiate Hyp-O-galactosylation for Hyp-arabinogalactan (AG) production in Arabidopsis thaliana. To assess the contributions of these Hyp-AGs to male reproductive function, we used a galt2galt5galt7galt8galt9 quintuple Hyp-GALT mutant for this study. Both anther size and pollen viability were compromised in the quintuple mutants. Defects in male gametogenesis were observed in later stages of maturing microspores after meiosis, accompanied by membrane blebbing and numerous lytic vacuoles. Cytological and ultramicroscopic observations revealed that pollen exine reticulate architecture and intine layer development were affected such that non-viable collapsed mature pollen grains were produced, which were devoid of cell content and nuclei, with virtually no intine. AGP immunolabeling demonstrated alterations in cell wall architecture of the anther, pollen grains, and pollen tube. Specifically, the LM2 monoclonal antibody (which recognized ß-GlcA epitopes on AGPs) showed a weak signal for the endothecium, microspores, and pollen tube apex. Pollen tube tips also displayed excessive callose deposition. Interestingly, expression patterns of pollen-specific AGPs, namely AGP6, AGP11, AGP23, and AGP40, were determined to be higher in the quintuple mutants. Taken together, our data illustrate the importance of type-II AGs in male reproductive function for successful fertilization.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Functional characterization of hydroxyproline-O-galactosyltransferases for Arabidopsis arabinogalactan-protein synthesis.

BACKGROUND: Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family. RESULTS: To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with ß-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to ß-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants. CONCLUSION: Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.

Three ß-Glucuronosyltransferase Genes Involved in Arabinogalactan Biosynthesis Function in Arabidopsis Growth and Development.

Arabinogalactan proteins (AGPs) contain arabinogalactan (AG) polysaccharides that are biologically relevant to plant growth processes. Here, the biochemical and physiological roles of three Golgi localized ß-glucuronosyltransferase genes (GLCAT14A, GLCAT14B and GLCAT14C) in Arabidopsis thaliana, responsible for the addition of glucuronic acid to AG chains, were further investigated using single, double and triple glcat14 mutant plants. These proteins were localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. Sugar analysis of AGP extracts from Arabidopsis stem, leaf and siliques showed a consistent reduction in glucuronic acid in glcat14 mutants relative to wild type, with concomitant effects resulting in tissue-specific alterations, especially in arabinose and galactose sugars. Although we observed defects in trichome branching in glca14a/b and glca14a/b/c mutants, scanning electron microscope analysis/energy dispersive microanalysis (SEM/EDX) showed no difference in the calcium content of trichomes in these mutants relative to wild type. Immunoblot analyses of the stem and leaf showed a reduction in AGPs as detected with the LM2 antibody in glcat14a/b and glcat14a/b/c mutants relative to wild type. The current work exemplifies the possibility of conducting structure-function assessment of cell wall biosynthetic genes to identify their physiological roles in plant growth and development.

Two ß-glucuronosyltransferases involved in the biosynthesis of type II arabinogalactans function in mucilage polysaccharide matrix organization in Arabidopsis thaliana.

BACKGROUND: Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of ß-glucuronosyltransferase genes/enzymes. RESULTS: Here, we have characterized two ß-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of ß-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size. CONCLUSIONS: These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis.

BACKGROUND: Arabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich proteins (HRGPs) that are heavily glycosylated (> 90%) with type II arabinogalactans (AGs). AGPs are implicated in various plant growth and development processes including cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, hormone signaling, male and female gametophyte development, and defense. To date, eight Hyp-O-galactosyltransferases (GALT2-6, HPGT1-3) have been identified; these enzymes are responsible for adding the first sugar, galactose, onto AGPs. Due to gene redundancy among the GALTs, single or double galt genetic knockout mutants are often not sufficient to fully reveal their biological functions. RESULTS: Here, we report the successful application of CRISPR-Cas9 gene editing/multiplexing technology to generate higher-order knockout mutants of five members of the GALT gene family (GALT2-6). AGPs analysis of higher-order galt mutants (galt2 galt5, galt3 galt4 galt6, and galt2 galt3 galt4 galt5 gal6) demonstrated significantly less glycosylated AGPs in rosette leaves, stems, and siliques compared to the corresponding wild-type organs. Monosaccharide composition analysis of AGPs isolated from rosette leaves revealed significant decreases in arabinose and galactose in all the higher-order galt mutants. Phenotypic analyses revealed that mutation of two or more GALT genes was able to overcome the growth inhibitory effect of ß-D-Gal-Yariv reagent, which specifically binds to ß-1,3-galactan backbones on AGPs. In addition, the galt2 galt3 galt4 galt5 gal6 mutant exhibited reduced overall growth, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Reciprocal crossing experiments demonstrated that galt2 galt3 galt4 galt5 gal6 mutants had defects in the female gametophyte which were responsible for reduced seed set. CONCLUSIONS: Our CRISPR/Cas9 gene editing/multiplexing approach provides a simpler and faster way to generate higher-order mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. Higher-order galt mutants produced and characterized in this study provide insight into the relationship between sugar decorations and the various biological functions attributed to AGPs in plants.

CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function.

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

Identification of Cis-Regulatory Sequences Controlling Pollen-Specific Expression of Hydroxyproline-Rich Glycoprotein Genes in Arabidopsis thaliana.

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall structural proteins that function in various aspects of plant growth and development, including pollen tube growth. We have previously characterized protein sequence signatures for three family members in the HRGP superfamily: the hyperglycosylated arabinogalactan-proteins (AGPs), the moderately glycosylated extensins (EXTs), and the lightly glycosylated proline-rich proteins (PRPs). However, the mechanism of pollen-specific HRGP gene expression remains unexplored. To this end, we developed an integrative analysis pipeline combining RNA-seq gene expression and promoter sequences to identify cis-regulatory motifs responsible for pollen-specific expression of HRGP genes in Arabidopsis thaliana. Specifically, we mined the public RNA-seq datasets and identified 13 pollen-specific HRGP genes. Ensemble motif discovery identified 15 conserved promoter elements between A.thaliana and A. lyrata. Motif scanning revealed two pollen related transcription factors: GATA12 and brassinosteroid (BR) signaling pathway regulator BZR1. Finally, we performed a regression analysis and demonstrated that the 15 motifs provided a good model of HRGP gene expression in pollen (R = 0.61). In conclusion, we performed the first integrative analysis of cis-regulatory motifs in pollen-specific HRGP genes, revealing important insights into transcriptional regulation in pollen tissue.

Systems identification and characterization of ß-glucuronosyltransferase genes involved in arabinogalactan-protein biosynthesis in plant genomes.

Utilizing plant biomass for bioethanol production requires an understanding of the molecular mechanisms involved in plant cell wall assembly. Arabinogalactan-proteins (AGPs) are glycoproteins that interact with other cell wall polymers to influence plant growth and developmental processes. Glucuronic acid, which is transferred to the AGP glycan by ß-glucuronosyltransferases (GLCATs), is the only acidic sugar in AGPs with the ability to bind calcium. We carried out a comprehensive genome-wide analysis of a putative GLCAT gene family involved in AGP biosynthesis by examining its sequence diversity, genetic architecture, phylogenetic and motif characteristics, selection pressure and gene expression in plants. We report the identification of 161 putative GLCAT genes distributed across 14 plant genomes and a widely conserved GLCAT catalytic domain. We discovered a phylogenetic clade shared between bryophytes and higher land plants of monocot grass and dicot lineages and identified positively selected sites that do not result in functional divergence of GLCATs. RNA-seq and microarray data analyses of the putative GLCAT genes revealed gene expression signatures that likely influence the assembly of plant cell wall polymers which is critical to the overall growth and development of edible and bioenergy crops.

Bioinformatic Identification of Plant Hydroxyproline-Rich Glycoproteins.

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: arabinogalactan-proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs). Hybrid and chimeric HRGPs also exist. A bioinformatic software program, BIO OHIO 2.0, was developed to expedite the genome-wide identification and classification of AGPs, EXTs, and PRPs based on characteristic HRGP motifs and biased amino acid compositions. This chapter explains the principles of identifying HRGPs and provides a stepwise tutorial for using the BIO OHIO 2.0 program with genomic/proteomic data. Here, as an example, the genome/proteome of the common bean (Phaseolus vulgaris) is analyzed using the BIO OHIO 2.0 program to identify and characterize its set of HRGPs.

Elucidating the roles of three ß-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis.

BACKGROUND: Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated. RESULTS: Three members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knock-out mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage. CONCLUSIONS: This research provides genetic evidence that GLCAT14A-C function in the transfer of GlcA to AGPs, which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation.

Three Decades of Advances in Arabinogalactan-Protein Biosynthesis.

Arabinogalactan-proteins (AGPs) are a large, complex, and highly diverse class of heavily glycosylated proteins that belong to the family of cell wall hydroxyproline-rich glycoproteins. Approximately 90% of the molecules consist of arabinogalactan polysaccharides, which are composed of arabinose and galactose as major sugars and minor sugars such as glucuronic acid, fucose, and rhamnose. About half of the AGP family members contain a glycosylphosphatidylinositol (GPI) lipid anchor, which allows for an association with the outer leaflet of the plasma membrane. The mysterious AGP family has captivated the attention of plant biologists for several decades. This diverse family of glycoproteins is widely distributed in the plant kingdom, including many algae, where they play fundamental roles in growth and development processes. The journey of AGP biosynthesis begins with the assembly of amino acids into peptide chains of proteins. An N-terminal signal peptide directs AGPs toward the endoplasmic reticulum, where proline hydroxylation occurs and a GPI anchor may be added. GPI-anchored AGPs, as well as unanchored AGPs, are then transferred to the Golgi apparatus, where extensive glycosylation occurs by the action of a variety glycosyltransferase enzymes. Following glycosylation, AGPs are transported by secretory vesicles to the cell wall or to the extracellular face of the plasma membrane (in the case of GPI-anchored AGPs). GPI-anchored proteins can be released from the plasma membrane into the cell wall by phospholipases. In this review, we present an overview of the accumulated knowledge on AGP biosynthesis over the past three decades. Particular emphasis is placed on the glycosylation of AGPs as the sugar moiety is essential to their function. Recent genetics and genomics approaches have significantly contributed to a broader knowledge of AGP biosynthesis. However, many questions remain to be elucidated in the decades ahead.

Bioinformatic Identification and Analysis of Hydroxyproline-Rich Glycoproteins in Populus trichocarpa.

BACKGROUND: Hydroxyproline-rich glycoproteins (HRGPs) constitute a plant cell wall protein superfamily that functions in diverse aspects of growth and development. This superfamily contains three members: the highly glycosylated arabinogalactan-proteins (AGPs), the moderately glycosylated extensins (EXTs), and the lightly glycosylated proline-rich proteins (PRPs). Chimeric and hybrid HRGPs, however, also exist. A bioinformatics approach is employed here to identify and classify AGPs, EXTs, PRPs, chimeric HRGPs, and hybrid HRGPs from the proteins predicted by the completed genome sequence of poplar (Populus trichocarpa). This bioinformatics approach is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs with a newly revised and improved BIO OHIO 2.0 program. Proteins detected by the program are subsequently analyzed to identify the following: 1) repeating amino acid sequences, 2) signal peptide sequences, 3) glycosylphosphatidylinositol lipid anchor addition sequences, and 4) similar HRGPs using the Basic Local Alignment Search Tool (BLAST). RESULTS: The program was used to identify and classify 271 HRGPs from poplar including 162 AGPs, 60 EXTs, and 49 PRPs, which are each divided into various classes. This is in contrast to a previous analysis of the Arabidopsis proteome which identified 162 HRGPs consisting of 85 AGPs, 59 EXTs, and 18 PRPs. Poplar was observed to have fewer classical EXTs, to have more fasciclin-like AGPs, plastocyanin AGPs and AG peptides, and to contain a novel class of PRPs referred to as the proline-rich peptides. CONCLUSIONS: The newly revised and improved BIO OHIO 2.0 bioinformatics program was used to identify and classify the inventory of HRGPs in poplar in order to facilitate and guide basic and applied research on plant cell walls. The newly identified poplar HRGPs can now be examined to determine their respective structural and functional roles, including their possible applications in the areas plant biofuel and natural products for medicinal or industrial uses. Additionally, other plants whose genomes are sequenced can now be examined in a similar way using this bioinformatics program which will provide insight to the evolution of the HRGP family in the plant kingdom.

Glycosylation of arabinogalactan-proteins essential for development in Arabidopsis.

Arabinogalactan-proteins (AGPs) are ubiquitous cell wall components present throughout the plant kingdom. They are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan (AG) polysaccharides to Hyp residues. Two small gene subfamilies within the CAZy GT31 family, referred to as Hyp-galactosyltransferases (Hyp-GALTs and HPGTs), encode enzymes that specifically add galactose to AGP protein backbones as revealed by heterologous expression of the genes coupled with an in vitro enzyme assay and by biochemical characterization of the genetic knock-out mutants. Biochemical analysis of galt2galt5 double and hpgt1hpgt2hpgt3 triple knockout mutants revealed significant reductions in both AGP-specific Hyp-GALT activity and ß-Gal-Yariv precipitable AGPs. Further analysis of these mutants demonstrated both overlapping and distinct pleiotropic growth and development phenotypes, indicating the important contributions of the carbohydrate moieties toward AGP function. Current research indicates that all 8 Hyp-GALT/HPGT genes encode enzymes that catalyze the initial step for AGP glycosylation and that AGP glycans play essential roles in plant growth and development.

Extensin and Arabinogalactan-Protein Biosynthesis: Glycosyltransferases, Research Challenges, and Biosensors.

Recent research, mostly in Arabidopsis thaliana, has led to the identification and characterization of the glycosyltransferases responsible for the biosynthesis of two of the most functionally important and abundant families of plant cell wall proteins, extensins, and arabinogalactan-proteins. Extensin glycosylation involves monogalactosylation of serine residues by O-α-serine galactosyltransferase and the addition of oligoarabinosides one to five arabinose units in length to contiguous hydroxyproline residues by a set of specific arabinosyltransferase enzymes, which includes hydroxyproline O-ß-arabinosyltransferases, ß-1,2-arabinosyltransferases, and at least one α-1,3-arabinosyltransferase. AGP glycosylation, however, is much more complex and involves the addition of large arabinogalactan polysaccharide chains to non-contiguous hydroxyproline residues. These arabinogalactan chains are composed of ß-1,3-galactan backbones decorated with ß-1,6-galactose side chains that are further modified with α-arabinose as well as other sugars, including ß-(methyl)glucuronic acid, α-rhamnose, and α-fucose. Specific sets of hydroxyproline O-ß-galactosyltransferases, ß-1,3-galactosyltransferases, ß-1,6-galactosyltransferases, α-arabinosyltransferases, ß-glucuronosyltransferases, α-rhamnosyltransferases, and α-fucosyltransferases are responsible for the synthesis of these complex structures. This mini-review summarizes the EXT and AGP glycosyltransferases identified and characterized to date along with corresponding genetic mutant data, which addresses the functional importance of EXT and AGP glycosylation. In one case, genetic mutant data indicate that the carbohydrate moiety of arabinogalactan-proteins may serve as an extracellular biosensor or signal for normal cellular growth. Finally, future research challenges with respect to understanding the function of these enzymes more completely and discovering and characterizing additional glycosyltransferases responsible for extensin and arabinogalactan-protein biosynthesis are also discussed.

Bioinformatic Identification and Analysis of Extensins in the Plant Kingdom.

Extensins (EXTs) are a family of plant cell wall hydroxyproline-rich glycoproteins (HRGPs) that are implicated to play important roles in plant growth, development, and defense. Structurally, EXTs are characterized by the repeated occurrence of serine (Ser) followed by three to five prolines (Pro) residues, which are hydroxylated as hydroxyproline (Hyp) and glycosylated. Some EXTs have Tyrosine (Tyr)-X-Tyr (where X can be any amino acid) motifs that are responsible for intramolecular or intermolecular cross-linkings. EXTs can be divided into several classes: classical EXTs, short EXTs, leucine-rich repeat extensins (LRXs), proline-rich extensin-like receptor kinases (PERKs), formin-homolog EXTs (FH EXTs), chimeric EXTs, and long chimeric EXTs. To guide future research on the EXTs and understand evolutionary history of EXTs in the plant kingdom, a bioinformatics study was conducted to identify and classify EXTs from 16 fully sequenced plant genomes, including Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Volvox carteri, Klebsormidium flaccidum, Physcomitrella patens, Selaginella moellendorffii, Pinus taeda, Picea abies, Brachypodium distachyon, Zea mays, Oryza sativa, Glycine max, Medicago truncatula, Brassica rapa, Solanum lycopersicum, and Solanum tuberosum, to supplement data previously obtained from Arabidopsis thaliana and Populus trichocarpa. A total of 758 EXTs were newly identified, including 87 classical EXTs, 97 short EXTs, 61 LRXs, 75 PERKs, 54 FH EXTs, 38 long chimeric EXTs, and 346 other chimeric EXTs. Several notable findings were made: (1) classical EXTs were likely derived after the terrestrialization of plants; (2) LRXs, PERKs, and FHs were derived earlier than classical EXTs; (3) monocots have few classical EXTs; (4) Eudicots have the greatest number of classical EXTs and Tyr-X-Tyr cross-linking motifs are predominantly in classical EXTs; (5) green algae have no classical EXTs but have a number of long chimeric EXTs that are absent in embryophytes. Furthermore, phylogenetic analysis was conducted of LRXs, PERKs and FH EXTs, which shed light on the evolution of three EXT classes.

Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis.

Fundamental processes that underpin plant growth and development depend crucially on the action and assembly of the cell wall, a dynamic structure that changes in response to both developmental and environmental cues. While much is known about cell wall structure and biosynthesis, much less is known about the functions of the individual wall components, particularly with respect to their potential roles in cellular signaling. Loss-of-function mutants of two arabinogalactan-protein (AGP)-specific galactosyltransferases namely, GALT2 and GALT5, confer pleiotropic growth and development phenotypes indicating the important contributions of carbohydrate moieties towards AGP function. Notably, galt2galt5 double mutants displayed impaired root growth and root tip swelling in response to salt, likely as a result of decreased cellulose synthesis. These mutants phenocopy a salt-overly sensitive mutant called sos5, which lacks a fasciclin-like AGP (SOS5/FLA4) as well as a fei1fei2 double mutant, which lacks two cell wall-associated leucine-rich repeat receptor-like kinases. Additionally, galt2gal5 as well as sos5 and fei2 showed reduced seed mucilage adherence. Quintuple galt2galt5sos5fei1fei2 mutants were produced and provided evidence that these genes act in a single, linear genetic pathway. Further genetic and biochemical analysis of the quintuple mutant demonstrated involvement of these genes with the interplay between cellulose biosynthesis and two plant growth regulators, ethylene and ABA, in modulating root cell wall integrity.

A small multigene hydroxyproline-O-galactosyltransferase family functions in arabinogalactan-protein glycosylation, growth and development in Arabidopsis.

BACKGROUND: Arabinogalactan-proteins (AGPs) are ubiquitous components of cell walls throughout the plant kingdom and are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan polysaccharides (AG) to Hyp residues. AGPs are implicated to function in various aspects of plant growth and development, but the functional contributions of AGP glycans remain to be elucidated. Hyp glycosylation is initiated by the action of a set of Hyp-O-galactosyltransferase (Hyp-O-GALT) enzymes that remain to be fully characterized. RESULTS: Three members of the GT31 family (GALT3-At3g06440, GALT4-At1g27120, and GALT6-At5g62620) were identified as Hyp-O-GALT genes by heterologous expression in tobacco leaf epidermal cells and examined along with two previously characterized Hyp-O-GALT genes, GALT2 and GALT5. Transcript profiling by real-time PCR of these five Hyp-O-GALTs revealed overlapping but distinct expression patterns. Transiently expressed GALT3, GALT4 and GALT6 fluorescent protein fusions were localized within Golgi vesicles. Biochemical analysis of knock-out mutants for the five Hyp-O-GALT genes revealed significant reductions in both AGP-specific Hyp-O-GALT activity and ß-Gal-Yariv precipitable AGPs. Further phenotypic analysis of these mutants demonstrated reduced root hair growth, reduced seed coat mucilage, reduced seed set, and accelerated leaf senescence. The mutants also displayed several conditional phenotypes, including impaired root growth, and defective anisotropic growth of root tips under salt stress, as well as less sensitivity to the growth inhibitory effects of ß-Gal-Yariv reagent in roots and pollen tubes. CONCLUSIONS: This study provides evidence that all five Hyp-O-GALT genes encode enzymes that catalyze the initial steps of AGP galactosylation and that AGP glycans play essential roles in both vegetative and reproductive plant growth.