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.

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.

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.

CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana.

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell-wall stiffness, cell-to-cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)-like proteins and an unrelated plant-specific protein, cotton Golgi-related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss-of-function mutants and over-expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell-wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over-expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell-wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.

Evidence for the involvement of the Arabidopsis SEC24A in male transmission.

Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility.

CGR3: a Golgi-localized protein influencing homogalacturonan methylesterification.

Plant cell walls are complex structures that offer structural and mechanical support to plant cells and are ultimately responsible for plant architecture and form. Pectins are a large group of complex polysaccharides of the plant cell wall that are made in the Golgi and secreted to the wall. The methylesterification of pectins is believed to be an important factor for the dynamic properties of the cell wall. Here, we report on a protein of unknown function discovered using an extensive proteomics analysis of cotton Golgi. Through bioinformatic analyses, we identified the ortholog of such protein, here named cotton Golgi-related 3 (CGR3) in Arabidopsis and found that it shares conserved residues with S-adenosylmethionine methyltransferases. We established that CGR3 is localized at the Golgi apparatus and that the expression of the CGR3 gene is correlated with that of several cell wall biosynthetic genes, suggesting a possible role of the protein in pectin modifications. Consistent with this hypothesis, immunofluorescence microscopy with antibodies for homogalacturonan pectins (HG) indicated that the cell walls of cgr3 knockout mutants and plants overexpressing CGR3 are decreased and increased in HG methylesterification, respectively. Our results suggest that CGR3 plays a role in the methylesterification of homogalacturonan in Arabidopsis.

Phosphate starvation responses are mediated by sugar signaling in Arabidopsis.

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be down-stream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.