Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. To date, to our knowledge, no direct evidence exists for the involvement of RGPs in a particular biochemical process. The Arabidopsis (Arabidopsis thaliana) genome contains five RGP genes out of which RGP1 and RGP2 share the highest sequence identity. We characterized the native expression pattern of Arabidopsis RGP1 and RGP2 and used reverse genetics to investigate their respective functions. Although both genes are ubiquitously expressed, the highest levels are observed in actively growing tissues and in mature pollen, in particular. RGPs showed cytoplasmic and transient association with Golgi. In addition, both proteins colocalized in the same compartments and coimmunoprecipitated from plant cell extracts. Single-gene disruptions did not show any obvious morphological defects under greenhouse conditions, whereas the double-insertion mutant could not be recovered. We present evidence that the double mutant is lethal and demonstrate the critical role of RGPs, particularly in pollen development. Detailed analysis demonstrated that mutant pollen development is associated with abnormally enlarged vacuoles and a poorly defined inner cell wall layer, which consequently results in disintegration of the pollen structure during pollen mitosis I. Taken together, our results indicate that RGP1 and RGP2 are required during microspore development and pollen mitosis, either affecting cell division and/or vacuolar integrity.     

A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.     

Glucosylation activity and complex formation of two classes of reversibly glycosylated polypeptides

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo- and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo- and heteromultimers in wheat endosperm.     

Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata

Maize and Arabidopsis thaliana class 1 reversibly glycosylated polypeptides (^C1RGPs) are plasmodesmata-associated proteins. Previously, overexpression of Arabidopsis ^C1RGP AtRGP2 in Nicotiana tabacum was shown to reduce intercellular transport of photoassimilate, resulting in stunted, chlorotic plants, and inhibition of local cell-to-cell spread of tobacco mosaic virus (TMV). Here, we used virus induced gene silencing to examine the effects of reduced levels of ^C1RGPs in Nicotiana benthamiana. Silenced plants show wild-type growth and development. Intercellular transport in silenced plants was probed using fluorescently labeled TMV and its movement protein, P30. P30 shows increased cell-to-cell movement and TMV exhibited accelerated systemic spread compared with control plants. These results support the hypothesis that ^C1RGPs act to regulate intercellular transport via plasmodesmata.     

Oligomerization of the reversibly glycosylated polypeptide: its role during rice plant development and in the regulation of self-glycosylation

A multigenic family of self-glycosylating proteins named reversibly glycosylated polypeptides, designated as RGPs, have been usually associated with carbohydrate metabolism, although they are an enigma both at the functional, as well as at the structural level. In this work, we used biochemical approaches to demonstrate that complex formation is linked to rice plant development, in which class 1 Oryza sativa RGP (OsRGP) would be involved in an early stage of growing plants, while class 2 OsRGP would be associated with a late stage linked to an active polysaccharide synthesis that occurs during the elongation of plant. Here, a further investigation of the complex formation of the Solanum tuberosum RGP (StRGP) was performed. Results showed that disulfide bonds are at least partially responsible for maintaining the oligomeric protein structure, so that the nonreduced StRGP protein showed an apparent higher molecular weight and a lower radioglycosylation of the monomer with respect to its reduced form. Hydrophobic cluster analysis and secondary structure prediction revealed that class 2 RGPs no longer maintained the Rossman fold described for class 1 RGP. A 3D structure of the StRGP protein resolved by homology modeling supports the possibility of intercatenary disulfide bridges formed by exposed cysteines residues C79, C303 and C251 and they are most probably involved in complex formation occurring into the cell cytoplasm.     

Complex formation regulates the glycosylation of the reversibly glycosylated polypeptide

Reversible glycosylated polypeptides (RGPs) are highly conserved plant-specific proteins, which can perform self-glycosylation. These proteins have been shown essential in plants yet its precise function remains unknown. In order to understand the function of this self-glycosylating polypeptide, it is important to establish what factors are involved in the regulation of the RGP activity. Here we show that incubation at high ionic strength produced a high self-glycosylation level and a high glycosylation reversibility of RGP from Solanum tuberosum L. In contrast, incubation at low ionic strength led to a low level of glycosylation and a low glycosylation reversibility of RGP. The incubation at low ionic strength favored the formation of high molecular weight RGP-containing forms, whereas incubation at high ionic strength produced active RGP with a molecular weight similar to the one expected for the monomer. Our data also showed that glycosylation of RGP, in its monomeric form, was highly reversible, whereas, a low reversibility of the protein glycosylation was observed when RGP was part of high molecular weight structures. In addition, glycosylation of RGP increased the occurrence of non-monomeric RGP-containing forms, suggesting that glycosylation may favor multimer formation. Finally, our results indicated that RGP from Arabidopsis thaliana and Pisum sativum are associated to golgi membranes, as part of protein complexes. A model for the regulation of the RGP activity and its binding to golgi membranes based on the glycosylation of the protein is proposed where the sugars linked to oligomeric form of RGP in the golgi may be transferred to acceptors involved in polysaccharide biosynthesis.     

Expression and genome-wide analysis of the xylogen-type gene family

In higher plants, many extracellular proteins are involved in developmental processes, including cell-cell signaling and cell wall construction. Xylogen is an extracellular arabinogalactan protein (AGP) isolated from Zinnia elegans xylogenic culture medium, which promotes xylem cell differentiation. Xylogen has a unique structure, containing a non-specific lipid transfer protein (nsLTP) domain and AGP domains. We searched for xylogen-type genes in the genomes of land plants, including Arabidopsis thaliana, to further our knowledge of xylogen-type genes as functional extracellular proteins in plants. We found that many xylogen-type genes, including 13 Arabidopsis genes, comprise a gene family in land plants, including Populus trichocarpa, Vitis vinifera, Lotus japonicus, Oryza sativa, Selaginella moellendorffii and Physcomitrella patens. The genes shared an N-terminal signal peptide sequence, a distinct nsLTP domain, one or more AGP domains and a glycosylphosphatidylinositol (GPI)-anchored sequence. We analyzed transgenic plants harboring promoter::GUS (β-glucuronidase) constructs to test expression of the 13 Arabidopsis xylogen-type genes, and detected a diversity of gene family members with related expression patterns. AtXYP2 was the best candidate as the Arabidopsis counterpart of the Zinnia xylogen gene. We observed two distinct expression patterns for several genes, with some anther specific and others preferentially expressed in the endodermis/pericycle. We conclude that xylogen-type genes, which may have diverse functions, form a novel chimeric AGP gene family with a distinct nsLTP domain.     

The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis

The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-D-Xyl to UDP-L-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-D-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-D-Xyl to UDP-L-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4-green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with >76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDP-L-Ara.     

Molecular characterization of a novel gene family encoding ACT domain repeat proteins in Arabidopsis

In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the "ACR" protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-beta-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.     

Regulation of self-glycosylation of reversibly glycosylated polypeptides from Solanum tuberosum

Reversibly glycosylated polypeptides (RGPs) belong to a family of self-glycosylating proteins believed to be involved in plant polysaccharide synthesis. The precise function of these enzymes remains to be elucidated. Our results showed that the RGP 38-kDa subunit is phosphorylated in potato extracts ( Solanum tuberosum L.). An increase in the self-glycosylation of Solanum tuberosum RGP (StRGP) 38-kDa subunit was observed after alkaline phosphatase (AP) treatment. Our results suggest that phosphorylation of StRGP appears to regulate its self-glycosylation. It was determined that when the StRGP reaction was carried out in the presence of UDP-[¹⁴C]Glc as the sugar donor and then 1 m M UDP was added in a chase-out experiment, radioactive UDP-Glc was obtained indicating that StRGP reaction seems to be reversible. The anomeric configuration of transferred sugars to StRGP protein was also studied.     

Members of the Arabidopsis dynamin-like gene family,ADL1, are essential for plant cytokinesis and polarized cell growth

Polarized membrane trafficking during plant cytokinesis and cell expansion are critical for plant morphogenesis, yet very little is known about the molecular mechanisms that guide this process. Dynamin and dynamin-related proteins are large GTP binding proteins that are involved in membrane trafficking. Here, we show that two functionally redundant members of the Arabidopsis dynamin-related protein family, ADL1A and ADL1E, are essential for polar cell expansion and cell plate biogenesis. adl1A-2 adl1E-1 double mutants show defects in cell plate assembly, cell wall formation, and plasma membrane recycling. Using a functional green fluorescent protein fusion protein, we show that the distribution of ADL1A is dynamic and that the protein is localized asymmetrically to the plasma membrane of newly formed and mature root cells. We propose that ADL1-mediated membrane recycling is essential for plasma membrane formation and maintenance in plants.