Cell wall proteome analysis of Arabidopsis thaliana mature stems

Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido‐reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido‐reductases.     

Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides

A high-throughput method is described by which Arabidopsis thaliana stems can be screened for variation in cell wall composition after hydrolysis with Driselase or trifluoroacetic acid (TFA). Driselase, a mixture of fungal enzymes, hydrolyses cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides); TFA hydrolyses the matrix polysaccharides, but not cellulose, to monosaccharides. Two different wild-type ecotypes, Columbia and Wassilewskija, showed only minor differences in wall carbohydrate composition. A small number of T-DNA-tagged populations that were screened contained individuals in which the proportion of cellulose, xyloglucan or xylan differed quantitatively from the wild-type. Differences from the wild-type were also observed in the susceptibility of the hemicelluloses to hydrolysis by Driselase, probably reflecting differences in wall architecture.     

Plant glycoside hydrolases involved in cell wall polysaccharide degradation.

The cell wall plays a key role in controlling the size and shape of the plant cell during plant development and in the interactions of the plant with its environment. The cell wall structure is complex and contains various components such as polysaccharides, lignin and proteins whose composition and concentration change during plant development and growth. Many studies have revealed changes in cell walls which occur during cell division, expansion, and differentiation and in response to environmental stresses; i.e. pathogens or mechanical stress. Although many proteins and enzymes are necessary for the control of cell wall organization, little information is available concerning them. An important advance was made recently concerning cell wall organization as plant enzymes that belong to the superfamily of glycoside hydrolases and transglycosidases were identified and characterized; these enzymes are involved in the degradation of cell wall polysaccharides. Glycoside hydrolases have been characterized using molecular, genetic and biochemical approaches. Many genes encoding these enzymes have been identified and functional analysis of some of them has been performed. This review summarizes our current knowledge about plant glycoside hydrolases that participate in the degradation and reorganisation of cell wall polysaccharides in plants focussing particularly on those from Arabidopsis thaliana.     

Comprehensive analysis of cytosolic Nudix hydrolases in Arabidopsis thaliana

Nudix hydrolases are a family of proteins that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Twenty-four genes of the Nudix hydrolase homologues (AtNUDTs) with predicted localizations in the cytosol, chloroplasts, and mitochondria exist in Arabidopsis thaliana. Here, we demonstrated the comprehensive analysis of nine types of cytosolic AtNUDT proteins (AtNUDT1, -2, -4, -5, -6, -7, -9, -10, and -11). The recombinant proteins of AtNUDT2, -6, -7, and -10 showed both ADP-ribose and NADH pyrophosphatase activities with significantly high affinities compared with those of animal and yeast enzymes. The expression of each AtNUDT is individually regulated in different tissues. These findings suggest that most cytosolic AtNUDTs may substantially function in the sanitization of potentially hazardous ADP-ribose and the regulation of the cellular NADH/NAD(+) ratio in plant cells. On the other hand, the AtNUDT1 protein had the ability to hydrolyze 8-oxo-dGTP with a K(m) value of 6.8 mum and completely suppress the increased frequency of spontaneous mutations in the Escherichia coli mutT(-) strain, indicating that AtNUDT1 is a functional homologue of E. coli MutT in A. thaliana and is involved in the prevention of spontaneous mutation. The results obtained here suggest that the plant Nudix family has evolved in a specific manner that differs from that of yeast and humans.     

Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis

Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.     

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.     

Assessment of cell wall porosity in Arabidopsis thaliana by NMR spectroscopy

The impact of homogalacturonans deficiency on the cell wall porosity of Arabidopsis thaliana QUA1 mutant was investigated using NMR measurements of protons mobility interpreted in terms of pore size variations at nanometer and micrometer scales. Isolation and purification of wild type and mutant stems and calli cell walls permitted to exacerbate the putative impact of the mutation on cellulose-hemicelluloses assembly in highly and poorly organised cell walls, respectively. NMR relaxation measurements of water and exchangeable biopolymer protons and self-diffusion processes of polyethylene glycol in walls informed about the porosity network density and heterogeneity. The role of pectins and proteins as well as the growth status of the cells on the wall porosity regulation are discussed.     

Mutants of Arabidopsis thaliana with altered cell wall polysaccharide composition

To analyze the synthesis, structure and function of the plant cell wall by a genetic approach, 5200 chemically mutagenized Arabidopsis plants were screened for changes in the monosaccharide composition of hydrolyzed cell wall material by gas chromatography of alditol acetates. This screening procedure identified 23 mutant lines representing 11 different loci designated mur1 to mur11 . The mur lines fall into essentially three groups: (1) complete absence of a monosaccharide, (2) significant reduction in the amount of a single monosaccharide, and (3) complex alterations in the relative amounts of several monosaccharides. All mutants in the first category represent alleles of the mur1 locus, and are deficient in the de novo synthesis of fucose. Mutants with reductions in a single monosaccharide have been identified for fucose ( mur2, mur3 ), arabinose ( mur4, mur5, mur6, mur7 ), and rhamnose ( mur8 ). Mutants with complex changes in monosaccharide composition are represented by the mur9 , mur10 and mur11 loci. Most of the mutant lines did not show obvious morphological or physiological alterations; however, lines mur1, mur9 and mur10 co-segregated with reduced vigor or dwarfism of the plants. These results demonstrate the feasibility of identifying plants with altered cell wall compositions via a biochemical screening procedure. The availability of these mutants provides novel opportunities to study the functions of cell wall polysaccharides, gain insight into the biosynthesis of cell wall material, and clone cell wall-related genes.     

Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil

Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well-defined localization of cell wall epitopes with highly esterified homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding (1) the ultrastructure of the pollen tube cell wall and (2) the immunolocalization of homogalacturonan and arabinan epitopes in 16-h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.Key words: arabinan, cell adhesion, cell wall, homogalacturonan, pistil, pollen tube growth, transmitting tractFertilization of flowering plants requires the delivery of the two sperm cells, carried by the fast growing tip-polarized pollen tube, to the egg cell. At every stage of the pollen tube development within the stigma, style and ovary, pollen tubes are guided to the ovules via multiple signals that need to pass through the cell wall of the pollen tube to reach their targets.^1–6The analysis of Arabidopsis pollen tube cell wall has recently been reported.⁷ Results showed a well-defined localization of cell wall epitopes with highly methylesterified homogalacturonan (HG) and arabinogalactan-protein (AGP) mainly in the tip region, weakly methylesterified HG back from the tip and xyloglucan and arabinan all along the tube. In addition, according to the one letter nomenclature of xyloglucan,⁸ the main motif of Arabidopsis pollen tube xyloglucan was XXFG harboring one O-acetyl group. In order to bring new information regarding the possible interaction between the pollen tubes and the female tissues, the ultrastructural organization of the pollen tube cell wall, the cytological staining and immunolocalization of the cell wall epitopes of the pistil and especially the transmitting tract (TT), a specialized tissue where pollen tubes grow, were carried out.     

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.     

Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.     

Measurement of diffusion within the cell wall in living roots of Arabidopsis thaliana

To quantify the diffusion constant of small molecules in the plant cell wall, fluorescence from carboxyfluorescein (CF) in the intact roots of Arabidopsis thaliana was recorded. Roots were immersed in a solution of the fluorescent dye and viewed through a confocal fluorescence microscope. These roots are sufficiently transparent that much of the apoplast can be imaged. The diffusion coefficient, D(cw), of CF in the cell wall was probed using two protocols: fluorescence recovery after photobleaching and fluorescence loss following perfusion with dye-free solution. Diffusion coefficients were obtained from the kinetics of the fluorescent transients and modelling apoplast geometry. Apoplastic diffusion constants varied spatially in the root. In the elongation zone and mature cortex, D(cw)=(3.2+/-1.4)x10(-11) m(2) s(-1), whereas in mature epidermis, D(cw)=(2.5+/-0.7)x10(-12) m(2) s(-1), at least an order of magnitude lower. Relative to the diffusion coefficient of CF in water, these represent reductions by approximately 1/15 and 1/195, respectively. The low value for mature epidermis is correlated with a suberin-like permeability barrier that was detected with either autofluorescence or berberine staining. This study provides a quantitative estimate of the permeability of plant cell walls to small organic acids-a class of compounds that includes auxin and other plant hormones. These measurements constrain models of solute transport, and are important for quantitative models of hormone signalling during plant growth and development.     

Arabidopsis thaliana myosin XIK is involved in root hair as well as trichome morphogenesis on stems and leaves

Myosins form a large superfamily of molecular motors that move along actin filaments. The functions of myosins in plant cells are thought to be related to various processes: cell division, movement of mitochondria and chloroplasts, cytoplasmic streaming, rearrangement of transvacuolar strands, and statolith positioning. Class VIII and XI myosins are represented in the Arabidopsis thaliana genome by 4 and 13 potential genes, respectively. The roles of individual class XI myosins and their cellular targets in A. thaliana are still unclear. In this work we implemented a reverse genetic approach to analyse the loss-of-function mutants of XIK, a representative of class XI myosins in A. thaliana. Three different T-DNA insertion mutants in the myosin XIK gene showed similar phenotypes: impaired growth of root hair cells, twisted shape of stem trichomes, and irregular size, branch positioning, and branch expansion of leaf trichomes. Morphometric analysis of mutant seedlings showed that the average length of root hairs was reduced up to 50% in comparison with wild-type root hairs, suggesting an involvement of the class XI myosin XIK in tip growth. On leaves, the proportion of trichomes with short branches was doubleed in mutant plants, and the mutant trichomes possessed a mildly twisted shape. Therefore, we concluded that myosin XIK is involved also in the elongation of stalks and branches of trichomes.     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.