Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose side chains block the degradation of the backbone, and a-xylosidase activity is necessary to remove them. Two Arabidopsis (Arabidopsis thaliana) mutant lines with insertions in the a-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of a-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in the accumulation of XXXG and XXLG in the liquid growth medium of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan b-galactosidase. In addition, the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the nonreducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments.

In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. In Arabidopsis (Arabidopsis thaliana), a significant proportion of xyloglucan side chains contain b-galactose linked to a-xylose at O2. In this work, we identified AtBGAL10 (At5g63810) as the gene responsible for the majority of b-galactosidase activity against xyloglucan. Xyloglucan from bgal10 insertional mutants was found to contain a large proportion of unusual subunits, such as GLG and GLLG. These subunits were not detected in a bgal10 xyl1 double mutant, deficient in both b-galactosidase and a-xylosidase. Xyloglucan from bgal10 xyl1 plants was enriched instead in XXLG/XLXG and XLLG subunits. In both cases, changes in xyloglucan composition were larger in the endoglucanase-accessible fraction. These results suggest that glycosidases acting on nonreducing ends digest large amounts of xyloglucan in wild-type plants, while plants deficient in any of these activities accumulate partly digested subunits. In both bgal10 and bgal10 xyl1, siliques and sepals were shorter, a phenotype that could be explained by an excess of nonreducing ends leading to a reinforced xyloglucan network. Additionally, AtBGAL10 expression was examined with a promoter-reporter construct. Expression was high in many cell types undergoing wall extension or remodeling, such as young stems, abscission zones, or developing vasculature, showing good correlation with a-xylosidase expression.

In several dicotyledonous species, NAC transcription factors act as master switches capable of turning on programmes of secondary cell-wall synthesis and cell death. This work used an oestradiol-inducible system to overexpress the NAC transcription factor BdSWN5 in the monocot model Brachypodium distachyon. This resulted in ectopic secondary cell-wall formation in both roots and shoots. Some of the genes upregulated in the process were a secondary cell-wall cellulose synthase (BdCESA4), a xylem-specific protease (BdXCP1) and an orthologue of AtMYB46 (BdMYB1). While activation of BdMYB1 may not be direct, this study showed that BdSWN5 is capable of transactivating the BdXCP1 promoter through two conserved binding sites. In the course of Brachypodium development, the BdXCP1 promoter was observed to be active in all types of differentiating tracheary elements. Together, these results suggest that Brachypodium SWNs can act as switches that turn on secondary cell-wall synthesis and programmed cell death.

A complete cDNA from Pinus pinaster Aiton, potentially coding for an a-xylosidase able to remove the xylose residue from xyloglucan oligosaccharides, has been cloned. Its sequence was homologous to previously published axylosidase genes from Arabidopsis and nasturtium. The protein also showed the two signature regions of family 31 of glycosyl hydrolases. The gene expression level was quantified by competitive RT-PCR, under different growth conditions, throughout seedling development, in different regions along the hypocotyls and in auxin-treated hypocotyl segments, and related with growth capacity and a-xylosidase activity. A role of a-xylosidase in regulating the level of xyloglucan oligosaccharides within the apoplast is proposed. The action of an a-xylosidase removing the xylose residue, would make possible the action of a b-glucosidase deblocking the xyloglucan oligosaccharide degradation and it could serve as a control point for the regulation of the apoplastic levels of xyloglucan oligosaccharides.

The hypothalamus exerts a predominantly inhibitory influence on prolactin secretion through dopamine. In addition, the expression of anterior pituitary hormone-gene products are regulated by intrapituitary growth factors. In particular, TGF-beta1 produced in the pituitary regulates lactotroph cell proliferation and prolactin gene-expression. This study characterized the regulation of in-vitro prolactin synthesis and secretion by TGF-beta1 using rat anterior pituitary cells in monolayer culture. Furthermore, we studied the interaction of TGF-beta1 with other signals involved in the neuroregulation of prolactin secretion, such as dopamine and TRH, as well as the importance of different signal transduction pathways in this response. TGF-beta1 inhibited prolactin secretion in a time- and concentration-dependent manner, with half-maximal inhibition occurring at the range of 15-30 pM. The inhibitory effect was observed after 4 h, being maximal after 4 days of exposure of the cells to the peptide. This inhibitory effect was mimicked by TGF-beta2 but not by inhibin, and was not influenced by oestrogens, being similar in male, normal female or oestradiol-treated rats. Prolonged pretreatment of the cells with TGF-beta1(4 days) did not modify GH or TSH secretion nor dopamine-induced inhibition of prolactin secretion, and blunted prolactin responses to TRH, Forskolin, But2-cAMP and to the calcium ionophore A23187. The effect observed after long-term treatment (24 h to 4 days) is essentially caused by a decrease in prolactin synthesis, since TGF-beta1 inhibited prolactin mRNA levels and de novo prolactin protein synthesis. However, in the short term (up to 12 h) TGF-beta1 inhibition of prolactin secretion was associated with an increase in intracellular prolactin content, dissecting a dual mechanism of action of TGF-beta1. The short-term TGF-beta1 effect did not modify Erk-2 phosphorylation, basal or TRH-induced increase in intracellular calcium concentration, but blunted basal and forskolin stimulated cAMP levels. But2-cAMP replacement did not revert the inhibition of prolactin secretion. However, pertussis toxin was able to recover a large percentage of TGF-beta1-induced inhibition of prolactin secretion. This study indicates that TGF-beta1 plays a crucial role as a modulator of lactotroph function, inhibiting prolactin biosynthesis after long-term treatment, as well as, after short-term exposure prolactin secretion at the level of the secretory process, through a mechanism pertussis toxin sensitive but independent of Erk-2 phosphorylation, calcium concentrations or intracellular cAMP.

Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams. Phenotypic, antigenic and genetic variability have been demonstrated, with three groups being established associated with host origin. In this work we analyze the variability of representative strains of these three groups, CECT 4600(T) and GR0202RD, isolated from Manila clam and carpet-shell clam, respectively, and HH6087, isolated from halibut, on the basis of the whole proteome analysis by 2D-PAGE and multilocus sequence analysis (MLSA). A quantitative analysis of the proteome match coefficient showed a similarity of 79% between the clam isolates, whereas fish isolate showed similarities lower than 70%. A preliminary mass spectrometry (MS) assay allowed the identification of 27 proteins including 50S ribosomal protein L9, riboflavin synthase β subunit, ribose-phosphate pyrophosphokinase and succinyl-CoA synthase α subunit. The MLSA approach gave similar results, showing a 99.4% similarity of the clam isolates, which was higher than that observed between the fish isolate and either clam strain (98.2%). The topology of the maximum parsimony tree, obtained from 2D-PAGE analysis, and the phylogenetic tree, constructed with the maximum likelihood algorithm from concatenated sequences of 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD), was very similar, confirming the closer relationship between the two clam isolates.

Xyloglucan is one of the main components of the primary cell wall in most species of plants.This protocol describes a method to analyze the composition of the enzyme-accessible and enzymeinaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks unsubstituted glucose residues in the backbone. The identities and relative amounts of released xyloglucan fragments are then determined using MALDI-TOF mass spectrometry.