Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part I: temporal and spatial distribution of xylans

We investigated the microdistribution of xylans in different cell types of Arabidopsis stem using immunolocalization methods with LM10 and LM11 antibodies. Xylan labeling in xylary fibers (fibers) was initially detected at the cell corner of the S(1) layer and increased gradually during fiber maturation, showing correlation between xylan labeling and general secondary cell wall formation processes in fibers. Metaxylem vessels (vessels) showed earlier development of secondary cell walls than fibers, but revealed almost identical labeling patterns to fibers during maturation. No difference in labeling patterns and intensity was detected in the cell wall of fibers, vessels and protoxylem vessels (proto-vessels) between LM10 and LM11, indicating that vascular bundle cells may be chemically composed of a highly homogeneous xylan type. Interestingly, interfascicular fibers (If-fibers) showed different labeling patterns between the two antibodies and also between different developmental stages. LM10 showed no labeling in primary cell walls and intercellular layers of If-fibers at the S(1) formation stage, but some labeling was detected in middle lamella cell corner regions at the S(2) formation stage. In contrast, LM11 revealed uniform labeling across the If-fiber cell wall during all developmental stages. These results suggest that If-fibers have different xylan deposition processes and patterns from vascular bundle cells. The presence of xylan was also confirmed in parenchyma cells following pectinase treatment. Together our results indicate that there are temporal and spatial differences in xylan labeling between cell types in Arabidopsis stem. Differences in xylan labeling between Arabidopsis stem and poplar are also discussed.     

Spatial and temporal variability of xylan distribution in differentiating secondary xylem of hybrid aspen

Xylans occupy approximately one-third of the cell wall components in hardwoods and their chemical structures are well understood. However, the microdistribution of xylans (O-acetyl-4-O-methylglucuronoxylans, AcGXs) in the cell wall and their correlation with functional properties of cells in hardwood xylem is poorly understood. We demonstrate here the spatial and temporal distribution of xylans in secondary xylem cells of hybrid aspen using immunolocalization with LM10 and LM11 antibodies. Xylan labeling was detected earliest in fibers at the cell corner of the S? layer, and then later in vessels and ray cells respectively. Fibers showed a heterogeneous labeling pattern in the mature cell wall with stronger labeling of low substituted xylans (lsAcGXs) in the outer than inner cell wall. In contrast, vessels showed uniform labeling in the mature cell wall with stronger labeling of lsAcGXs than fibers. Xylan labeling in ray cells was detected much later than that in fibers and vessels, but was also detected at the beginning of secondary cell wall formation as in fibers and vessels with uniform labeling in the cell wall regardless of developmental stage. Interestingly, pit membranes including fiber-, vessel- and ray-vessel pits showed strong labeling of highly substituted xylans (hsAcGXs) during differentiation, although this labeling gradually disappeared during pit maturation. Together our observations indicate that there are temporal and spatial variations of xylan deposition and chemical structure of xylans between cells in aspen xylem. Differences in xylan localization between aspen (hardwood) and cedar (softwood) are also discussed.     

Cloning and expression analysis of a wood-associated xylosidase gene (PtaBXL1) in poplar tension wood

In stems of woody angiosperms responding to mechanical stress, imposed for instance by tilting the stem or formation of a branch, tension wood (TW) forms above the affected part, while anatomically distinct opposite wood (OW) forms below it. In poplar TW the S3 layer of the secondary walls is substituted by a “gelatinous layer” that is almost entirely composed of cellulose and has much lower hemicellulose contents than unstressed wood. However, changes in xylan contents (the predominant hemicelluloses), their interactions with other wall components and the mechanisms involved in TW formation have been little studied. Therefore, in the study reported here we determined the structure and distribution of xylans, cloned the genes encoding the xylan remodeling enzymes β-xylosidases (PtaBXLi), and examined their expression patterns during tension wood, normal wood and opposite wood xylogenesis in poplar. We confirm that poplar wood xylans are substituted solely by 4-O-methylglucuronic acid in both TW and OW. However, although glucuronoxylans are strongly represented in both primary and secondary layers of OW, no 4-O-methylGlcA xylan was found in G-layers of TW. Four full-length BXL cDNAs encoding putative β-xylosidases were cloned. One, PtaBXL1, for which xylosidase activity was confirmed by heterologous expression in Escherichia coli, exhibited a wood-specific expression pattern in TW. In conclusion, xylan as PtaBXL1, encoding β4-xylosidase activity, are down-regulated in TW.     

Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part II: Mannan deposition is regulated by phase of development and its patterns of temporal and spatial distribution differ between cell types

Microdistribution of mannans in Arabidopsis stem was examined using immunolocalization with mannan-specific monoclonal antibodies (LM21 and LM22). Mannan labeling in secondary xylem cells (except for protoxylem vessels) was initially detected in the cell wall during S₂ formation and increased gradually during development. Labeling in metaxylem vessels (vessels) was detected earlier than that in xylary fibers (fibers), but was much weaker than fibers. The S₁ layer of vessels and fibers showed much less labeling than the S₂ layer. Some strong labeling was also detected in pit membranes of vessel pits. Interfascicular fibers (If-fibers) showed more heterogeneous labeling patterns than fibers by LM21. Unlike fibers, If-fibers also revealed some strong labeling in the cell corner of the S₁ layer, indicating different mannan labeling patterns between If-fibers and fibers. Interestingly, protoxylem vessels (proto-vessels) showed strong labeling at the early stage of secondary xylem formation with more intense labeling in the outer- than inner cell wall even though fibers and vessels showed no or very low labeling at this stage. Labeling intensity of proto-vessels was also much stronger than vessels and stronger or slightly weaker than fibers by LM21 and LM22, respectively. Using pectinase and mild alkali treatment, the presence of mannans in parenchymatous cells was also confirmed. Together our observations indicate that there are temporal and spatial variations in mannan labeling between cell types in the secondary xylem of Arabidopsis stems. Some similar features of mannan labeling between Arabidopsis and poplar are also discussed.     

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides × P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofiuorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.     

The poplar glycosyltransferase GT47C is functionally conserved with Arabidopsis Fragile fiber8

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba x tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wild-type level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation.     

Hypergravity stimulus enhances primary xylem development and decreases mechanical properties of secondary cell walls in inflorescence stems of Arabidopsis thaliana

BACKGROUND AND AIMS: The xylem plays an important role in strengthening plant bodies. Past studies on xylem formation in tension woods in poplar and also in clinorotated Prunus tree stems lead to the suggestion that changes in the gravitational conditions affect morphology and mechanical properties of xylem vessels. The aim of this study was to examine effects of hypergravity stimulus on morphology and development of primary xylem vessels and on mechanical properties of isolated secondary wall preparations in inflorescence stems of arabidopsis. METHODS: Morphology of primary xylem was examined under a light microscope on cross-sections of inflorescence stems of arabidopsis plants, which had been grown for 3-5 d after exposure to hypergravity at 300 g for 24 h. Extensibility of secondary cell wall preparation, isolated from inflorescence stems by enzyme digestion of primary cell wall components (mainly composed of metaxylem elements), was examined. Plants were treated with gadolinium chloride, a blocker of mechanoreceptors, to test the involvement of mechanoreceptors in the responses to hypergravity. KEY RESULTS: Number of metaxylem elements per xylem, apparent thickness of the secondary thickenings, and cross-section area of metaxylem elements in inflorescence stems increased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on the increase both in the thickness of secondary thickenings and in the cross-section area of metaxylem elements, while it did not suppress the effect of hypergravity on the increase in the number of metaxylem elements. Extensibility of secondary cell wall preparation decreased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on cell wall extensibility. CONCLUSIONS: Hypergravity stimulus promotes metaxylem development and decreases extensibility of secondary cell walls, and mechanoreceptors were suggested to be involved in these processes.     

Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells

A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels.     

Comparative subcellular immunolocation of polypeptides associated with xylan and callose synthases in French bean (Phaseolus vulgaris) during secondary wall formation

The Golgi apparatus of plant cells is thought to be the main site of synthesis of cell wall matrix polysaccharides and the terminal glycosylation of glycoproteins. Much of this evidence still depends on earlier biochemical studies employing subcellular fractionation. However acquiring pure Golgi membranes is still difficult and the question of spatial organisation of glycosyl transferases can be addressed by immunolocation of the enzymes. An antibody to a xylan synthase-associated polypeptide from French bean, the enzyme which synthesises the core polysaccharide for secondary wall xylan, has been raised and shown to inhibit its activity. Xylan is deposited in secondary thickenings and the xylan synthase was only detected in appreciable amounts in developing xylem cells. The location within the Golgi stack was observed throughout the dictyosomes. Some enzyme subunits were also detected in post-Golgi vesicles. A second antibody to a non-catalytic M(r) 65000 subunit of beta 1,3- glucan (callose) synthase was used for a comparative study. Although the bulk of this enzyme has been detected in previous studies at plasmamembrane-wall interfaces in sieve plates and stressed tissue, a Golgi-location can be observed in root tip meristematic cells during cell plate formation. The enzyme was present throughout the stacks. Callose was also immunolocated in a similar manner to xylan in secondary walls and thickenings and in pits in developing xylem. In these cells, the callose synthase was detected at the surface of the growing thickenings and the plasmamembrane within the pits.     

Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees

The activity of a -(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000–100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min^–1 mg^–1 of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min^–1 cell^–1) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Cell wall modifications in Arabidopsis plants with altered alpha-L-arabinofuranosidase activity

Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.     

Gibberellin signaling

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.