Plant cells encase themselves within a complex polysaccharide wall, which constitutes the raw material that is used to manufacture textiles, paper, lumber, films, thickeners and other products. The plant cell wall is also the primary source of cellulose, the most abundant and useful biopolymer on the Earth. The cell wall not only strengthens the plant body, but also has key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. Recent discoveries have uncovered how plant cells synthesize wall polysaccharides, assemble them into a strong fibrous network and regulate wall expansion during cell growth.

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Growth of the plant cell wall

In most tree-breeding programs worldwide, increasing the trees' growth rates and stem volumes and shortening their rotation times are important aims. Such trees would yield more biomass per unit area. Here we show that overexpressing a key regulatory gene in the biosynthesis of the plant hormone gibberellin (GA) in hybrid aspen (Populus tremula x P. tremuloides) improves growth rate and biomass. In addition, these transgenic trees have more numerous and longer xylem fibers than unmodified wild-type (wt) plants. Long fibers are desirable in the production of strong paper, but it has not as yet proved possible to influence this trait by traditional breeding techniques. We also show that GA has an antagonistic effect on root initiation, as the transgenic lines showed poorer rooting than the control plants when potted in soil. However, the negative effect on rooting efficiencies in the initial establishment of young plantlets in the growth chamber did not significantly affect root growth at later stages.

Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length.

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.

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Unravelling cell wall formation in the woody dicot stem.

Numerous examples have been presented of enzyme activities, assayed in vitro, that appear relevant to the synthesis of structural polysaccharides, and to their assembly and subsequent degradation in the primary cell walls (PCWs) of higher plants. The accumulation of the corresponding mRNAs, and of the (immunologically recognized) proteins, has often also (or instead) been reported. However, the presence of these mRNAs, antigens and enzymic activities has rarely been shown to correspond to enzyme action in the living plant cell. In some cases, apparent enzymic action is observed in vivo for which no enzyme activity can be detected in in-vitro assays; the converse also occurs. Methods are reviewed by which reactions involving structural wall polysaccharides can be tracked in vivo. Special attention is given to xyloglucan endotransglucosylase (XET), one of the two enzymic activities exhibited in vitro by xyloglucan endotransglucosylase/hydrolase (XTH) proteins, because of its probable importance in the construction and restructuring of the PCW's major hemicellulose. Attention is also given to the possibility that some reactions observed in the PCW in vivo are not directly enzymic, possibly involving the action of hydroxyl radicals. It is concluded that some proposed wall enzymes, for example XTHs, do act in vivo, but that for other enzymes this is not proven. Contents I. Primary cell walls: composition, deposition and roles 642 II. Reactions that have been proposed to occur in primary cell walls 645 III. Tracking the careers of wall components in vivo: evidence for action of enzymes in the walls of living plant cells 656 IV. Evidence for the occurrence of nonenzymic polymer scission in vivo? 666 VI. Conclusion 667 References 667.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.2004.00980.x

Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells

Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.

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Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.

Xyloglucan Endotransglycosylases Have a Function during the Formation of Secondary Cell Walls of Vascular Tissues

Tension wood is a specialized tissue of deciduous trees that functions in bending woody stems to optimize their position in space. Tension wood fibers that develop on one side of the stem have an increased potency to shrink compared with fibers on the opposite side, thus creating a bending moment. It is believed that the gelatinous (G) cell wall layer containing almost pure cellulose of tension wood fibers is pivotal to their shrinking. By analyzing saccharide composition and linkage in isolated G-layers of poplar, we found that they contain some matrix components in addition to cellulose, of which xyloglucan is the most abundant. Xyloglucan, xyloglucan endo-transglycosylase (XET) activity and xyloglucan endo-transglycosylase/hydrolase (XTH) gene products were detected in developing G-layers by labeling using CCRC-M1 monoclonal antibody, in situ incorporation of XXXG-SR and the polyclonal antibody to poplar PttXET16-34, respectively, indicating that xyloglucan is incorporated into the G-layer during its development. Moreover, several XTH transcripts were altered and were generally up-regulated in developing tension wood compared with normal wood. In mature G-fibers, XTH gene products were detected in the G-layers while the XET activity was evident in the adjacent S 2 wall layer. We propose that XET activity is essential for G-fiber shrinking by repairing xyloglucan cross-links between G- and S 2 -layers and thus maintaining their contact. Surprisingly, XTH gene products and XET activity persisted in mature G-fibers for several years, suggesting that the enzyme functions after cell death repairing the cross-links as they are being broken during the shrinking process.

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Xyloglucan Endo-transglycosylase (XET) Functions in Gelatinous Layers of Tension Wood Fibers in Poplar—A Glimpse into the Mechanism of the Balancing Act of Trees

Summary Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC::uidA, rolC::iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1999.00536.x

Veronica Bourquin

Papers

Xyloglucan Endotransglycosylases Have a Function during the Formation of Secondary Cell Walls of Vascular Tissues

Xyloglucan Endo-transglycosylase (XET) Functions in Gelatinous Layers of Tension Wood Fibers in Poplar—A Glimpse into the Mechanism of the Balancing Act of Trees

Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues.