Elsevier

Phytochemistry

Volume 61, Issue 1, September 2002, Pages 7-14
Phytochemistry

Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana

https://doi.org/10.1016/S0031-9422(02)00199-1Get rights and content

Abstract

In the Arabidopsis mutant irx3, truncation of the AtCesA7 gene encoding a xylem-specific cellulose synthase results in reduced cellulose synthesis in the affected xylem cells and collapse of mature xylem vessels. Here we describe spectroscopic experiments to determine whether any cellulose, normal or abnormal, remained in the walls of these cells and whether there were consequent effects on other cell-wall polysaccharides. Xylem cell walls from irx3 and its wild-type were prepared by anatomically specific isolation and were examined by solid-state NMR spectroscopy and FTIR microscopy. The affected cell walls of irx3 contained low levels of crystalline cellulose, probably associated with primary cell walls. There was no evidence that crystalline cellulose was replaced by less ordered glucans. From the molecular mobility of xylans and lignin it was deduced that these non-cellulosic polymers were cross-linked together in both irx3 and the wild-type. The disorder previously observed in the spatial pattern of non-cellulosic polymer deposition in the secondary walls of irx3 xylem could not be explained by any alteration in the structure or cross-linking of these polymers and may be attributed directly to the absence of cellulose microfibrils which, in the wild-type, scaffold the organisation of the other polymers into a coherent secondary cell wall.

Spectroscopic evidence was produced that the Arabidopsis mutant irx3 produces no detectable cellulose and that matrix polymers are unable to compensate for this loss.

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Introduction

Cellulose in higher plants is synthesised at the plasma membrane by a multi-enzyme complex comprising multiple catalytic subunits and other proteins (Delmer, 1999). The CesA gene family, with sequence similarity to genes encoding the catalytic polypeptide of bacterial cellulose synthase, probably has about 30 members in Arabidopsis (Taylor et al., 2000). Some of these may encode other polysaccharide synthases but about one-third of the Arabidopsis CesA genes for which sequence and functional evidence is available form a homologous group, within which evidence of involvement in cellulose synthesis is strong (Pear et al., 1996, Arioli et al., 1998, Taylor et al., 1999, Fagard et al., 2000a, Fagard et al., 2000b).

It seems reasonable that products of different CesA genes involved in cellulose synthesis are targeted to different cell types, with different types of cell wall (Fagard et al., 2000b). It has also been suggested that the synthesis of each microfibril requires the concerted action of two or more CesA gene products (Taylor et al., 2000). A number of Arabidopsis mutants at CesA loci have phenotypes that support these suppositions. They include rsw1 (Arioli et al., 1998) with a mutation in AtCesA1, and procuste (Fagard et al., 2000a) with a mutation in AtCesA6, both of which appear to affect primary cell walls in rapidly elongating tissues. In irx3, a mutant in AtCesA7, cellulose synthesis is defective in the thickened secondary cell walls of xylem and interfascicular tissues in the stem (Turner and Somerville, 1997). In the collapsed xylem phenotype which results, the walls of xylem vessels lose cohesion and, in mature plants, collapse inwards under hydrostatic pressure (Turner and Somerville, 1997). The cellulose content of mature irx3 stems is some 30% of that of the wild-type (Turner and Somerville, 1997, Taylor et al., 1999) when determined by the Updegraff method (Updegraff, 1969). This may of course include cellulose present in other tissues not affected by the irx3 mutation. It is therefore not certain whether cellulose is absent from the affected cell walls nor whether, if any cellulose is present, it is normal or altered in structure or supramolecular assembly.

Arioli et al. (1998) suggested that rsw1 accumulates atypical soluble glucans whose relationship to crystalline cellulose is not clear. They hypothesised that in rsw1 the catalytic subunit is functional but is not integrated into the synthetic complex in such a way as to permit normal assembly of the synthetic ‘rosette’ complexes or hence the assembly of normal microfibrils (Arioli et al., 1998). There is also evidence for release of relatively soluble glucan-containing fractions when the rosettes are disrupted by herbicide action (Peng et al., 2001). Recent studies (Nicol et al., 1998, Lane et al., 2001, Sato et al., 2001) have identified a putative endoglucanase that is required for cellulose synthesis and is deficient in kor plants, which also contain a soluble glucan (Lane et al., 2001). Soluble glucans would presumably not be determined as cellulose by the Updegraff procedure, which requires a degree of crystallinity to provide resistance to acid hydrolysis, and they might therefore be absent or present in irx3 on existing evidence (Turner and Somerville, 1997). Taken together these results invite the question whether or not a soluble glucan is present in all cellulose deficient plants, and how it might be synthesised.

In the study of the irx3 phenotype by Turner and Somerville (1997) xylan and lignin as well as cellulose contents were measured in whole mature stems. Since the visible phenotype was restricted to xylem and interfascicular tissues these measurements must have underestimated the extent of differences from the wild-type, to a degree that is uncertain. In a number of systems where cellulose synthesis in primary cell walls is impaired by mutation (Peng et al., 2000, Fagard et al., 2000a, His et al., 2001) or by herbicide action (Shedletzky et al., 1990), a less branched, more acidic pectin matrix is produced. This may assume part of the load-bearing function of the missing cellulose. It is not clear if there are any similar, compensatory alterations in the non-cellulosic polymers of the cellulose-depleted secondary cell walls in irx3. Lignin levels measured by Turner and Somerville (1997) were similar to the wild-type stems but electron-dense material, apparently lignin, was visible by TEM as diffuse deposits in the lumina of collapsed xylem vessels (Taylor et al., 1992). This raises several questions. Are the non-cellulosic polymers of irx3 altered in structure or only in the spatial pattern of their deposition? Does the deposition pattern result merely from absence of the spatial order imposed on wild-type wall architecture by cellulose? Would any structural alterations restore limited mechanical strength to cellulose-deficient cell walls?

Previous experiments on irx3 and other cellulose mutants have involved either chemical studies on whole plant organs or classical histology. To focus on the wall structure of the cells that express the irx3 mutation, and only these cells, we adopted two new approaches. Firstly we isolated cell walls specifically from the vascular ring of inflorescence stems of irx3. We examined these cell walls in solid state NMR experiments that are sensitive to the crystalline form of any cellulose present, and to the molecular rigidity of the non-cellulosic matrix. Secondly we examined the distribution and structure of crystalline cellulose in specific tissues of inflorescence stems by FTIR-microspectroscopy of deuterated thin sections.

Section snippets

Cell-wall isolation

Controlled mechanical disintegration followed by differential sieving is an established method for isolating anatomically specific cell-wall preparations from brassica stems (Wilson et al., 1988). This technique was applied to mature Arabidopsis stems after removal of the epidermis by freezing and peeling. The resulting cell walls were derived from the xylem and interfascicular tissues with a high degree of specificity, although they were not anatomically homogeneous as these tissues contain a

Residual cellulose in irx3 sclerenchyma cell walls

Isolating sclerenchyma cell walls allowed us to assess the effects of the irx3 mutation on the tissues where the collapsed xylem phenotype is expressed. Our results confirm the ultrastructural observations of Turner and Somerville (1997) that cellulose synthesis in the cell walls of the xylem and interfascicular tissues is severely disrupted by the mutation to AtCesA7. Little cellulose remained in the sclerenchyma cell-wall preparations from irx3.

The cellulose content of these cell-wall

Plant materials

Plants were grown at 22 °C in continuous light at light intensity of 120–150 μE m−2. Stem material was harvested from inflorescences 10–20 cm tall, frozen in liquid nitrogen and freeze dried overnight.

Isolation of sclerenchyma cell walls

Freeze-dried Arabidopsis stems were rehydrated, frozen and thawed. Epidermal and cortical tissues were removed using a scalpel blade. The residual stem material was homogenised (6×15 s) in a Waring blender in 2% Triton X100, and wet-sieved through stacked stainless steel square-mesh sieves to

Acknowledgements

This work was financially supported by BBSRC, the Royal Society, the EC and EPSRC.

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