Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana
Spectroscopic evidence was produced that the Arabidopsis mutant irx3 produces no detectable cellulose and that matrix polymers are unable to compensate for this loss.
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.
References (34)
- et al.
Investigation of macromolecular orientation in dry and hydrated walls of single onion epidermal cells by FTIR microspectroscopy
J. Mol. Struct.
(1997) - et al.
Alfalfa stem tissuescell-wall development and lignification
Ann. Bot.
(1998) - et al.
Cell wall mutants
Plant Physiol. Biochem.
(2000) - et al.
Methyl-esterification, de-esterification and gelation of pectins in the primary cell wall
Solid-state 13C-n.m.r. spectra of Vigna primary cell walls and their polysaccharide components
Carbohydr. Res.
(1990)- et al.
Cell wall biophysicsconcepts and methodology
Plant Biochem. Physiol.
(2000) - et al.
A CP/MAS C-13 NMR investigation of molecular ordering in celluloses
Carbohydr. Res.
(1997) Semimicro detemination of cellulose in biological materials
Anal. Biochem.
(1969)- et al.
Lignified and non-lignified cell walls from kale
Plant Sci.
(1988) - et al.
Molecular analysis of cellulose synthesis in Arabidopsis
Science
(1998)
Cellulose biosynthesisexciting times for a difficult field of study
Ann. Rev. Plant Physiol. Plant Mol. Biol.
PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis
Plant Cell
Fine structure in cellulose microfibrilsNMR evidence from onion and quince
Plant J.
Molecular rigidity in dry and hydrated onion cell walls
Plant Physiol.
Lignin-polysaccharide interactions in woody plants
ACS Symp. Ser.
Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-beta-glucanase
Planta
Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-β-glucanase to cellulose synthesis and cytokinesis in Arabidopsis
Plant Physiol.
Cited by (45)
Genome-wide transcriptomic analysis during rhizome development of ginger (Zingiber officinale Roscoe.) reveals hormone and transcriptional regulation involved in cellulose production
2020, Scientia HorticulturaeCitation Excerpt :There is 10 CESA isoforms exist in Arabidopsis, of them, CESA1, CESA3, and CESA6 required for primary cell wall synthesis, while CESA4, CESA7, and CESA8 involved in secondary cell wall production (Watanabe et al., 2015). The cellulose content in the mature stems of mutant irx3, truncation of the AtCesA7 gene, is some 30 % of that in the wild-type (Ha et al., 2002). In Populus, suppression of PtrCesA3D and PtrCesA7A dramatically elevated and reduced cellulose content, respectively (Xi et al., 2017).
Unraveling the biochemical and molecular networks involved in maize cell habituation to the cellulose biosynthesis inhibitor dichlobenil
2010, Molecular PlantCitation Excerpt :Other Arabidopsis CesA, such as CesA2, CesA5, CesA6, and CesA9, are also involved in primary cell wall formation but their functions are partially redundant (Desprez et al., 2007; Persson et al., 2007). In contrast, characterization of Arabidopsis mutants for AtCesA4 (IRX5: irregular xylem 5), AtCesA7 (IRX3), and AtCesA8 (IRX1) revealed that these three proteins are essential for secondary cell wall formation (Taylor et al., 1999, 2000, 2003; Ha et al., 2002) and do not affect cellulose biosynthesis in primary cell walls (Turner and Somerville, 1997; Ha et al., 2002). As cellulose represents the main load-bearing polysaccharide in cell walls, the habituation of plant cell cultures to lethal concentrations of cellulose biosynthesis inhibitors has emerged as a valuable tool for the study of important mechanisms involved in plant survival, such as cell wall plasticity (both structural and compositional) to cope with cell wall integrity disrupting factors (Acebes et al., 2010).
NMR Studies on Cellulose and Plant Cell Walls
2023, Plant Cell Walls: Research Milestones and Conceptual InsightsMultimodal Imaging of Silicified Sorghum Leaves
2022, Analysis and Sensing