Secondary Wall Cellulose Synthases Research Articles

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms.

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

Involvement of CesA4, CesA7-A/B and CesA8-A/B in secondary wall formation in Populus trichocarpa wood.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

Domain swaps of Arabidopsis secondary wall cellulose synthases to elucidate their class specificity.

Cellulose microfibrils are synthesized by membrane‐embedded cellulose synthesis complexes (CSCs), currently modeled as hexamers of cellulose synthase (CESA) trimers. The three paralogous CESAs involved in secondary cell wall (SCW) cellulose biosynthesis in Arabidopsis (CESA4, CESA7, CESA8) are similar, but nonredundant, with all three isoforms required for assembly and function of the CSC. The molecular basis of protein–protein recognition among the isoforms is not well understood. To investigate the locations of the interfaces that are responsible for isoform recognition, we swapped three domains between the Arabidopsis CESAs required for SCW synthesis (CESA4, CESA7, and CESA8): N‐terminus, central domain containing the catalytic core, and C‐terminus. Chimeric genes with all pairwise permutations of the domains were tested for in vivo functionality within knockout mutant backgrounds of cesa4, cesa7, and cesa8. Immunoblotting with isoform‐specific antibodies confirmed the anticipated protein expression in transgenic plants. The percent recovery of stem height and crystalline cellulose content was assayed, as compared to wild type, the mutant background lines, and other controls. Retention of the native central domain was sufficient for CESA8 chimeras to function, with neither its N‐terminal nor C‐terminal domains required. The C‐terminal domain is required for class‐specific function of CESA4 and CESA7, and CESA7 also requires its own N‐terminus. Across all isoforms, the results indicate that the central domain, as well as the N‐ and C‐terminal regions, contributes to class‐specific function variously in Arabidopsis CESA4, CESA7, and CESA8.

Pathway-specific genetic pretreatment strategy to improve bioenergy feedstock

The plant secondary wall is composed of a complex mixture of cellulose, hemicellulose (e.g., xylan), and lignin. The transcription factor MYB46 (At5g12870) has been reported as a central regulator of the secondary wall formation. However, constitutive overexpression of MYB46 results in a severe growth penalty, possibly due to ectopic lignification in the parenchymatous and photosynthetic cells. To test this hypothesis, we constitutively overexpressed MYB46 in the genetic background of a T-DNA insertion mutant 4cl1-2 of 4-coumarate-CoA ligase 1 (4CL1), a key lignin biosynthesis gene. As expected, the transgenic Arabidopsis plants 35S::MYB46/4cl1-2 grow normally, similarly to the 4cl1-2 mutant, with elevated expressions of three secondary wall cellulose synthase genes and xylan biosynthesis genes. Consistent with the gene expression changes, the immunohistological staining of stem tissues showed the increased accumulation of both cellulose and xylan contents in the 35S::MYB46/4cl1-2 plants. Further biochemical analyses confirmed that 35S::MYB46/4cl1-2 plants have higher level accumulations of crystalline cellulose and xylan in both leaf and stem tissues than wild-type control plants as well as the 4cl1-2 mutant, up to 1.18- and 1.13-fold respectively, with considerably reduced lignin content (up to 0.40-fold). Subsequent analysis of enzymatic glucose release from cell wall materials revealed that 35S::MYB46/4cl1-2 plants have higher yields up to 1.27-fold increase in both leaf and stem tissues than that of control plants and 4cl1-2 mutant. Our results showed that overexpression of MYB46 with simultaneous reduction of lignin biosynthesis significantly increases both fermentable sugar contents and cell wall digestibility without growth penalty associated with MYB46 overexpression.

Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Transcription factor MYB46 is an obligate component of the transcriptional regulatory complex for functional expression of secondary wall-associated cellulose synthases in Arabidopsis thaliana

Cellulose, the most abundant biopolymer on Earth, is a central component in plant cell walls and highly abundant (up to 50%) in the secondary walls. In Arabidopsis thaliana, the cellulose biosynthesis in the secondary walls is catalyzed by three cellulose synthases CESA4, CESA7 and CESA8. The transcription factor MYB46 and its close homolog MYB83 directly regulate the expression of the three secondary wall cellulose synthases (CESAs). However, it is not known whether MYB46 is the necessary regulator for functional expression of the secondary wall CESAs or one of the multiple transcriptional factors involved in the transcriptional regulatory program. To address this question, we used a series of genetic complementation experiments of the cesa knock-out mutants with the CESA coding sequence driven by either native- or mutated promoter of the genes. The mutant promoters have two nucleotide point mutations in the MYB46 binding cis element (M46RE) such that MYB46 cannot bind to the promoter, while the binding of other known secondary wall transcription factors is not affected. The mutant complementation results showed that MYB46 is essential to restore normal phenotype from the cesa mutants. We conclude that MYB46 is an obligate component of the transcriptional regulatory complex toward the commitment of secondary wall cellulose synthesis in Arabidopsis.

A Cellulose Synthase-Containing Compartment Moves Rapidly Beneath Sites of Secondary Wall Synthesis

The woody secondary walls of plants represent the major sites of cellulose deposition. The polymerization of cellulose occurs at the plasma membrane by the secondary wall cellulose synthase complex (CSC). In the long, cylindrical cells that make up the xylem, secondary wall deposition is confined to discrete regions of the cell, and yellow fluorescent protein (YFP)-labeled CSCs are also localized to these regions. Using fluorescence loss in photobleaching (FLIP) of complete hoops containing YFP-CSCs, we demonstrate movement of the complexes beneath the nascent secondary wall in developing xylem vessels. We have devised a method for determining particle velocities for particles moving around a cylindrical object using data from FLIP. By applying this method to the hoops of YFP-CSCs of the developing vessels, we have obtained the first estimates of speed of these complexes. These speeds are calculated to be in excess of 7 microm s(-1) and are far higher than those speeds previously reported for the primary wall complex. These high speeds are unlikely to be consistent with CSC movement being attributed to cellulose synthesis alone, and suggest the existence of a highly motile compartment beneath the nascent secondary wall.