Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Gradients in Wall Mechanics and Polysaccharides along Growing Inflorescence Stems.

At early stages of Arabidopsis (Arabidopsis thaliana) flowering, the inflorescence stem undergoes rapid growth, with elongation occurring predominantly in the apical ∼4 cm of the stem. We measured the spatial gradients for elongation rate, osmotic pressure, cell wall thickness, and wall mechanical compliances and coupled these macroscopic measurements with molecular-level characterization of the polysaccharide composition, mobility, hydration, and intermolecular interactions of the inflorescence cell wall using solid-state nuclear magnetic resonance spectroscopy and small-angle neutron scattering. Force-extension curves revealed a gradient, from high to low, in the plastic and elastic compliances of cell walls along the elongation zone, but plots of growth rate versus wall compliances were strikingly nonlinear. Neutron-scattering curves showed only subtle changes in wall structure, including a slight increase in cellulose microfibril alignment along the growing stem. In contrast, solid-state nuclear magnetic resonance spectra showed substantial decreases in pectin amount, esterification, branching, hydration, and mobility in an apical-to-basal pattern, while the cellulose content increased modestly. These results suggest that pectin structural changes are connected with increases in pectin-cellulose interaction and reductions in wall compliances along the apical-to-basal gradient in growth rate. These pectin structural changes may lessen the ability of the cell wall to undergo stress relaxation and irreversible expansion (e.g. induced by expansins), thus contributing to the growth kinematics of the growing stem.

Role of (1,3)(1,4)-ß-glucan in cell walls: interaction with cellulose.

(1,3)(1,4)-ß-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-ß-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-ß-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.

Grazing-incidence diffraction reveals cellulose and pectin organization in hydrated plant primary cell wall.

The primary cell wall is highly hydrated in its native state, yet many structural studies have been conducted on dried samples. Here, we use grazing-incidence wide-angle X-ray scattering (GIWAXS) with a humidity chamber, which enhances scattering and the signal-to-noise ratio while keeping outer onion epidermal peels hydrated, to examine cell wall properties. GIWAXS of hydrated and dried onion reveals that the cellulose ([Formula: see text]) lattice spacing decreases slightly upon drying, while the (200) lattice parameters are unchanged. Additionally, the ([Formula: see text]) diffraction intensity increases relative to (200). Density functional theory models of hydrated and dry cellulose microfibrils corroborate changes in crystalline properties upon drying. GIWAXS also reveals a peak that we attribute to pectin chain aggregation. We speculate that dehydration perturbs the hydrogen bonding network within cellulose crystals and collapses the pectin network without affecting the lateral distribution of pectin chain aggregates.

Detecting the orientation of newly-deposited crystalline cellulose with fluorescent CBM3.

Cellulose microfibril patterning influences many of the mechanical attributes of plant cell walls. We developed a simple, fluorescence microscopy-based method to detect the orientation of newly-synthesized cellulose microfibrils in epidermal peels of onion and Arabidopsis. It is based on Alexa Fluor 488-tagged carbohydrate binding module 3a (CBM3a) from Clostridium thermocellum which displayed a nearly 4-fold greater binding to cell walls at pH 5.5 compared with pH 8. Binding to isolated cellulose did not display this pH dependence. At pH 7.5 fibrillar patterns at the surface of the epidermal peels were visible, corresponding to the directionality of surface cellulose microfibrils, as verified by atomic force microscopy. The fibrillar pattern was not visible as the labeling intensity increased at lower pH. The pH of greatest cell wall labeling corresponds to the isoelectric point of CBM3a, suggesting that electrostatic forces limit CBM3a penetration into the wall. Consistent with this, digestion of the wall with pectate lyase to remove homogalacturonan increased labeling intensity. We conclude that electrostatic interactions strongly influence labeling of cell walls with CBM3 and potentially other proteins, holding implications for any work that relies on penetration of protein probes such as CBMs, antibodies, or enzymes into charged polymeric substrates.

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.

Preferred crystallographic orientation of cellulose in plant primary cell walls.

Cellulose, the most abundant biopolymer on earth, is a versatile, energy rich material found in the cell walls of plants, bacteria, algae, and tunicates. It is well established that cellulose is crystalline, although the orientational order of cellulose crystallites normal to the plane of the cell wall has not been characterized. A preferred orientational alignment of cellulose crystals could be an important determinant of the mechanical properties of the cell wall and of cellulose-cellulose and cellulose-matrix interactions. Here, the crystalline structures of cellulose in primary cell walls of onion (Allium cepa), the model eudicot Arabidopsis (Arabidopsis thaliana), and moss (Physcomitrella patens) were examined through grazing incidence wide angle X-ray scattering (GIWAXS). We find that GIWAXS can decouple diffraction from cellulose and epicuticular wax crystals in cell walls. Pole figures constructed from a combination of GIWAXS and X-ray rocking scans reveal that cellulose crystals have a preferred crystallographic orientation with the (200) and (110)/([Formula: see text]) planes preferentially stacked parallel to the cell wall. This orientational ordering of cellulose crystals, termed texturing in materials science, represents a previously unreported measure of cellulose organization and contradicts the predominant hypothesis of twisting of microfibrils in plant primary cell walls.

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

Dehydration-induced physical strains of cellulose microfibrils in plant cell walls.

The effect of dehydration of plant cell walls on the physical status of cellulose microfibrils (CMFs) interspersed in pectin matrices was studied. Vibrational sum frequency generation (SFG) spectroscopy analysis of cellulose revealed reversible changes in spectral features upon dehydration and rehydration of onion epidermal walls used as a model primary cell wall (PCW). Combined with microscopic imaging and indentation modulus data, such changes could be attributed to local strains in CMFs due to the collapse of the pectin matrix upon dehydration. X-ray diffraction (XRD) showed that the (200) spacing of cellulose in dried PCWs is larger than that of cellulose Iß obtained from tunicates. Thus, the modulus of CMFs in PCWs would be lower than those of highly-crystalline cellulose Iß and inhomogeneous local bending or strain of CMFs could occur readily during the physical collapse of pectin matrix due to dehydration.

Resonant soft X-ray scattering reveals cellulose microfibril spacing in plant primary cell walls.

Cellulose microfibrils are crucial for many of the remarkable mechanical properties of primary cell walls. Nevertheless, many structural features of cellulose microfibril organization in cell walls are not yet fully described. Microscopy techniques provide direct visualization of cell wall organization, and quantification of some aspects of wall microstructure is possible through image processing. Complementary to microscopy techniques, scattering yields structural information in reciprocal space over large sample areas. Using the onion epidermal wall as a model system, we introduce resonant soft X-ray scattering (RSoXS) to directly quantify the average interfibril spacing. Tuning the X-ray energy to the calcium L-edge enhances the contrast between cellulose and pectin due to the localization of calcium ions to homogalacturonan in the pectin matrix. As a consequence, RSoXS profiles reveal an average center-to-center distance between cellulose microfibrils or microfibril bundles of about 20 nm.

Investigating Biochemical and Developmental Dependencies of Lignification with a Click-Compatible Monolignol Analog in Arabidopsis thaliana Stems.

Lignin is a key structural component of plant cell walls that provides rigidity, strength, and resistance against microbial attacks. This hydrophobic polymer also serves a crucial role in water transport. Despite its abundance and essential functions, several aspects of lignin biosynthesis and deposition remain cryptic. Lignin precursors are known to be synthesized in the cytoplasm by complex biosynthetic pathways, after which they are transported to the apoplastic space, where they are polymerized via free radical coupling reactions into polymeric lignin. However, the lignin deposition process and the factors controlling it are unclear. In this study, the biochemical and developmental dependencies of lignification were investigated using a click-compatible monolignol analog, 3-O-propargylcaffeyl alcohol (3-OPC), which can incorporate into both in vitro polymerized lignin and Arabidopsis thaliana tissues. Fluorescence labeling of 3-OPC using click chemistry followed by confocal fluorescence microscopy enabled the detection and imaging of 3-OPC incorporation patterns. These patterns were consistent with endogenous lignification observed in different developmental stages of Arabidopsis stems. However, the concentration of supplied monolignols influenced where lignification occurred at the subcellular level, with low concentrations being deposited in cell corners and middle lamellae and high concentrations also being deposited in secondary walls. Experimental inhibition of multiple lignification factors confirmed that 3-OPC incorporation proceeds via a free radical coupling mechanism involving peroxidases/laccases and reactive oxygen species (ROS). Finally, the presence of peroxide-producing enzymes determined which cell walls lignified: adding exogenous peroxide and peroxidase caused cells that do not naturally lignify in Arabidopsis stems to lignify. In summary, 3-OPC accurately mimics natural lignification patterns in different developmental stages of Arabidopsis stems and allows for the dissection of key biochemical and enzymatic factors controlling lignification.

CELL-WALL DEVELOPMENT AND BIPOLAR GROWTH IN THE DESMID PENIUM MARGARITACEUM (ZYGNEMATOPHYCEAE, STREPTOPHYTA). ASYMMETRY IN A SYMMETRIC WORLD(1).

Cell-wall (CW) development in the desmid Penium margaritaceum (Ehrenb.) Bréb. was studied using immunofluorescence labeling of living cells with the monoclonal antibodies (mAbs) JIM5 and JIM7, which recognize unesterified and methyl-esterified homogalacturonan (HG), respectively. During cell expansion, HG was secreted in a high-esterified form at a narrow band, called the HG secretion band or HGSB, at the isthmus or the polar tip of a daughter semicell. As newly secreted HG is displaced outward on the cell surface, deesterification and subsequent calcium (Ca(2+) )-complexing occurred to yield a rigid covering. HG secretion and CW/cell expansion were reversibly inhibited by dark, brefeldin A (BFA), and incubation in 0.24-0.36 M sucrose but were not altered by treatment with actin/microfilament drugs. The HGSB was detected near the nucleus during most cell-cycle events. Centrifugation displaced the nucleus away from the HGSB, but HG synthesis was not affected. HGSB activity was preceded by, and coordinated with, Calcofluor labeling, which suggests that cellulose production in CW/cell-expansion sites was critical to expansion control. In many first-cell-division products, asymmetric patterning of HG was noted in the CW. These asymmetric patterns most likely were a result of timing mechanisms and displacement of the nucleus-HGSB during the cell cycle.