Yong Bum Park

A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this “tethered network” model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.

Sensitivity-enhanced solid-state NMR detection of expansin’s target in plant cell walls

Significance The protein expansin loosens the cell walls of plants for cell growth, but its carbohydrate target of binding has been elusive because of the difficulty of studying the noncrystalline plant cell wall by most structural biology techniques and the trace amount of expansin needed for wall loosening. We have now combined dynamic nuclear polarization sensitivity-enhanced solid-state NMR spectroscopy with 13C labeling of Arabidopsis thaliana plants and 13C, 15N labeling of expansin, to determine that expansin binds cellulose microfibrils to loosen the plant cell wall. The expansin binding site is enriched in the hemicellulose xyloglucan and has a different cellulose structure from bulk cellulose, shedding light on the mechanism of wall loosening. Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of 13C spin diffusion from trace concentrations of 13C, 15N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short 13C-13C distances of 4–6 Å between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D 13C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets.

Selective Detection of Crystalline Cellulose in Plant Cell Walls with Sum-Frequency-Generation (SFG) Vibration Spectroscopy

The selective detection of crystalline cellulose in biomass was demonstrated with sum-frequency-generation (SFG) vibration spectroscopy. SFG is a second-order nonlinear optical response from a system where the optical centrosymmetry is broken. In secondary plant cell walls that contain mostly cellulose, hemicellulose, and lignin with varying concentrations, only certain vibration modes in the crystalline cellulose structure can meet the noninversion symmetry requirements. Thus, SFG can be used to detect and analyze crystalline cellulose selectively in lignocellulosic biomass without extraction of noncellulosic species from biomass or deconvolution of amorphous spectra. The selective detection of crystalline cellulose in lignocellulosic biomass is not readily achievable with other techniques such as XRD, solid-state NMR, IR, and Raman analyses. Therefore, the SFG analysis presents a unique opportunity to reveal the cellulose crystalline structure in lignocellulosic biomass.

Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary Cell Walls: Evidence from Solid-State Nuclear Magnetic Resonance

Subnanometer pectin-cellulose contacts are an inherent organizational feature of the Arabidopsis primary cell wall. The structural role of pectins in plant primary cell walls is not yet well understood because of the complex and disordered nature of the cell wall polymers. We recently introduced multidimensional solid-state nuclear magnetic resonance spectroscopy to characterize the spatial proximities of wall polysaccharides. The data showed extensive cross peaks between pectins and cellulose in the primary wall of Arabidopsis (Arabidopsis thaliana), indicating subnanometer contacts between the two polysaccharides. This result was unexpected because stable pectin-cellulose interactions are not predicted by in vitro binding assays and prevailing cell wall models. To investigate whether the spatial contacts that give rise to the cross peaks are artifacts of sample preparation, we now compare never-dried Arabidopsis primary walls with dehydrated and rehydrated samples. One-dimensional 13C spectra, two-dimensional 13C-13C correlation spectra, water-polysaccharide correlation spectra, and dynamics data all indicate that the structure, mobility, and intermolecular contacts of the polysaccharides are indistinguishable between never-dried and rehydrated walls. Moreover, a partially depectinated cell wall in which 40% of homogalacturonan is extracted retains cellulose-pectin cross peaks, indicating that the cellulose-pectin contacts are not due to molecular crowding. The cross peaks are observed both at −20°C and at ambient temperature, thus ruling out freezing as a cause of spatial contacts. These results indicate that rhamnogalacturonan I and a portion of homogalacturonan have significant interactions with cellulose microfibrils in the native primary wall. This pectin-cellulose association may be formed during wall biosynthesis and may involve pectin entrapment in or between cellulose microfibrils, which cannot be mimicked by in vitro binding assays.

Quantification of crystalline cellulose in lignocellulosic biomass using sum frequency generation (SFG) vibration spectroscopy and comparison with other analytical methods

The non-centrosymmetry requirement of sum frequency generation (SFG) vibration spectroscopy allows the detection and quantification of crystalline cellulose in lignocellulose biomass without spectral interferences from hemicelluloses and lignin. This paper shows a correlation between the amount of crystalline cellulose in biomass and the SFG signal intensity. Model biomass samples were prepared by mixing commercially available cellulose, xylan, and lignin to defined concentrations. The SFG signal intensity was found sensitive to a wide range of crystallinity, but varied non-linearly with the mass fraction of cellulose in the samples. This might be due to the matrix effects such as light scattering and absorption by xylan and lignin, as well as the non-linear density dependence of the SFG process itself. Comparison with other techniques such as XRD, FT-Raman, FT-IR and NMR demonstrate that SFG can be a complementary and sensitive tool to assess crystalline cellulose in biomass.

Cellulose microfibril orientation in onion (Allium cepa L.) epidermis studied by atomic force microscopy (AFM) and vibrational sum frequency generation (SFG) spectroscopy

Cellulose microfibril orientation in plant cell walls changes during cell expansion and development. The cellulose microfibril orientation in the abaxial epidermis of onion scales was studied by atomic force microscopy (AFM) and sum frequency generation (SFG) vibrational spectroscopy. Onion epidermal cells in all scales are elongated along the onion bulb axis. AFM images showed that cellulose microfibrils exposed at the innermost surface of the abaxial epidermis are oriented perpendicular to the bulb axis in the outer scales and more dispersed in the inner scales of onion bulb. SFG analyses can determine the orientation of cellulose microfibrils averaged over the entire thickness of the cell wall. We found that the average orientation of cellulose microfibrils inside onion abaxial epidermal cell walls as revealed by SFG is similar to the orientation observed at the innermost cell wall surface by AFM. The capability to determine the average orientation of cellulose microfibrils in intact cell walls will be useful to study how cellulose microfibril orientation is related to biomechanical properties and the growth mechanism of plant cell walls.

Water-polysaccharide interactions in the primary cell wall of Arabidopsis thaliana from polarization transfer solid-state NMR.

Polysaccharide-rich plant cell walls are hydrated under functional conditions, but the molecular interactions between water and polysaccharides in the wall have not been investigated. In this work, we employ polarization transfer solid-state NMR techniques to study the hydration of primary-wall polysaccharides of the model plant, Arabidopsis thaliana. By transferring water (1)H polarization to polysaccharides through distance- and mobility-dependent (1)H-(1)H dipolar couplings and detecting it through polysaccharide (13)C signals, we obtain information about water proximity to cellulose, hemicellulose, and pectins as well as water mobility. Both intact and partially extracted cell wall samples are studied. Our results show that water-pectin polarization transfer is much faster than water-cellulose polarization transfer in all samples, but the extent of extraction has a profound impact on the water-polysaccharide spin diffusion. Removal of calcium ions and the consequent extraction of homogalacturonan (HG) significantly slowed down spin diffusion, while further extraction of matrix polysaccharides restored the spin diffusion rate. These trends are observed in cell walls with similar water content, thus they reflect inherent differences in the mobility and spatial distribution of water. Combined with quantitative analysis of the polysaccharide contents, our results indicate that calcium ions and HG gelation increase the amount of bound water, which facilitates spin diffusion, while calcium removal disrupts the gel and gives rise to highly dynamic water, which slows down spin diffusion. The recovery of spin diffusion rates after more extensive extraction is attributed to increased water-exposed surface areas of the polysaccharides. Water-pectin spin diffusion precedes water-cellulose spin diffusion, lending support to the single-network model of plant primary walls in which a substantial fraction of the cellulose surface is surrounded by pectins.

Monitoring Meso-Scale Ordering of Cellulose in Intact Plant Cell Walls Using Sum Frequency Generation Spectroscopy

Sum frequency generation spectroscopy is sensitive to the ordering of cellulose microfibrils in plant cell walls at the meso scale (nm to μm) that is important for cell wall architecture but cannot be probed by other spectroscopic or diffraction techniques. Sum frequency generation (SFG) vibration spectroscopy can selectively detect crystalline cellulose without spectral interference from cell wall matrix components. Here, we show that the cellulose SFG spectrum is sensitive to cellulose microfibril alignment and packing within the cell wall. SFG intensity at 2,944 cm−1 correlated well with crystalline cellulose contents of various regions of the Arabidopsis (Arabidopsis thaliana) inflorescence, while changes in the 3,320/2,944 cm−1 intensity ratio suggest subtle changes in cellulose ordering as tissues mature. SFG analysis of two cellulose synthase mutants (irx1/cesa8 and irx3/cesa7) indicates a reduction in cellulose content without evidence of altered cellulose structure. In primary cell walls of Arabidopsis, cellulose exhibited a characteristic SFG peak at 2,920 and 3,320 cm−1, whereas in secondary cell walls, it had peaks at 2,944 and 3,320 cm−1. Starch (amylose) gave an SFG peak at 2,904 cm−1 (CH methine) whose intensity increased with light exposure prior to harvest. Selective removal of matrix polysaccharides from primary cell walls by acid hydrolysis resulted in an SFG spectrum resembling that of secondary wall cellulose. Our results show that SFG spectroscopy is sensitive to the ordering of cellulose microfibrils in plant cell walls at the meso scale (nm to μm) that is important for cell wall architecture but cannot be probed by other spectroscopic or diffraction techniques.

Effects of Plant Cell Wall Matrix Polysaccharides on Bacterial Cellulose Structure Studied with Vibrational Sum Frequency Generation Spectroscopy and X-ray Diffraction

The crystallinity, allomorph content, and mesoscale ordering of cellulose produced by Gluconacetobacter xylinus cultured with different plant cell wall matrix polysaccharides were studied with vibrational sum frequency generation (SFG) spectroscopy and X-ray diffraction (XRD). Crystallinity and ordering were assessed as the intensity of SFG signals in the CH/CH2 stretch vibration region (and confirmed by XRD), while Iα content was assessed by the relative intensity of the OH stretch vibration at 3240 cm(-1). A key finding is that the presence of xyloglucan in the culture medium greatly reduced Iα allomorph content but with a relatively small effect on cellulose crystallinity, whereas xylan resulted in a larger decrease in crystallinity with a relatively small decrease in the Iα fraction. Arabinoxylan and various pectins had much weaker effects on cellulose structure as assessed by SFG and XRD. Homogalacturonan with calcium ion reduced the SFG signal, evidently by changing the ordering of cellulose microfibrils. We propose that the distinct effects of matrix polysaccharides on cellulose crystal structure result, at least in part, from selective interactions of the backbone and side chains of matrix polysaccharides with cellulose chains during the formation of the microfibril.

The Identification of Two Arabinosyltransferases from Tomato Reveals Functional Equivalency of Xyloglucan Side Chain Substituents

Expression of xyloglucan arabinofuranosyltransferases, identified from tomato using a comparative genomics approach, rescues growth and mechanical defects of an Arabidopsis mutant deficient for xyloglucan galactosylation. Xyloglucan (XyG) is the dominant hemicellulose present in the primary cell walls of dicotyledonous plants. Unlike Arabidopsis (Arabidopsis thaliana) XyG, which contains galactosyl and fucosyl substituents, tomato (Solanum lycopersicum) XyG contains arabinofuranosyl residues. To investigate the biological function of these differing substituents, we used a functional complementation approach. Candidate glycosyltransferases were identified from tomato by using comparative genomics with known XyG galactosyltransferase genes from Arabidopsis. These candidate genes were expressed in an Arabidopsis mutant lacking XyG galactosylation, and two of them resulted in the production of arabinosylated XyG, a structure not previously found in this plant species. These genes may therefore encode XyG arabinofuranosyltransferases. Moreover, the addition of arabinofuranosyl residues to the XyG of this Arabidopsis mutant rescued a growth and cell wall biomechanics phenotype, demonstrating that the function of XyG in plant growth, development, and mechanics has considerable flexibility in terms of the specific residues in the side chains. These experiments also highlight the potential of reengineering the sugar substituents on plant wall polysaccharides without compromising growth or viability.