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|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
|Enzyme Activity:||Pectate Lyase|
|Synonyms:||pectate lyase; (1→4)-alpha-D-galacturonan lyase|
|Concentration:||Supplied at ~ 500 U/mL|
|Expression:||Recombinant from Cellvibrio japonicus|
|Specificity:||Eliminative cleavage of (1,4)-α-D-galacturonan to give oligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at their non-reducing ends.|
|Specific Activity:||~ 500 U/mg (40oC, pH 10.0 on polygalacturonic acid)|
|Unit Definition:||One Unit of pectate lyase activity is defined as the amount of enzyme required to release one µmole of 4,5-unsaturated product per minute from polygalacturonic acid (1.25 mg/mL) in the presence of calcium chloride (1 mM) in CAPS buffer (50 mM), pH 10.0 at 40oC.|
|Application examples:||Applications in carbohydrate and biofuels research.|
High-resolution imaging of cellulose organization in cell walls by field emission scanning electron microscopy.
Zheng, Y., Ning, G. & Cosgrove, D. J. (2020). “The Plant Cell Wall”, Humana, New York, NY, pp. 225-237.
Field emission scanning electron microscopy (FESEM) is a powerful tool for analyzing surface structures of biological and nonbiological samples. However, when it is used to study fine structures of nanometer-sized microfibrils of epidermal cell walls, one often encounters tremendous challenges to acquire clear and undistorted images because of two major issues: (1) Preparation of samples suitable for high resolution imaging; due to the delicateness of some plant materials, such as onion epidermal cell walls, many things can happen during sample processing, which subsequently result in damaged samples or introduce artifacts. (2) Difficulties to acquire clear images of samples which are electron-beam sensitive and prone to charging artifacts at magnifications over 100,000×. In this chapter we described detailed procedures for sample preparation and conditions for high-resolution FESEM imaging of onion epidermal cell walls. The methods can be readily adapted for other wall materials.Hide Abstract
Changes in the orientations of cellulose microfibrils during the development of collenchyma cell walls of celery (Apium graveolens L.).
Chen, D., Melton, L. D., McGillivray, D. J., Ryan, T. M. & Harris, P. J. (2019). Planta, 250(6), 1819-1832.
Collenchyma cells have thickened primary cell walls and provide mechanical support during plant growth. During their development, these cells elongate and their walls thicken considerably. We used microscopy and synchrotron small-angle X-ray scattering to study changes in the orientations of cellulose microfibrils that occur during development in the walls of collenchyma cells present in peripheral strands in celery (Apium graveolens) petioles. Transmission electron microscopy showed that the walls consisted of many lamellae (polylamellate), with lamellae containing longitudinally oriented cellulose microfibrils alternating with microfibrils oriented at higher angles. The lamellae containing longitudinally oriented microfibrils predominated at later stages of development. Nevertheless, transmission electron microscopy of specially stained, oblique sections provided evidence that the cellulose microfibrils were ordered throughout development as crossed-polylamellate structures. These results are consistent with our synchrotron small-angle X-ray scattering results that showed the cellulose microfibrils become oriented increasingly longitudinally during development. Some passive reorientation of cellulose microfibrils may occur during development, but extensive reorientation throughout the wall would destroy ordered structures. Atomic force microscopy and field emission scanning electron microscopy were used to determine the orientations of newly deposited cellulose microfibrils. These were found to vary widely among different cells, which could be consistent with the formation of crossed-polylamellate structures. These newly deposited cellulose microfibrils are deposited in a layer of pectic polysaccharides that lies immediately outside the plasma membrane. Overall, our results show that during development of collenchyma walls, the cellulose microfibrils become increasingly longitudinal in orientation, yet organized, crossed-polylamellate structures are maintained.Hide Abstract
Huang, S., Makarem, M., Kiemle, S. N., Zheng, Y., He, X., Ye, D., Gomaz, E. W., Gomaz, E. D., Cosgrove, D. J. & Kim, S. H. (2018). Carbohydrate Polymers, 197, 337-348.
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 Iβ obtained from tunicates. Thus, the modulus of CMFs in PCWs would be lower than those of highly-crystalline cellulose Iβ and inhomogeneous local bending or strain of CMFs could occur readily during the physical collapse of pectin matrix due to dehydration.Hide Abstract
Hernandez-Gomez, M. C., Rydahl, M. G., Rogowski, A., Morland, C., Cartmell, A., Crouch, L., Labourel, A., Fontes, C. M. G. A., Willats, W. G. T., Gilbert, H. J. & Knox, J. P. (2015). FEBS Letters, 589(18), 2297-2303.
Type A non-catalytic carbohydrate-binding modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan (XG), a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces. Xyloglucan and cellulose were shown to bind to the same planar surface on CBM3acipA. A range of type A CBMs from different families were shown to bind to xyloglucan in solution with ligand binding driven by enthalpic changes. The nature of CBM-polysaccharide interactions is discussed.Hide Abstract
Eeckhout, S., Leroux, O., Willats, W. G. T., Popper, Z. A. & Viane, R. L. L. (2014). Annals of Botany, mcu039.
Background and Aims Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii ‘C-Fern’, a widely used model system for ferns. Methods Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of ‘C-Fern’ sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. Key Results While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. Conclusions The differences and similarities in glycan cell-wall composition between ‘C-Fern’ gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte.Hide Abstract
Domozych, D. S., Sørensen, I., Popper, Z. A., Ochs, J., Andreas, A., Fangel, J. U., Pielach, A., Sacks, C., Brechka, H., Ruisi-Besares, P., Willats, W. G. & Rose, J. K. C. (2014). Plant Physiology, 165(1), 105-18.
The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its co-localization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens and Arabidopsis (Arabidopsis thaliana) further suggested conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of Penium, a member of the Charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. The calcium-HG gel at the cell surface may therefore represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.Hide Abstract
Park, Y. B. & Cosgrove, D. J. (2012). Plant Physiology, 158(1), 465-475.
The main load-bearing network in the primary cell wall of most land plants is commonly depicted as a scaffold of cellulose microfibrils tethered by xyloglucans. However, a xyloglucan-deficient mutant (xylosyltransferase1/xylosyltransferase2 [xxt1/xxt2]) was recently developed that was smaller than the wild type but otherwise nearly normal in its development, casting doubt on xyloglucan’s role in wall structure. To assess xyloglucan function in the Arabidopsis (Arabidopsis thaliana) wall, we compared the behavior of petiole cell walls from xxt1/xxt2 and wild-type plants using creep, stress relaxation, and stress/strain assays, in combination with reagents that cut or solubilize specific components of the wall matrix. Stress/strain assays showed xxt1/xxt2 walls to be more extensible than wild-type walls (supporting a reinforcing role for xyloglucan) but less extensible in creep and stress relaxation processes mediated by α-expansin. Fusicoccin-induced “acid growth” was likewise reduced in xxt1/xxt2 petioles. The results show that xyloglucan is important for wall loosening by α-expansin, and the smaller size of the xxt1/xxt2 mutant may stem from the reduced effectiveness of α-expansins in the absence of xyloglucan. Loosening agents that act on xylans and pectins elicited greater extension in creep assays of xxt1/xxt2 cell walls compared with wild-type walls, consistent with a larger mechanical role for these matrix polymers in the absence of xyloglucan. Our results illustrate the need for multiple biomechanical assays to evaluate wall properties and indicate that the common depiction of a cellulose-xyloglucan network as the major load-bearing structure is in need of revision.Hide Abstract
Tabuchi, A., Li, L. C. & Cosgrove, D. J. (2011). The Plant Journal, 68(3), 546-559.
Beta-expansins accumulate to high levels in grass pollen, a feature apparently unique to grasses. These proteins, which are major human allergens, facilitate pollen tube penetration of the maize stigma and style (the silk). Here we report that treatment of maize silk cell walls with purified β-expansin from maize pollen led to solubilization of wall matrix polysaccharides, dominated by feruloyated highly substituted glucuronoarabinoxylan (60%) and homogalacturonan (35%). Such action was selective for cell walls of grasses, and indicated a target preferentially found in grass cell walls, probably the highly substituted glucuronoarabinoxylan. Several tests for lytic activities by β-expansin were negative and polysaccharide solubilization had weak temperature dependence, which indicated a non-enzymatic process. Concomitant with matrix solubilization, β-expansin treatment induced creep, reduced the breaking force and increased the plastic compliance of wall specimens. From comparisons of the pH dependencies of these processes, we conclude that matrix solubilization was linked closely to changes in wall plasticity and breaking force, but not so closely coupled to cell wall creep. Because matrix solubilization and increased wall plasticity have not been found with other expansins, we infer that these novel activities are linked to the specialized role of grass pollen β-expansins in promotion of penetration of the pollen tube through the stigma and style, most likely by weakening the middle lamella.Hide Abstract
Park, S., Szumlanski, A. L., Gu, F., Guo, F. & Nielsen, E. (2011). Nature Cell Biology, 13(8), 973-980.
In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP–CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.Hide Abstract