Plant cell enlargement and the action of expansins

Plant cells are caged within a distended polymeric network (the cell wall), which enlarges by a process of stress relaxation and slippage (creep) of the polysaccharides that make up the load-bearing network of the wall. Protein mediators of wall creep have recently been isolated and characterized. These proteins, called expansins, appear to disrupt the noncovalent adhesion of matrix polysaccharides to cellulose microfibrils, thereby permitting turgor-driven wall enlargement. Expansin activity is specifically expressed in the growing tissues of dicotyledons and monocotyledons. Sequence analysis of cDNAs indicates that expansins are novel proteins, without previously known functional motifs. Comparison of expansin cDNAs from cucumber, pea, Arabidopsis and rice shows that the proteins are highly conserved in size and amino acid sequence. Phylogenetic analysis of expansin sequences suggests that this multigene family diverged before the evolution of angiosperms. Speculation is presented about the role of this gene family in plant development and evolution.     

How do expansins control plant growth? A model for cell wall loosening via defect migration in cellulose microfibrils

Expansins are plant cell wall-loosening proteins that promote cell growth and are essential for many critical developmental processes and stress responses. The molecular basis for expansin action is uncertain. Recently, it has been proposed that expansins loosen the wall by means of the generation of mobile conformational defects at the surface of cellulose microfibrils. The present work addresses this hypothesis by elaborating three assumptions: (1) microfibril–matrix interfaces cause steep stress gradients on the microfibril surface, (2) stress gradients drive the motion of conformational defects along the microfibril surface toward the microfibril–matrix interfaces, and (3) the approach of the defects to the microfibril–matrix interfaces facilitates the dissociation of matrix polysaccharides from cellulose microfibrils.     

Distribution of expansins in graviresponding maize roots

To test if expansins, wall loosening proteins that disrupt binding between microfibrils and cell wall matrix, participate in the differential elongation of graviresponding roots, Zea mays L. cv. Merit roots were gravistimulated and used for immunolocalization with anti-expansin. Western blots showed cross-reaction with two proteins of maize, one of the same mass as cucumber expansin (29 kDa), the second slightly larger (32 kDa). Maize roots contained mainly the larger protein, but both were found in coleoptiles. The expansin distribution in cucumber roots and hypocotyls was similar to the distribution in maize. Roots showed stronger expansin signals on the expanding convex side than the concave flank as early as 30 min after gravistimulation. Treatment with brefeldin A, a vesicle transport inhibitor, or the auxin transport inhibitor, naphthylphthalamic acid, showed delayed graviresponse and the appearance of differential staining. Our results indicate that expansins may be transported and secreted to cell walls via vesicles and function in wall expansion.     

Expansins in growing tomato leaves

An expansin-like protein from growing tomato leaves was identified by its ability to restore the 'acid-growth' response to heat-inactivated tomato walls and by its similarity to expansins from cucumber hypocotyls. Native walls from growing tomato leaves exhibit an endogenous acid-induced extension (creep) that resembles in various biochemical characteristics the acid-growth activity of cucumber hypocotyls. For example, the acid-growth activity is lost when the walls of tomato leaves are briefly heated and is largely restored by addition of a crude protein extract from the walls of growing leaves. Wall proteins from growing leaves enhance the stress relaxation spectrum of tomato walls in a fashion characteristic of cucumber expansins. HPLC fractionation of the crude wall protein from tomato leaves yielded an active fraction containing a major 27 kDa protein that cross-reacts with an antibody raised against cucumber expansin. The results show that tomato leafwalls possess at least one expansin that is responsible for the acid-growth property of leaves and indicate that cell wall extension in leaves shares an underlying protein mechanism common to cell wall expansion in stems.     

Expansins

Biochemical dissection of the "acid-growth" process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit meristems and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.     

Expanisns

Biochemical dissection of the “acid-growth” process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril-matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit, meristerms and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.     

The expansin superfamily

The expansin superfamily of plant proteins is made up of four families, designated α-expansin, β-expansin, expansin-like A and expansin-like B. α-Expansin and β-expansin proteins are known to have cell-wall loosening activity and to be involved in cell expansion and other developmental events during which cell-wall modification occurs. Proteins in these two families bind tightly to the cell wall and their activity is typically assayed by their stimulation of cell-wall extension and stress relaxation; no bona fide enzymatic activity has been detected for these proteins. α-Expansin proteins and some, but not all, β-expansin proteins are implicated as catalysts of 'acid growth', the enlargement of plant cells stimulated by low extracellular pH. A divergent group of β-expansin genes are expressed at high levels in the pollen of grasses but not of other plant groups. They probably function to loosen maternal cell walls during growth of the pollen tube towards the ovary. All expansins consist of two domains; domain 1 is homologous to the catalytic domain of proteins in the glycoside hydrolase family 45 (GH45); expansin domain 2 is homologous to group-2 grass pollen allergens, which are of unknown biological function. Experimental evidence suggests that expansins loosen cell walls via a nonenzymatic mechanism that induces slippage of cellulose microfibrils in the plant cell wall.     

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as ''acid growth'', by which we mean they are more extensible at low pH (< 5) ¹. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism ^{2, 3}. Auxin activates a H⁺ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins ⁴, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This ''acid growth'' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins ⁵. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.Open in a separate windowClick here to view.^{(58M, flv)}     

Structure-function analysis of the bacterial expansin EXLX1

We made use of EXLX1, an expansin from Bacillus subtilis, to investigate protein features essential for its plant cell wall binding and wall loosening activities. We found that the two expansin domains, D1 and D2, need to be linked for wall extension activity and that D2 mediates EXLX1 binding to whole cell walls and to cellulose via distinct residues on the D2 surface. Binding to cellulose is mediated by three aromatic residues arranged linearly on the putative binding surface that spans D1 and D2. Mutation of these three residues to alanine eliminated cellulose binding and concomitantly eliminated wall loosening activity measured either by cell wall extension or by weakening of filter paper but hardly affected binding to whole cell walls, which is mediated by basic residues located on other D2 surfaces. Mutation of these basic residues to glutamine reduced cell wall binding but not wall loosening activities. We propose domain D2 as the founding member of a new carbohydrate binding module family, CBM63, but its function in expansin activity apparently goes beyond simply anchoring D1 to the wall. Several polar residues on the putative binding surface of domain D1 are also important for activity, most notably Asp82, whose mutation to alanine or asparagine completely eliminated wall loosening activity. The functional insights based on this bacterial expansin may be extrapolated to the interactions of plant expansins with cell walls.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

How Does Hemicelluloses Removal Alter Plant Cell Wall Nanoscale Architecture and Correlate with Enzymatic Digestibility?

Thorough understanding of how hemicelluloses removal influences cell wall nanoscale architecture and cellulose digestion is of crucial importance for enabling low-cost industrial conversion of lignocellulosic biomass to renewable biofuels. In this work, delignified poplar cell walls, after various degrees of hemicelluloses removal, were characterized by Fourier transform infrared imaging spectroscopy and atomic force microscopy to evaluate enhancement in cell wall digestibility. There was a gradual decrease in hemicelluloses content with dilute alkali treatment, which resulted in alterations in the nanoscale architecture and crystallinity of cell walls. Removal of hemicelluloses did not disrupt the integrity of microfibrils but resulted in exposure of microfibrils and a decrease in the diameter of microfibrils. X-ray analysis indicated that the increase in crystallinity beyond natural variations in the crystallinity of cellulose was mainly attributable to removal of hemicelluloses. In conclusion, alterations in the architecture and crystallinity of cell walls facilitated enzymatic digestion of delignified poplar, enhancing cellulose conversion from 68.24 to 75.16 %.     

Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy

Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.     

How do cell walls regulate plant growth?

The cell wall of growing plant tissues has frequently been interpreted in terms of inextensible cellulose microfibrils 'tethered' by hemicellulose polymers attached to the microfibril surface by hydrogen bonds, with growth occurring when tethers are broken or 'peeled' off the microfibril surface by expansins. This has sometimes been described as the 'sticky network' model. In this paper, a number of theoretical difficulties with this model, and discrepancies between predicted behaviour and observations by a number of researchers, are noted. (i) Predictions of cell wall moduli, based upon the sticky network model, suggest that the cell wall should be much weaker than is observed. (ii) The maximum hydrogen bond energy between tethers and microfibrils is less than the work done in expansion and therefore breakage of such hydrogen bonds is unlikely to limit growth. (iii) Composites of bacterial cellulose with xyloglucan are weaker than pellicles of pure cellulose so that it seems unlikely that hemicelluloses bind the microfibrils together. (iv) Calcium chelators promote creep of plant material in a similar way to expansins. (v) Reduced relative 'permittivities' inhibit the contraction of cell wall material when an applied stress is decreased. Revisions of the sticky network model that might address these issues are considered, as are alternatives including a model of cell wall biophysics in which cell wall polymers act as 'scaffolds' to regulate the space available for microfibril movement. Experiments that support the latter hypothesis, by demonstrating that reducing cell wall free volume decreases extensibility, are briefly described.     

Expansins in deepwater rice internodes.

Cell walls of deepwater rice (Oryza sativa L.) internodes undergo long-term extension (creep) when placed under tension in acidic buffers. This is indicative of the action of the cell wall-loosening protein expansin. Wall extension had a pH optimum of around 4.0 and was abolished by boiling. Acid-induced extension of boiled cell walls could be reconstituted by addition of salt-extracted rice or cucumber cell wall proteins. Cucumber expansin antibody recognized a single protein band of 24.5-kD apparent molecular mass on immunoblots of rice cell wall proteins. Expansins were partially purified by concanavalin A affinity chromatography and sulfopropyl (SP) cation-exchange chromatography. The latter yielded two peaks with extension activity (SP20 and SP29), and immunoblot analysis showed that both of these active fractions contained expansin of 24.5-kD molecular mass. The N-terminal amino acid sequence of SP20 expansin is identical to that deduced from the rice expansin cDNA Os-EXP1. The N-terminal amino acid sequence of SP29 expansin matches that deduced from the rice expansin cDNA Os-EXP2 in six of eight amino acids. Our results show that two expansins occur in the cell walls of rice internodes and that they may mediate acid-induced wall extension.     

Is acid-induced extension in seed plants only protein-mediated?

Cell wall extensibility controls the rate of plant cell growth. It is determined by intrinsic mechanical properties of wall polymers and by wall proteins modifying these polymers and their interactions. Heat-inactivation of endogenous cell wall proteins inhibited acid-induced extension of onion epidermis peels transverse to the net cellulose alignment in the cell wall but not parallel to it. In the former case the acid-induced extension could be controlled by expansins and in the latter case by pectins restricting shear between microfibrils. Heat-inactivated cell walls stretched transversely to the net cellulose orientation extended faster at pH 5.7 and slower at pH 4.5 compared to native walls. Expansins seem to be inactive at pH 5.7, so that faster extension may result from heat-induced viscous flow of pectins and conformational changes in the cuticle of the epidermis. This stimulation of wall extension is not seen at pH 4.5 as it is outweighed by the inhibitory effect of expansin heat-inactivation. Thus, cell wall extension in higher plants might be controlled by a complex interplay between protein-dependent and protein-independent mechanisms, the result of which depends on pH and preferential orientation of main wall polymers.     

A cross-polarization, magic-angle-spinning, 13C-nuclear-magnetic-resonance study of polysaccharides in sugar beet cell walls

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T_1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.     

Structure and interactions of plant cell-wall polysaccharides by two- and three-dimensional magic-angle-spinning solid-state NMR

The polysaccharide-rich cell walls (CWs) of plants perform essential functions such as maintaining tensile strength and allowing plant growth. Using two- and three-dimensional magic-angle-spinning (MAS) solid-state NMR and uniformly (13)C-labeled Arabidopsis thaliana, we have assigned the resonances of the major polysaccharides in the intact and insoluble primary CW and determined the intermolecular contacts and dynamics of cellulose, hemicelluloses, and pectins. Cellulose microfibrils showed extensive interactions with pectins, while the main hemicellulose, xyloglucan, exhibited few cellulose cross-peaks, suggesting limited entrapment in the microfibrils rather than extensive surface coating. Site-resolved (13)C T(1) and (1)H T(1ρ) relaxation times indicate that the entrapped xyloglucan has motional properties that are intermediate between the rigid cellulose and the dynamic pectins. Xyloglucan absence in a triple knockout mutant caused the polysaccharides to undergo much faster motions than in the wild-type CW. These results suggest that load bearing in plant CWs is accomplished by a single network of all three types of polysaccharides instead of a cellulose-xyloglucan network, thus revising the existing paradigm of CW structure. The extensive pectin-cellulose interaction suggests a central role for pectins in maintaining the structure and function of plant CWs. This study demonstrates the power of multidimensional MAS NMR for molecular level investigation of the structure and dynamics of complex and energy-rich plant materials.