Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.Cellulose is the primary polysaccharide of plant cell wall and the most abundant renewable biomass resource. Biological degradation of cellulose to soluble sugars has long been considered an alternative to the use of starch feedstocks for bioethanol production. Natural cellulose is an ordered, linear polymer of thousands of d-glucose residues linked by β-1,4-glucosidic bonds. Spontaneous crystallization of cellulose molecules due to chemical uniformity of glucose units and the high degree of hydrogen bonding in cellulose can often result in the formation of tightly packed microfibrils (8), which remain inaccessible to cellulolytic enzymes. No single enzyme is able to hydrolyze crystalline cellulose microfibrils completely. Synergistic effects of cellulase mixtures on crystalline cellulose degradation are well known (1, 7, 21). Nevertheless, cost-competitive technology for overcoming the recalcitrance of cellulosic biomass to enhance enzymatic saccharification is still a major impediment to the utilization of cellulosic materials in bioenergy generation.Expansins are plant cell wall proteins that cause cell wall enlargement by a unique loosening effect in an acid-induced manner (15, 20). They are also involved in many physiological processes where cell wall extension occurs, such as pollination, fruit ripening, organ abscission, and seed germination (13, 14). It has been proposed that plant expansins disrupt hydrogen bonding between cellulose microfibrils and other cell wall polysaccharides without hydrolytic activity, causing sliding of cellulose fibers or expansion of the cell wall (18, 19, 27). Swollenin, an expansin-like protein, was isolated and characterized from the cellulolytic filamentous fungus Trichoderma reesei. It has a bimodular structure consisting of a carbohydrate-binding module family 1 (CBM1) domain and an expansin-like domain connected by a linker region rich in serine and threonine. Swollenin exhibits disruption activity on cellulosic materials such as cotton and algal cell walls without releasing any detectable reducing sugars (23). However, effects of cellulose disruption activity on degradation/saccharification of crystalline cellulose have not yet been reported.Here, we report cloning a swollenin-like gene (designated Afswo1) from the filamentous fungus Aspergillus fumigatus. We also report its production by Aspergillus oryzae and characterization of the purified AfSwo1.     

Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β‐expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface‐binding carbohydrate‐binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. Biotechnol. Bioeng. 2013; 110: 401–407. © 2012 Wiley Periodicals, Inc.     

PhEXPA1, a Petunia hybrida expansin,is involved in cell wall metabolism and in plant architecture specification

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.     

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353. © 2008 Wiley Periodicals, Inc.     

Cerato-platanin shows expansin-like activity on cellulosic materials

Cerato-platanin (CP) is a non-catalytic protein with a double ψβ-barrel fold located in the cell wall of the phytopathogenic fungus Ceratocystis platani. CP is released during growth and induces defence-related responses in plants. CP is also the first member of the “cerato-platanin family” (CPF) (Pfam PF07249). In the CPF, the molecular mechanism of action on plants and above all the biological role in fungal life are little-known aspects. However, an expansin-like function has recently been suggested concerning CP. Expansin-like proteins have the ability to act non-hydrolytically on cellulose. In the present work, the expansin-like activity of CP and Pop1, a CP family member, was investigated. Like expansins, CP and Pop1 were able to weaken filter paper in a concentration-dependent manner and without the production of reducing sugars. A metal-dependent polysaccharide monooxygenase-like activity was excluded. The optimum of activity was pH?5.0, 38 °C. CP was also able to cause fragmentation of the crystalline cellulose Avicel and the breakage and defibration of cotton fibres. However, the interaction did not involve a stable bond with the substrates and CP did not significantly enhance the hydrolytic activity of cellulase. On the other hand, CP and Pop1 bound quickly to chitin. We consider CP as a novel one-domain expansin-like protein. We propose a structural role for CP in the fungal cell wall due to the ability to bind chitin, and hypothesize a functional role in the interaction of the fungus with the plant for the weakening activity shown on cellulose.     

Electrostatic analysis of bacterial expansins

Expansins are a family of proteins with plant cell wall remodeling‐activity, which bind cell wall components through hydrophobic and electrostatic interactions. A shallow area on the surface of the protein serves as the polysaccharide binding site (PBS) and it is composed of conserved residues. However, electric charge differences on the opposite face of the PBS produce basic, neutral, or acidic proteins. An analysis of forty‐four bacterial expansins, homologues of BsEXLX1, revealed two main groups defined by: (a) the presence or absence of disulfide bonds; and (b) by the proteins isoelectric point (pI). We determined the location of the residues responsible for the pI on the structure of representative expansins. Our results suggest that the electric charge at the opposite site of the PBS may help in substrate differentiation among expansins from different species; in addition, electrostatic polarization between the front and the back of the molecule could affect expansin activity on cellulose. Proteins 2015; 83:215–223. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Functionalization of Recombinant Amelogenin Nanospheres Allows Their Binding to Cellulose Materials

Protein engineering to functionalize the self‐assembling enamel matrix protein amelogenin with a cellulose binding domain (CBD) is used. The purpose is to examine the binding of the engineered protein, rh174CBD, to cellulose materials, and the possibility to immobilize self‐assembled amelogenin nanospheres on cellulose. rh174CBD assembled to nanospheres ≈35 nm in hydrodynamic diameter, very similar in size to wild type amelogenin (rh174). Uniform particles are formed at pH 10 for both rh174 and rh174CBD, but only rh174CBD nanospheres showes significant binding to cellulose (Avicel). Cellulose binding of rh174CBD is promoted when the protein is self‐assembled to nanospheres, compared to being in a monomeric form, suggesting a synergistic effect of the multiple CBDs on the nanospheres. The amount of bound rh174CBD nanospheres reached ≈15 mg/g Avicel, which corresponds to 4.2 to 6.3 × 10^?7 mole/m². By mixing rh174 and rh174CBD, and then inducing self‐assembly, composite nanospheres with a high degree of cellulose binding can be formed, despite a lower proportion of rh174CBD. This demonstrates that amelogenin variants like rh174 can be incorporated into the nanospheres, and still retain most of the binding to cellulose. Engineered amelogenin nanoparticles can thus be utilized to construct a range of new cellulose based hybrid materials, e.g. for wound treatment.     

The effect of removing cellulase(s) from a commercial pectinase on maceration and liquefaction of carrots

Rohament P, a macerating enzyme preparation from Aspergillus alleaceus containing an endo-polygalacturonase as the major activity but also substantial amounts of cellulase(s), was purified by affinity chromatography on Avicel cellulose. Treating Rohament P with Avicel at different protein:cellulose ratios was more efficient in columns than in batch. An Avicel column, with an enzyme:substrate ratio of 1:80, retained 94% of cellulase from Rohament P in 60 min at 40–45°C, pH 4.4. Treated enzyme containing 6% residual cellulase, when incubated with fresh carrot rasps, released the maximum amount of cells with intact cell walls. Untreated enzyme did not macerate the carrot tissue but liquified under same conditions. Degradation of a washed carrot preparation by treated enzyme was 44% compared with 55% for the untreated enzyme. Gas chromatographic analysis of sugars revealed that treated Rohament P liberated less glucose and others sugars than did untreated enzyme. Enzyme visualization studies of treated and untreated Rohament P reflected a quantitative difference in protein bands in a pH gradient of 4–6 in miniature isoelectric focusing. Application of treated and untreated Rohament P to disks of carrot tissue led to ultrastructural changes. Untreated Rohament P dissolved the middle lamella and disintegrated the fibrillar material predominantly throughout the cell wall, resulting mainly in cell breakage. Treated Rohament P preferentially dissolved the middle lamellar region of the cell wall without touching the fibrillar part. This indicates that pectin is confined to the middle lamellar region.     

Expression and characterization of the Neurospora crassa endoglucanase GH5-1

Filamentous fungi secrete a wide range of enzymes, including cellulases and hemicellulases, with potential applications in the production of lignocellulosic biofuels. Of the cellulolytic fungi, Hypocrea jecorina (anamorph Trichoderma reesei) is the best characterized in terms of cellulose degradation, but other cellulolytic fungi, such as the model filamentous fungus Neurospora crassa, can serve a crucial role in building our knowledge about the fungal response to biomass due to the many molecular and genetic tools available for this organism. Here we cloned and expressed GH5-1 (NCU00762), a secreted endoglucanase in N. crassa. The protein was produced using a ccg-1 promoter under conditions in which no other cellulases are present. Native GH5-1 (nGH5-1) and this recombinant GH5-1 (rGH5-1) were purified to gauge differences in glycosylation and activity; both rGH5-1 and nGH5-1 were similarly glycosylated, with an estimated molecular weight of 52 kDa. On azo-carboxymethylcellulose, rGH5-1 activity was equal to that of nGH5-1, and on cellulose (Avicel) rGH5-1 was 20% more active. The activity of a GH5-1-GFP fusion protein (rGH5-1-GFP-6xHis) was similar to rGH5-1 and nGH5-1. To determine the binding pattern of catalytically active rGH5-1-GFP-6xHis to plant cell walls, Arabidopsis seedlings were incubated with rGH5-1-GFP-6xHis or Pontamine Fast Scarlet 4B (S4B), a cellulose-specific dye. Confocal microscopy showed that rGH5-1-GFP-6xHis bound in linear, longitudinal patterns on seedling roots, similar to S4B. The functional expression and characterization of rGH5-1 and its GFP fusion derivative set important precedents for further investigation of biomass degradation by filamentous fungi, especially N. crassa, with applications for characterization and manipulation of novel enzymes.     

The carbohydrate-binding module of Fragaria × ananassa expansin 2 (CBM-FaExp2) binds to cell wall polysaccharides and decreases cell wall enzyme activities “in vitro”

A putative carbohydrate binding module (CBM) from strawberry (Fragaria × ananassa Duch.) expansin 2 (CBM-FaExp2) was cloned and the encoding protein was over-expressed in Escherichia coli and purified in order to evaluate its capacity to bind different cell wall polysaccharides “in vitro”. The protein CBM-FaExp2 bound to microcrystalline cellulose, xylan and pectin with different affinities (K_ad = 33.6 ± 0.44 mL g^?1, K_ad = 11.37 ± 0.87 mL g^?1, K_ad = 10.4 ± 0.19 mL g^?1, respectively). According to “in vitro” enzyme assays, this CBM is able to decrease the activity of cell wall degrading enzymes such as polygalacturonase, endo-glucanase, pectinase and xylanase, probably because the binding of CBM-FaExp2 to the different substrates interferes with enzyme activity. The results suggest that expansins would bind not only cellulose but also a wide range of cell wall polymers.     

Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding

The biochemical mechanisms underlying cell wall expansion in plants have long been a matter of conjecture. Previous work in our laboratory identified two proteins (named "expansins") that catalyze the acid-induced extension of isolated cucumber cell walls. Here we examine the mechanism of expansin action with three approaches. First, we report that expansins did not alter the molecular mass distribution or the viscosity of solutions of matrix polysaccharides. We conclude that expansins do not hydrolyze the major pectins or hemicelluloses of the cucumber wall. Second, we investigated the effects of expansins on stress relaxation of isolated walls. These studies show that expansins account for the pH-sensitive and heat-labile components of wall stress relaxation. In addition, these experiments show that expansins do not cause a progressive weakening of the walls, as might be expected from the action of a hydrolase. Third, we studied the binding of expansins to the cell wall and its components. The binding characteristics are consistent with this being the site of expansin action. We found that expansins bind weakly to crystalline cellulose but that this binding is greatly increased upon coating the cellulose with various hemicelluloses. Xyloglucan, either solubilized or as a coating on cellulose microfibrils, was not very effective as a binding substrate. Expansins were present in growing cell walls in low quantities (approximately 1 part in 5000 on a dry weight basis), suggesting that they function catalytically. We conclude that expansins bind at the interface between cellulose microfibrils and matrix polysaccharides in the wall and induce extension by reversibly disrupting noncovalent bonds within this polymeric network. Our results suggest that a minor structural component of the matrix, other than pectin and xyloglucan, plays an important role in expansin binding to the wall and, presumably, in expansin action.     

Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.     

Effect of physical conditions and chemicals on the binding of a mini-CbpA from Clostridium cellulovorans to a semi-crystalline cellulose ligand

Aims:  To investigate the effect that environmental factors have on Clostridium cellulovorans cellulose binding domain (CBD) binding to a semi-crystalline cellulose ligand, namely Avicel.
Methods and Results:  The behaviour of a 58 kDa mini-CbpA protein containing the CBD from the scaffoldin protein of C. cellulovorans was studied in the presence of various environmental factors, in order to determine whether such factors promote or reduce CBD binding to its ligand, thus potentially affecting its activity on the substrate. The amount of binding was found to be dependent on the Avicel concentration and optimal binding occurred when the ligand concentration was 15 mg ml⁻¹. Optimal CBD binding occurred at pH 7·0 and at an incubation temperature of 28°C. The effects of dithiothreitol (DTT), 2-mercaptoethanol, acetone, butanol, ethanol and butyric acid were also investigated.
Conclusions:  Temperature, pH, DTT, 2-mercaptoethanol and solvents were shown to affect the binding of C. cellulovorans CBD to Avicel.
Significance and Impact of the Study:  Clostridium cellulovorans CBD binding to Avicel is affected by physical conditions and chemicals.     

Regulation of stomatal opening by the guard cell expansin AtEXPA1

Stomatal movement is strictly regulated by various intracellular and extracellular factors in response environmental signals. In our recent study, we found that an Arabidopsis guard cell expressed expansin, AtEXPA1, regulates stomatal opening by altering the structure of the guard cell wall. This addendum proposes a mechanism by which guard cell expansins regulate stomatal movement.Key words: expansin, stomatal movement, AtEXPA1, guard cell, wall looseningStomatal movement is the most popular model system for cellular signaling transduction research. A complicated complex containing many proteins has been proposed to control stomatal responses to outside stimuli. The known regulation factors are primarily located in the nucleus, cytoplasm, plasma membrane and other intracellular organelles.^1,2 Although the cell wall structure of the stomata is different from that of other cells,^3,4 the presence of stomatal movement regulation factors in the cell wall has seldom been reported in reference ⁵. In our previous work, we found that extracellular calmodulin stimulates a cascade of intracellular signaling events to regulate stomatal movement.⁶ The involvement of this signaling pathway is the first evidence that cell wall proteins play an important role in regulation of stomatal opening. Cell wall-modifying factors constitute a major portion of cell wall proteins. However, the role of these factors in the regulation of stomatal movement is not yet known.Expansins are nonenzymatic proteins that participate in cell wall loosening.^7–9 Expansins were first identified as “acid-growth” factors because they have much higher activities at acidic pHs.^10,11 It has been reported that expansins play important roles in plant cell growth, fruit softening, root hair emergence and other developmental processes in which cell wall loosening is involved.^7–9,12,13 Wall loosening is an essential step in guard cell swelling and the role of stomatal expansins was investigated. AtEXPA1 is an Arabidopsis guard-cell-specific expansin.^13,14 Over-expressing AtEXPA1 increases the rate of light-induced stomatal opening,^14,15 while a potential inhibitor of expansin activity, AtEXPA1 antibody, reduces the sensitivity of stomata to stimuli.¹⁴ We showed that the transpiration rate and the photosynthesis rate in plant lines overexpressing AtEXPA1 were nearly two times the rates for wild-type plants (Fig. 1). These in plant data revealed that expansins accelerated stomatal opening under normal physiological conditions. In addition, the increases in the transpiration and photosynthesis rates strongly suggested the possibility of exploiting expansin-regulated stomatal sensitivity to modify plant drought tolerance. Compared with the effect of hydrolytic cell wall enzymes, the destruction of cell wall structures induced by expansins is minimal. In addition, it is very difficult to directly observe the changes in the guard cell wall structure caused by expansins during stomatal movement. Our recent work showed that, in AtEXPA1-overexpressing plants, the volumetric elastic modulus is lower than in wild-type plants,¹⁴ which indicates the wall structure was loosened and that the cell wall was easier to extend. Taken together, our data suggest that expansins participate in the regulation of stomatal movement by modifying the cell walls of guard cells.Open in a separate windowFigure 1Effects of AtEXPA1 overexpression on transpiration rates and photosynthesis rates. The transpiration rate (left) and photosynthesis rate (right) of wild-type and transgenic AtEXPA1 lines were measured at 10:00 AM in the greenhouse after being watered overnight. The illumination intensity was 180 µmol/m²·s. Bars represent the standard error of the mean of at least five plants per line.It is well known that the activation of proton-pumping ATPase (H⁺-ATPase) in the plasma membrane is an early and essential step in stomatal opening.¹⁶ The action of the pump results in an accumulation of H⁺ outside of the cell, increases the inside-negative electrical potential across the plasma membrane and drives potassium uptake through the voltage-gated, inward-rectifying K⁺ channels.^17–19 The main function of the H⁺ pump is well accepted to create an electrochemical gradient across the plasma membrane; however, the other result is the acidification of the guard cell wall, which may also contribute to stomatal opening. A possible mechanism responsible for this effect is as follows. Expansins are in an inactive state when the stomata are in the resting state. Stomatal opening signals induce wall acidification and activate expansins. Then, the expansins move along with cellulose microfibrils and transiently break down hydrogen bonding between hemicellulose and the surface of cellulose microfibrils,^20,21 facilitating the slippage of cell wall polymers under increasing guard cell turgor pressure. The guard cell then swells and the stomata open (Fig. 2).Open in a separate windowFigure 2Model of how guard cell wall expansins regulate stomatal opening. Environmental stimuli, e.g., light, activate guard cell plasma membrane H⁺-ATPases to pump H⁺ into the extracellular wall space. The accumulation H⁺ acidifies the cell wall and induces the activation of expansin. The active expansin disrupts non-covalent bonding between cellulose microfibrils and matrix glucans to enable the slippage of the cell wall. The wall is loosened coincident with guard cell swelling and without substantial breakdown of the structure.Although our results indicate that AtEXPA1 regulates stomatal movement, the biochemical and structural mechanism by which AtEXPA1 loosens the cell wall remains to be discovered. It remains to figure out the existing of other expansins or coordinators involving in this process. In addition, determining the roles of expansins and the guard cell wall in stomatal closing is another main goal of future research.     

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.