Enhancement of growth by expression of poplar cellulase in Arabidopsis thaliana

To study the role of cellulose and cellulase in plant growth, we expressed poplar cellulase (PaPopCel1) constitutively in Arabidopsis thaliana. Expression increased the size of the rosettes due to increased cell size. The change in growth was accompanied by changes in biomechanical properties due to cell wall structure indicative of decrease in xyloglucan cross-linked with cellulose microfibrils by chemical analysis and nuclear magnetic resonance (NMR) spectra. The result supports the concept that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase to loosen xyloglucan intercalation and this irreversible wall modification promotes the enlargement of plant cells.     

涝渍胁迫对杨树苗期叶片生长及其生理性状的影响

以３种典型的美洲黑杨苗木（Ｉ６９、ＮＬ８０１０５和ＮＬ８０３５１）在不同的涝渍胁迫条件下,苗木叶面积生长明显减慢;叶片气孔开度显著减小;叶片水势和丙二醛（ＭＤＡ）含量有所增加;超氧化物歧化酶（ＳＯＤ）活性无明显变化;叶片中的全Ｎ、全Ｐ和全Ｋ含量发生变化。综合分析认为,Ｉ６９杨在强涝渍胁迫下抗耐能力较高。ＮＬ８０３５１杨在弱涝渍胁迫下适应性较强,短期涝渍对杨树无性系苗木无明显影响,３０ｄ以上的涝渍对其影响显著。     

光诱导下杂种杨无性系叶角和叶绿体的运动

自然光结合人工光源对几个杂种杨无性系叶片进行处理后,对其在两种光环境下３种叶角（方位角、方向角、悬挂角）和叶绿体的运动方式进行了研究。结果发现：各无性系的叶角运动在晴天比比阴天强烈,特别是三倍体无性系最为明显,晴天上午９：００－１１：００,三倍体无性系ＺＨ６和Ｂ３４６通过方位角运动来避免强光胁迫,各无性系主要通过方向角和悬挂角的变化来调节获得最佳太阳辐射,在中午受到强光胁迫时存在明显的“避光运动”。三倍体无性系和某些二倍体无性系在避光运动方式上是不同的,三倍体无性系ＺＨ６和Ｂ３４６采用叶片下垂形式,而二倍体无性系Ｂ１１则采用叶片竖立方式,无论是晴天还是阴天,植物的节律性运动可能参与了叶角运动。受光胁迫时,栅栏组织的叶绿通过不同的运动排列方式来实现对光辐射的最佳吸收。强光胁迫下叶绿体沿径向细胞壁排列,以尽量养活接受过量的太阳辐射,处于弱光条件时,叶绿体则充满足整个细胞,以扩大上太阳辐射的表面积,三倍体无性系对光诱导的敏感程度程度要高于二倍体无性系。     

The rice dynamin‐related protein DRP2B mediates membrane trafficking,and thereby plays a critical role in secondary cell wall cellulose biosynthesis

Membrane trafficking between the plasma membrane (PM) and intracellular compartments is an important process that regulates the deposition and metabolism of cell wall polysaccharides. Dynamin‐related proteins (DRPs), which function in membrane tubulation and vesiculation are closely associated with cell wall biogenesis. However, the molecular mechanisms by which DRPs participate in cell wall formation are poorly understood. Here, we report the functional characterization of Brittle Culm3 (BC3), a gene encoding OsDRP2B. Consistent with the expression of BC3 in mechanical tissues, the bc3 mutation reduces mechanical strength, which results from decreased cellulose content and altered secondary wall structure. OsDRP2B, one of three members of the DRP2 subfamily in rice (Oryza sativa L.), was identified as an authentic membrane‐associated dynamin via in vitro biochemical analyses. Subcellular localization of fluorescence‐tagged OsDRP2B and several compartment markers in protoplast cells showed that this protein not only lies at the PM and the clathrin‐mediated vesicles, but also is targeted to the trans‐Golgi network (TGN). An FM4‐64 uptake assay in transgenic plants that express green fluorescent protein‐tagged OsDRP2B verified its involvement in an endocytic pathway. BC3 mutation and overexpression altered the abundance of cellulose synthase catalytic subunit 4 (OsCESA4) in the PM and in the endomembrane systems. All of these findings lead us to conclude that OsDRP2B participates in the endocytic pathway, probably as well as in post‐Golgi membrane trafficking. Mutation of OsDRP2B disturbs the membrane trafficking that is essential for normal cellulose biosynthesis of the secondary cell wall, thereby leading to inferior mechanical properties in rice plants.     

Cellulase activity on decomposing leaf litter in microcosms

In this paper, we describe cellulase and cellobiose dehydrogenase (CBDH) dynamics in relation to incubation time, mass loss and chemical composition of decomposing deciduous leaf litter. Cellulose disappearance from litter coincided with periods of maximum cellulase activity. CBDH activity peaked later in decomposition after cellulase activity had declined. Enzyme activity patterns differed among litter types when expressed on the basis of decomposition time or cumulative mass loss. The patterns converged when expressed on the basis of chemical composition as indexed by the fraction of cellulose in the lignocellulose complex. We present a three-stage model of decomposition based on temporal changes in cellulase activities and coincident changes in litter chemical composition.     

Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013     

Variations in leaf anatomical characteristics drive the decrease of mesophyll conductance in poplar under elevated ozone

Decline in mesophyll conductance (g_m) plays a key role in limiting photosynthesis in plants exposed to elevated ozone (O₃). Leaf anatomical traits are known to influence g_m, but the potential effects of O₃-induced changes in leaf anatomy on g_m have not yet been clarified. Here, two poplar clones were exposed to elevated O₃. The effects of O₃ on the photosynthetic capacity and anatomical characteristics were assessed to investigate the leaf anatomical properties that potentially affect g_m. We also conducted global meta-analysis to explore the general response patterns of g_m and leaf anatomy to O₃ exposure. We found that the O₃-induced reduction in g_m was critical in limiting leaf photosynthesis. Changes in liquid-phase conductance rather than gas-phase conductance drive the decline in g_m under elevated O_3, and this effect was associated with thicker cell walls and smaller chloroplast sizes. The effects of O₃ on palisade and spongy mesophyll cell traits and their contributions to g_m were highly genotype-dependent. Our results suggest that, while anatomical adjustments under elevated O₃ may contribute to defense against O₃ stress, they also cause declines in g_m and photosynthesis. These results provide the first evidence of anatomical constraints on g_m under elevated O₃.     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

杨树和桤木落叶混合分解对土壤微生物生物量的影响

通过室内培养,研究了杨树和江南桤木落叶混合分解过程中两种落叶的混合比例及落叶添加方式对土壤微生物生物量的影响.结果表明:落叶混合比例对土壤微生物生物量碳(MBC)、氮(MBN)均有显著的影响.培养30 d,江南桤木落叶比例为50％以上的土壤MBC和MBN显著高于纯杨树落叶处理及对照;75 d时,江南桤木落叶比例≥40％的土壤MBC和≥30％的土壤MBN均显著高于纯杨树落叶处理及对照;135 d后,江南桤木落叶比例≥20％的土壤MBC和≥40％的土壤MBN均显著高于纯杨树落叶处理及对照.不同混合比例的土壤MBC/MBN无显著差异,总体呈前期增长后期下降的变化趋势.杨树和江南桤木落叶混合分解对土壤MBC和MBN有显著的协同促进作用.在整个培养过程中,落叶添加方式对土壤MBC、MBN和MBC/MBN无显著影响.     

Endosidin20-1 is more potent than endosidin20 in inhibiting plant cellulose biosynthesis and molecular docking analysis of cellulose biosynthesis inhibitors on modeled cellulose synthase structure

Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs and identified nine active analogs named ES20-1 to ES20-9. Among these, endosidin20-1 (ES20-1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. At the biochemical level, we demonstrated that ES20-1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20-1 likely targets the catalytic site of CESA. However, through molecular docking analysis using a modeled structure of full-length CESA6, we found that both ES20 and ES20-1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as isoxaben, C17, and flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are also valuable resources for the development of herbicides. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on a modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20-1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20-1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants, and both ES20 and ES20-1 show an inhibitory effect on bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.     

Effect of surfactants on cellulose hydrolysis

The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low. (c) 1993 John Wiley & Sons, Inc.     

Surface architecture of the plant cell: Biogenesis of the cell wall,with special emphasis on the role of the plasma membrane in cellulose biosynthesis

Cell wall structure and biogenesis in the unicellular green alga, Oocystis apiculata, is described. The wall consists of an outer amourphous primary layer and an inner secondary layer of highly organized cellulosic microfibrils. The primary wall is deposited immediately after cytokinesis. Golgi-derived products contribute to this layer. Cortical microtubules underlie the plasma membrane immediately before and during primary wall formation. They function in maintaining the elliptical cell shape. Following primary wall synthesis, Golgi-derived materials accumulate on the cell surface to form the periplasmic layer. This layer functions in the deposition of coating and cross-linking substances which associate with cellulosic microfibrils of the incipient secondary wall. Secondary wall microfibrils are assembled in association with the plasma membrane. Freeze-etch preparations of untreated, living cells reveal linear terminal complexes in association with growing cellulosic microfibrils. These complexes are embedded in the EF fracture face of the plasma membrane. The newly synthesized microfibril lies in a groove of the outer leaflet of the plasma membrane. The groove is decorated on the EF fracture face by perpendicular structures termed “ridges.” The ridges interlink with definitive rows of particles associated with the PF fracture face of the inner leaflet of the plasma membrane. These particles are termed “granule bands,” and they function in the orientation of the newly synthesized microfibrils. Microfibril development in relation to a coordinated multienzyme complex is discussed. The process of cell wall biogenesis in Oocystis is compared to that in higher plants.     

Nitrogen deficiency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates

Nitrogen deficiency severely inhibits leaf growth. This response was analysed at the cellular level by growing Lolium perenne L. under 7.5 mM (high) or 1 mM (low) nitrate supply, and performing a kinematic analysis to assess the effect of nitrogen status on cell proliferation and cell growth in the leaf blade epidermis. Low nitrogen supply reduced leaf elongation rate (LER) by 43% through a similar decrease in the cell production rate and final cell length. The former was entirely because of a decreased average cell division rate (0.023 versus 0.032 h(-1)) and thus longer cell cycle duration (30 versus 22 h). Nitrogen status did not affect the number of division cycles of the initial cell's progeny (5.7), and accordingly the meristematic cell number (53). Meristematic cell length was unaffected by nitrogen deficiency, implying that the division and mitotic growth rates were equally impaired. The shorter mature cell length arose from a considerably reduced post-mitotic growth rate (0.033 versus 0.049 h(-1)). But, nitrogen stress did not affect the position where elongation stopped, and increased cell elongation duration. In conclusion, nitrogen deficiency limited leaf growth by increasing the cell cycle duration and decreasing mitotic and post-mitotic elongation rates, delaying cell maturation.     

Enzymatic digestion of liquid hot water pretreated hybrid poplar

Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200^oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot‐washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot‐washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar‐to‐ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The role of auxin-binding protein 1 in the expansion of tobacco leaf cells

Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor.