Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

小麦根系对铝毒的反应及其与根细胞壁组分和细胞壁对铝的吸附-解吸性能的关系

研究了小麦根系对铝毒的反应与不同根段细胞壁的组分及细胞壁对铝的吸附解吸性能的关系。结果表明,30μmol/LAlCl3可迅速抑制根系伸长,在铝处理30h时其根长仅为对照的30.2%;小麦根系相对伸长率随着铝浓度的提高而急剧降低,30μmol/LAlCl3处理24h对根系伸长的抑制率高达70.9%。小麦根系中距根尖0~10mm根段的铝含量和细胞壁中果胶糖醛酸含量明显高于距根尖10~20mm根段;距根尖0~10mm根段细胞壁对铝的吸附量明显大于距根尖10~20mm根段,而前者吸附态铝的解吸率低于后者;铝浓度从10μmol/L提高到20μmol/L时细胞壁对铝的吸附量增加,但对铝的解吸没有明显影响。采用1.0mol/LNH3·H2O对细胞壁预处理2h降低果胶甲基酯化程度后,铝吸附量降低了20.9%,但对铝解吸率没有影响。由此可见,小麦根尖是铝毒的主要位点,细胞壁果胶含量和果胶甲基酯化程度对小麦不同根段细胞壁对铝的吸附、积累具有重要作用,铝与细胞壁的结合是根系对铝毒胁迫反应的重要原因。     

Changes in cell wall composition associated with maturation in the gymnosperm Araucaria angustifolia

A general structural characterization and an investigation on the dynamics of formation of cell wall polysaccharides was performed, using plantlets stem samples from a typical gymnosperm from southern Brazil, Araucaria angustifolia, as experimental model. Microscopic examination and monosaccharide composition of plantlet segments at different heights were carried out to show the representative portions of stem cell wall development. The plantlets were divided in portions (tip, middle and base) which were submitted to sequential extractions. The extraction with water gave rise to large amounts of pectic material in the three portions and more highly substituted pectins occurred in the tip portion of the stems. Increase in alkali concentration extracted, respectively, higher amounts of xyloglucan structurally similar to those from dicotyledons. However, oligosaccharides containing galactose and fucose where found in higher amounts in base than tip portion. The changes in cell wall composition suggest that the development in gymnosperm cell walls follow the same key events as found in dicotyledon walls (type I).     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Distribution of isoflavones in lupin hypocotyls. Possible control of cell wall peroxidase activity involved in lignification

The distribution of 4 key isoflavones (luteone, genistein, 2'-hydroxygenistein and wighteone) in lupin ( Lupinus albus L. cv. multolupa) hypocotyls shows a gradient that diminishes from young to old tissues. A spatial gradient occurs within the hypocotyl, and a temporal gradient in both the outermost vascular and epidermal tissues. Not only does a gradient exist in respect to the quantity of isoflavones, but there is also a gradient in respect to the type of isoflavone. Thus, wighteone is mainly associated with the non-meristematic zones of the lupin hypocotyl. A close relationship was found between the distribution and the localization in the walls of phloem cells of both peroxidase (EC 1.11.1.7) and isoflavones. This observation suggests an in vivo peroxidase-isoflavone interconnection. In fact, lupin isoflavones are able to inhibit the peroxidase-catalyzed oxidation of the lignin precursor coniferyl alcohol, probably due to the co-oxidation of isoflavones in the reaction media. The results are discussed on the basis of a possible role for isoflavones in controlling cell wallperoxidase activity involved in the lignification of phloem cells.     

Relation of cell wall peroxidase activity with growth in epicotyls of Cicer arietinum. Effects of calmodulin inhibitors

Sánchez, O.J., Pan, A., Nicolás, G. and Labrador, E. 1989. Relation of cell wall peroxidase activity with growth in epicotyls of Cicer arietinum. Effects of calmodulin inhibitors.
Peroxidases are bound ionically to cell walls in epicotyls of Cicer arietinum L. cv. Castellana. The cell wall peroxidase activity increases during the growth of epicotyls, being the lowest in 3-day-old epicotyls with high growth capacity. The cell wall phenolic compounds, postulated natural substrates of cell wall peroxidases, also increase during growth.
The calmodulin inhibitors chlorpromazine and trifluoperazine decrease the elongation rate of epicotyls of Cicer arietinum. These inhibitors also cause an increase in the cell wall peroxidase activity and in the level of phenolic compounds. A possible regulatory effect of calmodulin on peroxidase activity is postulated.     

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.     

Kinetin induced changes in cell wall composition of tobacco callus

Culture of tobacco callus on high or low kinetin in light or darkness leads to changed tissue texture and associated changes in cell wall composition. In particular, friable callus (low kinetin, darkness) cell walls have a greater extensin content and an altered composition of arabinose and xylose containing hemicelluloses compared with cell walls of compact callus (high kinetin, darkness). The possible importance of these differences in determining callus friability is discussed.     

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207and/or EC 3.2.1.15 [EC] 1) are enzymes involved in the modificationof cell wall structure by cleaving and, often, also re-joiningxyloglucan molecules in primary plant cell walls. Using a poolof antibodies raised against an enriched cell wall protein fraction,a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNAexpression library obtained from the elongation zone of themaize root. The predicted protein has a putative N-terminalsignal peptide and possesses the typical domains of this enzymefamily, such as a catalytic domain that is homologous to thatof Bacillus macerans β-glucanase, a putative N-glycosylationmotif, and four cysteine residues in the central and C terminalregions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1reveals that it belongs to subgroup 4, so far only reportedfrom Poaceae monocot species. ZmXTH1 has been expressed in Pichiapastoris (a methylotrophic yeast) and the recombinant enzymeshowed xyloglucan endotransglucosylase but not xyloglucan endohydrolaseactivity, representing the first enzyme belonging to subgroup4 characterized in maize so far. Expression data indicate thatZmXTH1 is expressed in elongating tissues, modulated by cultureconditions, and induced by gibberellins. Transient expressionassays in onion cells reveal that ZmXTH1 is directed to thecell wall, although weakly bound. Finally, Arabidopsis thalianaplants expressing ZmXTH1 show slightly increased xyloglucanendohydrolase activity and alterations in the cell wall structureand composition. Key words: Cell elongation, cell wall, plant transformation, XEH, XET, XTH, Zea mays     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

Changes in physical properties and cell wall polysaccharides of tomato (Lycoperskon esculentum) pericarp tissues

Green and red tomato pericarp tissues were subjected to stress-relaxation analyses to evaluate their physical properties. Significant decreases in the initial stress, minimum stress-relaxation and maximum stress-relaxation times in the red tissues predict the losses of both viscosity and elasticity in the tissue. Cell walls of red fruit yielded more water-soluble polysaccharides and less pectin, hemicelluloses and cellulose. Average molecular mass of pectin determined by gel filtration chromatography was similar in the green and red, but molecular mass of hemicellulose of red fruit walls was reduced to 50% of that of the green fruit. The decreases in the amount of hemicellulose B and in the average molecular mass were associated primarily with the degradation of xylo-glucans. These data demonstrate that pectin solubilization, depolymerization of xyloglucans and over-all changes in the quantity of cell wall polysaccharide fractions contribute to tomato fruit softening.     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.     

Investigation of cell wall composition related to stem lodging resistance in wheat (Triticum aestivum L.) by FTIR spectroscopy

We explored the rapid qualitative analysis of wheat cultivars with good lodging resistances by Fourier transform infrared resonance (FTIR) spectroscopy and multivariate statistical analysis. FTIR imaging showing that wheat stem cell walls were mainly composed of cellulose, pectin, protein, and lignin. Principal components analysis (PCA) was used to eliminate multicollinearity among multiple peak absorptions. PCA revealed the developmental internodes of wheat stems could be distributed from low to high along the load of the second principal component, which was consistent with the corresponding bands of cellulose in the FTIR spectra of the cell walls. Furthermore, four distinct stem populations could also be identified by spectral features related to their corresponding mechanical properties via PCA and cluster analysis. Histochemical staining of four types of wheat stems with various abilities to resist lodging revealed that cellulose contributed more than lignin to the ability to resist lodging. These results strongly suggested that the main cell wall component responsible for these differences was cellulose. Therefore, the combination of multivariate analysis and FTIR could rapidly screen wheat cultivars with good lodging resistance. Furthermore, the application of these methods to a much wider range of cultivars of unknown mechanical properties promises to be of interest.     

The use of lanthanum to characterize cell wall permeability in relation to deep supercooling and extracellular freezing in woody plants: II. Intrageneric comparisons betweenBetula lenta andBetula papyrifera

Summary The permeability and porosity of xylem cell walls are believed to play a major role in defining the ability of a cell or tissue to exhibit deep supercooling. Lanthanum nitrate, was utilized to contrast the permeability of stem tissues inB. lenta, which exhibits deep supercooling, withB. papyrifera, which exhibits equilibrium freezing. Although the two species differed greatiy in their response to low temperature, distribution of lanthanum deposits was quite similar. Primary cell walls of all xylem cell types appeared permeable although lanthanum deposition was patchy. Secondary cell walls of fiber cells were also permeable to lanthanum whereas the secondary wall of vessel elements and xylem parenchyma appeared impermeable to the lanthanum. Pit membranes, in all cell types and the protective layer in xylem parenchyma frequently exhibited deposits of lanthanum. Results of this study indicate that the porosity and permeability of the pit membrane, rather than the entire cell wall may determine the rate of water loss from xylem parenchyma to sites of extracellular ice. If differences exist between the species in the physical structure of these sites, they may explain differences observed in their response to freezing.Abbreviations DTA differential thermal analysis - HTE high temperature exotherm - LTE low temperature exoterm - F fiber cell - V vessel element     

Ectopic expression of a novel OsExtensin‐like gene consistently enhances plant lodging resistance by regulating cell elongation and cell wall thickening in rice

Plant lodging resistance is an important integrative agronomic trait of grain yield and quality in crops. Although extensin proteins are tightly associated with plant cell growth and cell wall construction, little has yet been reported about their impacts on plant lodging resistance. In this study, we isolated a novel extensin‐like (OsEXTL) gene in rice, and selected transgenic rice plants that expressed OsEXTL under driven with two distinct promoters. Despite different OsEXTL expression levels, two‐promoter‐driven OsEXTL‐transgenic plants, compared to a rice cultivar and an empty vector, exhibited significantly reduced cell elongation in stem internodes, leading to relatively shorter plant heights by 7%–10%. Meanwhile, the OsEXTL‐transgenic plants showed remarkably thickened secondary cell walls with higher cellulose levels in the mature plants, resulting in significantly increased detectable mechanical strength (extension and pushing forces) in the mature transgenic plants. Due to reduced plant height and increased plant mechanical strength, the OsEXTL‐transgenic plants were detected with largely enhanced lodging resistances in 3 years field experiments, compared to those of the rice cultivar ZH11. In addition, despite relatively short plant heights, the OsEXTL‐transgenic plants maintain normal grain yields and biomass production, owing to their increased cellulose levels and thickened cell walls. Hence, this study demonstrates a largely improved lodging resistance in the OsEXTL‐transgenic rice plants, and provides insights into novel extensin functions in plant cell growth and development, cell wall network construction and wall structural remodelling.     

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37°C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.     

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.     

Infrared microspectroscopy: Sampling heterogeneity in plant cell wall composition and architecture

The use of probes such as monoclonal and polyclonal antibodies to specific cell wall components, at both the light and electron microscope levels, has demonstrated the diversity in cell wall composition between species, between tissues, between different regions of the cell surface, and even within a single wall. Traditional methods of cell wall analysis have provided valuable information on wall composition and architecture, but, by having to rely on the use of bulk samples, have averaged out this intrinsic heterogeneity. Fourier Transform Infrared (FTIR) microspectroscopy addresses this problem by providing chemical information from an area as small as 10×10 μm of a single cell wall fragment or area of a tissue section that has been imaged with a microscope accessory.
We have used FTIR microspectroscopy as a powerful and extremely rapid assay for wall components and putative cross-links in muro. The spectra are sensitive to polymer conformation, and the use of polarisers in the microscope accessory allows the orientation of particular functional groups to be determined, with respect to the long axis of elongating cells. The spectra constitute species and tissue-specific 'fingerprints', and the use of classical discriminant analysis may provide the opportunity for correlating spectral features with chemical, architectural or rheological wall properties. Spectral mapping of an area of a specimen allows the morphological features resulting from cell growth and differentiation to be characterised chemically at the single cell level.     

Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis

Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.