Pectins as mediators of wall porosity in soybean cells

The non-invasive technique of fluorescence redistribution after photobleaching was employed on soybean (Glycine max (L.) Merr.) root cells grown in suspension culture to examine macromolecular transport across plant cell walls. Using both fluorescently derivatized dextrans and proteins of graded size, a functional range of diameters for putative trans-wall channels was determined to be 6.6–8.6 nm. A mild treatment with pectinase apparently enlarged the channels, without adversely affecting cell viability, enabling significantly larger molecules to pass through the wall. Treatment of the cells with cellulysin or protease did not have this enlargement effect. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Fl-dextran fluorescein-derivatized dextran - FRAP fluorescence redistribution after photobleaching - kDa kilodalton     

Cell wall proteomics of sugarcane cell suspension cultures

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl₂ and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second‐generation ethanol production.     

植物激素在植物细胞壁扩展中的作用

细胞壁不仅是植物细胞结构的重要组成部分,而且控制着细胞的大小、形状和生长。细胞经有丝分裂后,原生质体吸水膨胀,细胞壁重塑,新生壁物质合成,纤维素定向沉积等引发细胞壁生长。在这些过程中,乙烯（ethylene,ET）、生长素（auxin）、赤霉素（gibberellin,GA）、油菜素甾醇（brassinosteroids,BR）等植物激素调控细胞壁生长相关酶类如纤维素合酶复合体（cellulose synthase A,CESA）、扩展素（expansin,EXP）、木葡聚糖内糖基转移酶／水解酶（xyloglucan endotran glucosylase／hydrolase,XET／XTH）的表达活性,进而调控细胞壁扩展,促使细胞壁的生长。     

Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

Shear stress effects on plant cell suspension cultures in a rotating wall vessel bioreactor

A rotating wall vessel, designed for growth of mammalian cells under microgravity, was used to study shear effects on Taxus cuspidata plant suspension cell cultures. Shear stress, as quantified by defined shear fields of Couette viscometers, improved specific cell growth rates and was detrimental to volumetric product formation rates. Received 5 January 1998/ Accepted in revised form 8 December 1998     

A method for growing stationary plant suspension cell cultures

Summary Stationary culture of plant cell suspensions has been achieved. Slurries, produced when small amounts of agar (0.1–0.4%) were added to culture media, were used to suspend plant cells. Growth proceeded more slowly than in standard shake culture, but cells remained viable for months of culture. This method of growing plant cells in stationary culture should be useful for general applications including long-term cell culture, shipment of cultures, and physiological, molecular biological, and pathological studies. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Editor’s Statement This procedure for growing stationary suspension cultures in an agar slurry should be useful for shipping suspensions and for long-term maintenance of little used or back-up cultures.     

Methylation reactions and the phytoalexin response in alfalfa suspension cultures

 In order to determine why the activated methyl cycle is up-regulated in plants undergoing defence responses to fungal pathogens we have monitored the utilisation of methyl groups derived from methionine in cell-suspension cultures of alfalfa (Medicago sativa L.) treated for various times with fungal elicitor, by carrying out a parallel labelling study with [³⁵S]methionine and [methyl-³H]methionine. The distribution of the two radiolabels among the medium, soluble cellular components and cell wall was then determined. In the absence of elicitor the utilisation of the two radiolabels was similar. However, in the presence of the elicitor the total incorporation of radioactivity from [methyl-³H]methionine into metabolites was far greater than from [³⁵S]methionine, indicating that the methyl label had been utilised in methylation reactions. Elicitor treatment resulted in up to a sixfold increase in the use of ³H-methyl groups in the methylation of hydrophobic metabolites. In the period 0–24 h after elicitor treatment, increased methylation was directed largely into the synthesis of the isoflavonoid phytoalexin medicarpin and related metabolites. Newly synthesized phytoalexins were exported into the medium, while a significant proportion of the medicarpin accumulating in the cell in the early stages of elicitation was derived from the hydrolysis of its respective conjugate. Elicitor treatment also modified the incorporation of ³H-methyl groups into the cell wall. Between 0 and 24 h after elicitor treatment the methylation of pectin in the cell wall declined. After 24 h, pectin methylation recovered and was associated with an increase in the methylation of other wall-bound polysaccharide components. Since no other major metabolic sink for the increased methylation was determined we conclude that the increased activity of the activated methyl cycle during defence interactions in alfalfa is required to support phytoalexin synthesis and cell wall modifications. Received: 1 August 1996 / Accepted: 24 October 1996     

Absolute measurement of cell expansion in plant cell suspension cultures

A method is described for the measurement of intracellular volume (V_i) in cell cultures. In principle, any stable compound that neither penetrates the plasma membrane nor binds to the cells can be used to trace the total extracellular (apoplastic) volume and hence to estimate the intracellular volume. No suitable coloured or UV-absorbing compound could be found among those tested; the main problems were binding to the cell surface and/or instability in the medium. However, [¹⁴C]mannitol was an acceptable apoplastic marker, by use of which we showed that 21–47% of total packed cell volume (PCV) was intracellular, and 14–33% of total settled cell volume (SCV) was intracellular. Therefore, measurements of PCV and SCV misrepresent cell expansion to a variable extent. Cultures of Acer, Rosa, Spinacia and Zea achieved final symplastic volumes of only 9, 14, 6 and 6%, respectively, of the total suspension culture volume.     

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30–40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans. Received: 26 November 1996 / Accepted: 30 January 1997     

Xyloglucan endotransglucosylase activity loosens a plant cell wall

BACKGROUND AND AIMS: Plant cells undergo cell expansion when a temporary imbalance between the hydraulic pressure of the vacuole and the extensibility of the cell wall makes the cell volume increase dramatically. The primary cell walls of most seed plants consist of cellulose microfibrils tethered mainly by xyloglucans and embedded in a highly hydrated pectin matrix. During cell expansion the wall stress is decreased by the highly controlled rearrangement of the load-bearing tethers in the wall so that the microfibrils can move relative to each other. Here the effect was studied of a purified recombinant xyloglucan endotransglucosylase/hydrolase (XTH) on the extension of isolated cell walls. METHODS: The epidermis of growing onion (Allium cepa) bulb scales is a one-cell-thick model tissue that is structurally and mechanically highly anisotropic. In constant load experiments, the effect of purified recombinant XTH proteins of Selaginella kraussiana on the extension of isolated onion epidermis was recorded. KEY RESULTS: Fluorescent xyloglucan endotransglucosylase (XET) assays demonstrate that exogeneous XTH can act on isolated onion epidermis cell walls. Furthermore, cell wall extension was significantly increased upon addition of XTH to the isolated epidermis, but only transverse to the net orientation of cellulose microfibrils. CONCLUSIONS: The results provide evidence that XTHs can act as cell wall-loosening enzymes.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

Changes in the pattern of phospholipid synthesis during the induction by cytokinin of cell division in soybean suspension cultures

A method is described for preparing fully viable, cytokinin-starved soybean (Glycine max (L.) Merr. cv. Acme) cells from a suspension-culture of callus tissue. The cells respond to kinetin treatment by re-initiating cell division. We present evidence, from the pattern of incorporation of ³²P-labelled inorganic phosphate into individual phospholipids during the first hour of this response, that the synthesis of phosphatidylinositol (PI) and of phosphatidic-acid head-groups is affected within 15 min. The polyphosphoinositide phosphatidylinositol 4-phosphate, but not phosphatidylinositol 4,5-bisphosphate, was detected in the tissue. The characteristics of cytokinin-induced PI synthesis in cytokinin-starved soybean cells appear to resemble the PI response of animal cells.Abbreviations DPG diphosphatidylglycerol - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PS phosphatidylserine - Pi inorganic phosphate - TLC thin-layer chromatography     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U-¹⁴C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP.     

Effects of perfluorinated oxygen carrier application in yeast, fungi and plant cell suspension cultures

The growth of the yeast Saccharomyces cerevisiae, the fungus Rhizopus nigricans and Nicotiana tabacum cells with perfluorodecalin as an oxygen carrier has been studied. The volumetric mass transfer coefficient (k_La) measured by the dynamic method was higher for the perfluorodecalin oxygenation system than for the conventional aeration system. The results show that perfluorocarbon can be successfully used as an efficient gas carrier, especially for the culture of delicate plant cells. The increase in yeast biomass in the suspension culture aerated by perfluorodecalin was as much as 110% higher than in the culture aerated by air. The fungus R. nigricans grew better when the conventional aeration system was used due to the fact that growth of the mycelium is limited by the transport of oxygen by diffusion in the pellets rather than by interfacial oxygen transport. In the case of isolated tobacco cells, an increase of over 350% in biomass growth was observed for the PFC aeration system.     

气体成分对植物细胞悬浮培养的影响

气体成分对植物悬浮培养细胞的生长和次生代谢物的产量有深刻的影响。就有关氧、二氧化碳、乙烯和一些未知成分作用的研究进行了综述。     

Dynamics of cell wall structure in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein-polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers' yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed.     

Low-molecular-weight precursors for defense-related cell wall hydroxycinnamoyl esters in elicited parsley suspension cultures

Cell walls from suspension cultures of parsley (Petroselinum crispum L.) induced with a fungal elicitor contained hydroxycinnamoyl ester groups presumably not bound to pectic polysaccharides. Extracts from these cells were separated into a range of low-molecular-weight compounds containing esterified ferulic and p-coumaric acid as well as glucose and some arabinose. Similar compounds also accumulated extracellularly in elicited cultures but only in the presence of the peroxidase inhibitor ascorbate, suggesting that they may represent the exported precursors for cell wall hydroxycinnamic acids. From cultures elicited in the presence of ascorbate, alkali released from the cell walls more ferulic, p-coumaric and p-hydroxybenzoic acid, as well as p-hydroxybenzaldehyde and vanillin, indicating that the corresponding wall phenolics can all become further cross-linked. Received: 6 September 1996 / Revision received: 10 March 1997 / Accepted: 10 April 1997