Cell wall development in maize coleoptiles

The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.

The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages.

     

Auxin-induced changes in cell wall extensibility of maize roots

The rheological properties of corn (Zea mays L. cv. Garant) root elongation zones were investigated by means of a computer-controlled extensiometer. Creep closely followed a logarithmic time function, which was used to quantify creep activity. Pretreatment with auxin, which inhibits extension growth in roots, lowered the creep activity and the apparent plastic extensibility. While the time course of the inhibition of apparent plastic extensibility lagged behind the cessation of elongation growth, the drop in creep activity matched the growth inhibition more closely. Creep activity and apparent plastic extensibility were not significantly affected by pH. These data support the view that the auxin-induced cell wall stiffening (e.g. by cross-linking processes), while causal for the growth inhibition, is not brought about by a cell wall alkalinization. Received: 10 December 1996 / Accepted: 19 August 1997     

Cell wall polysaccharide interactions in maize bran

Sequential extractions with alkali have been carried out in order to study the nature of linkages which hold heteroxylans in maize bran cell walls. Treatment with 0.5 sodium hydroxide at 30 °C for 2 h released all the phenolic acids (p-coumaric, ferulic, and diferulic) but extracted only ˜30% of heteroxylans (S₁); further treatment with 1.5 potassium hydroxide at 100 °C for 2 h released the remaining heteroxylans (S₂). The heteroxylans from S₁ and S₂ had a similar neutral sugar composition and structure, but their weight average molecular weights were 270 kDa (M_w/M_n = 2) and 370 kDa (M_w/M_n = 2.8), respectively. Proteins (5%) are found with polysaccharides from S₁ and S₂, with different amino acid composition. The results suggest that covalent linkages through phenolic acids are only partly responsible for the associations of maize bran heteroxylans in the cell wall and that linkages to structural cell wall proteins were probably the main cause of heteroxylan insolubility.     

Cell wall-associated malate dehydrogenase activity from maize roots

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1 M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn²⁺ and Cu²⁺ in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn²⁺ and Cu²⁺ in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.     

Cell wall-bound trans- and cis-ferulic acids in growing maize roots

The levels of cell wall-bound trans - and cis -ferulic acids in roots of dark grown Zea mays cv. LG11 plants were measured. They were quantified after alkaline hydrolysis of purified cell walls by reversed phase HPLC using trans -cinnamic acid as internal standard. The total amount of ferulic acid ( trans - and cis -ferulic acid) in the root base was 3–4 times higher than in the root tip. Cis -ferulic acid represented between 2% (tip) and 18% (base) of the total ferulic acid content. The total content of trans - and cis -ferulic acids was approximately the same in the stele and the cortex, but the level of cis -ferulic acid in the stele was 5–6 times higher than in the cortex. Trans - and cis -ferulic acid levels as well as the percentage of cis -ferulic acid in the elongation zone were steady between 48 and 96 h after the beginning of germination. Slowly growing roots contained more wall-bound ferulic acids, particularly cis -ferulic acid, than fast growing roots. This relationship was found in the differentiation zone but not in the elongation zone. The importance of cell wall-bound trans - and cis -ferulic acids is discussed in the context of root growth and differentiation.     

Cell wall architecture of the elongating maize coleoptile

The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed.     

Metabolic adaptations to environmental changes in Caenorhabditis elegans

Metabolic adaptations to environmental changes were studied in Caenorhabditis elegans. To assess adjustments in enzyme function, maximum activities of key enzymes of main metabolic pathways were determined. After a 12 h incubation at varying temperatures (10, 20°C) and oxygen supplies (normoxia or anoxia), the activities of the following enzymes were determined at two measuring temperatures in tissue extracts: lactate dehydrogenase (LDH; anaerobic glycolysis), 3-hydroxyacyl-CoA-dehydrogenase (HCDH; fatty acid oxidation), isocitrate dehydrogenases (NAD-IDH, NADP-IDH; tricarboxylic acid cycle) and isocitrate lyase (ICL; glyoxylate cycle). Incubation at 20°C induced a strong increase in maximum LDH activity. Anoxic incubation caused maximum HCDH and NADP-IDH activities and, at 10°C incubation, LDH activity to increase. Maximum NAD-IDH and ICL activities were not influenced by any type of incubation. In order to study the time course of metabolic adaptations to varying oxygen supplies, relative quantities of free and protein-bound NADH were determined in living C. elegans using time-resolved fluorescence spectroscopy. During several hours of anoxia, free and protein-bound NADH showed different time courses. One main result was that just at the moment when the protein-bound NADH had reached a constant level, and the free NADH started to increase rapidly, the worms fell into a rigor state. The data on enzyme activity and NADH fluorescence can be interpreted on the basis of a two-stage model of anaerobiosis.     

Transgenic rice plants conferring increased tolerance to rice blast and multiple environmental stresses

We isolated a rice gene (denoted YK1), which showed78 percent amino acid sequence homology to the maize HM1gene. A chimeric gene consisting of a promoter and first intron of maizeubiquitin gene and the cDNA of YK1 was introduced intorice via Agrobacterium mediated transformation. Transgenic riceplants overexpressing this chimeric gene were resistant to rice blast(Magnaporthe grisea) disease, which is one of the mostserious pathogens in rice. Furthermore, the same transgenic plants conferredhigh tolerance to several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide.     

Cell wall synthesis in cotton roots after infection with Fusarium oxysporum

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.     

Patterns of incorporation of d-galactose into cell wall polysaccharide of growing maize roots

Summary ¹⁴C-1-d-galactose was rapidly taken up by excised corn root-tips and efficiently converted to hexose units in cell wall polysaccharides. The label recovered in both hydrolysed pectin and hemicellulose was predominantly in galactose and only the -cellulose contained appreciable amounts of labelled glucose. There was no evidence for breakdown of labelled units after incorporation into the cell wall. It is suggested that the utilisation of this free galactose has not appreciably affected the normal metabolic pathway by which galactose is incorporated into plant cell walls.Advantage was taken of the specificity of this labelling to follow patterns of galactosyl incorporation in roots. Autoradiographs were prepared from adjacent longitudinal sections that had been extracted with ammonium oxalate solution and 24% (w/v) KOH respectively. The distribution of silver grains over these sections was compared with that over an unextracted section. Galactosyl units of pectin were incorporated in young cell walls in all tissues investigated. The pattern closely resembled that noted in earlier work for uronosyl and pentosyl incorporation. In pith and cortical cells, galactosyl units of hemicellulose were deposited at a maximum rate in walls approaching the end of their growth when pentose incorporation was low. Because branched alkali-soluble polysaccharides containing galactose and pentose have been isolated from several tissues of corn, similar compounds are likely to exist in the root. It is proposed that the continued elaboration of such a polysaccharide might continue after deposition, and the addition of galactosyl units may be a factor which limits further plastic extension of the wall.     

Aerenchyma formation in maize roots

Maize (Zea mays L.) is generally considered to be a plant with aerenchyma formation inducible by environmental conditions. In our study, young maize plants, cultivated in various ways in order to minimise the stressing effect of hypoxia, flooding, mechanical impedance or nutrient starvation, were examined for the presence of aerenchyma in their primary roots. The area of aerenchyma in the root cortex was correlated with the root length. Although 12 different maize accessions were used, no plants without aerenchyma were acquired until an ethylene synthesis inhibitor was employed. Using an ACC-synthase inhibitor, it was confirmed that the aerenchyma formation is ethylene-regulated and dependent on irradiance. The presence of TUNEL-positive nuclei and ultrastructural changes in cortical cells suggest a connection between ethylene-dependent aerenchyma formation and programmed cell death. Position of cells with TUNEL-positive nuclei in relation to aerenchyma-channels was described.     

Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago sativa L

Alfalfa (Medicago sativa) roots were treated with mercuric ions in a concentration- and time-dependent manner, and lipid peroxidation was studied biochemically as well as histochemically along with other physiological responses. Histochemical staining with Schiff's reagent and Evans blue revealed that the peroxidation of membrane lipids and loss of plasma membrane integrity in Hg-treated roots occurred in the meristem and the elongation zone. The histochemical observations were supported by the quantitative determinations of thiobarbituric acid reactive substances (TBARS). However, under the mercuric ions stress, the alfalfa plants showed no significant alteration of hydrogen peroxide in roots. Analysis of lipoxygenase activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that there were two isoforms in the root of alfalfa plants, but they showed quite different patterns under the Hg exposure. Also, using non-denaturing PAGE, activities of superoxide dismutase (SOD) and peroxidase (POD) were determined in roots after treatment with Hg ions. The total activities of SOD and POD increased in roots after Hg treatment of roots. Activity of ascorbate peroxides (APX) was stimulated at relatively high concentration of Hg (40microM), and after prolonged Hg exposure (20microM, 24h). In contrast, glutathione reductase activity was depressed at higher concentrations of Hg (10-20microM). Treatments of seedlings with 10-40microM Hg decreased the ascorbate and glutathione amounts but increased total non-protein thiols. The above results indicated that Hg exerted its toxic effect on the root growth of alfalfa by induction of oxidative stress.     

Responses of cariogenic streptococci to environmental stresses

To persist in the oral cavity, bacteria must be able to tolerate rapid and substantial environmental fluctuations, particularly in pH and nutrient source and availability. Various species of Streptococcus, one of the most abundant genera in the mouth, are associated with oral health, as well as with dental caries. Cariogenic streptococci depend on a biofilm lifestyle for survival and persistence in the oral cavity and have developed sophisticated mechanisms to cope with environmental stresses. Here, we analyze the primary factors that allow these bacteria to emerge as significant members of tooth biofilms during adverse conditions. Our focus is on the molecular mechanisms of biofilm formation, stress tolerance and sugar metabolism by pathogenic oral streptococci, mainly Streptococcus mutans. Overlaps in the roles and regulation of these virulence attributes are highlighted and areas of research that deserve further investigation are proposed.     

Cell wall composition as a maize defense mechanism against corn borers

European and Mediterranean corn borers are two of the most economically important insect pests of maize (Zea mays L.) in North America and southern Europe, respectively. Cell wall structure and composition were evaluated in pith and rind tissues of resistant and susceptible inbred lines as possible corn borer resistance traits. Composition of cell wall polysaccharides, lignin concentration and composition, and cell wall bound forms of hydroxycinnamic acids were measured. As expected, most of the cell wall components were found at higher concentrations in the rind than in the pith tissues, with the exception of galactose and total diferulate esters. Pith of resistant inbred lines had significantly higher concentrations of total cell wall material than susceptible inbred lines, indicating that the thickness of cell walls could be the initial barrier against corn borer larvae attack. Higher concentrations of cell wall xylose and 8-O-4-coupled diferulate were found in resistant inbreds. Stem tunneling by corn borers was negatively correlated with concentrations of total diferulates, 8-5-diferulate and p-coumarate esters. Higher total cell wall, xylose, and 8-coupled diferulates concentrations appear to be possible mechanisms of corn borer resistance.