Maize Coleoptiles Research Articles

Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth.

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.

Laser-Capture Microdissection, a Tool for the Global Analysis of Gene Expression in Specific Plant Cell Types: Identification of Genes Expressed Differentially in Epidermal Cells or Vascular Tissues of Maize[W

Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers (>10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid-fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing approximately 8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type-specific biological processes.

The role of β-glucosidase in phototropism of maize coleoptiles

The role of β-glucosidase in phototropism of maize coleoptiles

Polysaccharide degradation by Fenton reaction- or peroxidase-generated hydroxyl radicals in isolated plant cell walls

The formation of hydroxyl radicals (OH ) by peroxidase was confirmed by EPR spectroscopy using ethanol/α-(4-pyridyl-1-oxide)- N-tert-butylnitrone as a spin-trapping system specific of OH . The effect of OH , generated either non-enzymatically with the Fenton reaction (H 2O 2+Fe 2+) or with horseradish peroxidase in the presence of O 2 and NADH, on cell walls isolated from maize ( Zea mays) coleoptiles or soybean ( Glycine max) hypocotyls was investigated. OH produced by these reactions attack polysaccharides in the wall, demonstrated by the release of a heterogeneous mixture of polymeric breakdown products into the incubation medium. The peroxidase-catalyzed degradation of cell-wall polysaccharides can be inhibited by KCN and superoxide radical (O 2 − ) or OH scavengers. These data support the hypothesis that OH , produced by cell-wall peroxidases in vivo, act as wall-loosening agents in plant extension growth.

Spermidine-binding proteins. Purification and expression analysis in maize.

Polyamine-binding proteins have been identified in a wide range of organisms, including mammals, yeasts, and bacteria. In this work, we have investigated specific spermidine binding to plant membrane proteins purified from microsomes of etiolated maize (Zea mays) coleoptiles. In the final purification step, specific spermidine-binding activity (K(d) 6.02 10(-7) M) was eluted from a HiTrapQ fast-protein liquid chromatography column at about 0.25 M NaCl, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction showed a major polypeptide of about 60 kD and another copurifying 18-kD protein. Competition experiments, performed on HiTrapQ active fractions, confirmed the specificity of the binding. Upon Sephadex G-100 gel filtration, spermidine binding was associated almost exclusively with the 18-kD protein. On the basis of the N-terminal sequences, degenerate oligonucleotide probes were designed and used to isolate, by reverse transcriptase-polymerase chain reaction and polymerase chain reaction, cDNA fragments of about 1 kb for the 60-kD protein, and 0.9 kb for the 18-kD protein. Northern-blot analysis performed on etiolated coleoptiles and different tissues from 10-d-old maize plants indicated the presence of two different mRNAs of 1.7 and 0.7 kb. Southern-blot analysis indicated that the genes encoding the 60- and 18-kD proteins are probably derived from differential processing of the same precursor mRNA. Using rabbit polyclonal antibodies raised against these proteins, affinity purification and dot-blot experiments detected analogous membrane proteins in monocot and dicot plants.

A proposed role for copper ions in cell wall loosening

We present the hypothesis that Cu2+, complexed with cell wall polymers (especially histidine-rich glycoproteins), can be reduced to Cu+ by apoplastic electron donors such as ascorbate and superoxide, and that the Cu+ can then undergo a Fenton reaction with apoplastic hydrogen peroxide (H2O2) to generate hydroxyl radicals (·OH). These can cause non-enzymic scission of wall polysaccharides and may loosen the cell wall. In tomato fruit cell walls, ·OH radicals cause pectin solubilisation. However, we show that acidic pectins do not sequester Cu2+ sufficiently strongly to prevent ·OH-attack on other molecules (xyloglucan or aromatics) present in the same solution. We describe two approaches by which apoplastic ·0H can be detected in vivo: (a) detection of a chemical ‘fingerprint’ characteristic of · OH-attacked cell wall polysaccharides; (b) infiltration into the apoplast of membrane-impermeant [3H]benzoyl-amides which react with ·OH to yield 3H2O. Data support apoplastic ·OH production in vivo, and its enhancement during pear (Pyrus communis) fruit ripening, but have not so far supported its proposed enhancement during auxin-stimulated cell expansion in maize (Zea mays) coleoptiles.

Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth.

Hydroxyl radicals (OH) are capable of unspecifically cleaving cell-wall polysaccharides in a site-specific reaction. I investigated the hypothesis that cell-wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load-bearing cell-wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre-loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin-induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2(-)), an OH precursor, in the growth-controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall-loosening factor acting in auxin-mediated elongation growth of plant species with widely differing cell-wall polysaccharide compositions.

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.

Cell Wall Architecture of the Elongating Maize Coleoptile

Cell Wall Architecture of the Elongating Maize Coleoptile

Auxin-induced elongation of short maize coleoptile segments is supported by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one.

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 microM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2-3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported longterm, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically "spare" the co-auxin in maize.

Zea mays CCaMK: autophosphorylation-dependent substrate phosphorylation and down-regulation by red light.

The role of protein kinases has been extensively studied in various signal transduction pathways and they are one of the most important components that link the signal perception to the final response. However, not many studies have been reported, especially from the plant systems, that show the regulation pattern of the kinase itself under different conditions. A calcium/calmodulin-dependent protein kinase has already been purified and characterized from etiolated maize coleoptiles. In this paper a detailed study of how the kinase itself is regulated at the autophosphorylation level is provided. Evidence is also given that the autophosphorylation of kinase effects its activity towards substrate phosphorylation. It is further shown that the kinase is an important component of the light signalling pathway as the level of the kinase itself decreases by red light irradiation.

Microinjection reveals cell-to-cell movement of green fluorescent protein in cells of maize coleoptiles.

During the evaluation of dual-purpose plant/fungal expression systems, we found that green fluorescent protein (GFP) has the ability to move from cell to cell in the epidermis of Zea mays L. cv. Mutator coleoptiles as well as into underlying cortical cells. Movement of GFP was observed both when DNA encoding GFP and bacterially expressed GFP were microinjected into epidermal cells. This suggests that GFP is capable of cell-to-cell movement. From experiments using dextrans of known molecular weight linked to fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate, we estimate that the plasmodesmata of these cells have a size exclusion limit < 4.4 kDa. Cell-to-cell GFP movement did not occur when GFP was altered to include a nucleus- or endoplasmic reticulum-retention sequence. The fact that these transcripts differ from that of cytoplasmic GFP by a small number of nucleotides suggests that the transcripts are not capable of movement, but movement of nucleic acid cannot be excluded. Since GFP is widely used to study cell-to-cell movement and to localize the expression of transgenes, caution should be exercised when interpreting results where GFP expression is used for localization.

Fusicoccin‐ and IAA‐induced elongation growth share the same pattern of K+ dependence

The dependence of growth induced by the fungal toxin fusicoccin (FC) on the K+ content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca2+ was included in the bathing medium, no FC-induced growth occurred in the absence of K+, whereas a strong response was detected in presence of K+. The optimal K+ concentration was in the range of 1-10 mM. With the exception of Rb+, none of the other alkali ions (Na+, Li+, Cs+) could replace for K+ in sustaining FC-induced growth. The potassium channel blocker tetraethylammonium (TEA) reversibly inhibited FC-induced growth. As shown earlier for auxin-induced growth, no strict potassium dependence of FC-triggered elongation was observed in Ca2+ -free media. However, TEA abolished this apparently K+ independent FC-induced growth. It is concluded that FC-induced growth, like auxin-induced growth, requires K+ uptake through K+ channels.

NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay.

Using the tetrazolium salt XTT (Na,3'-[(phe-nylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O2*-) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O2*- in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an O2*--synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the O2*--producing activity of coleoptiles. The maize NADH-dependent O2*--synthase activity could clearly be differentiated from peroxidase-mediated O2*--synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O2. Formation of O2*-, and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O2*- and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.

Gravitropic microtubule reorientation can be uncoupled from growth.

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90 degrees), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation.

Ca(2)+-stimulated exocytosis in maize coleoptile cells.

Changes in membrane capacitance (C(m)) after photolysis of the caged Ca(2)+ compound dimethoxynitrophenamine were studied in protoplasts from maize coleoptiles. Changes in C(m) values resulting from increased concentrations of free Ca(2)+ in the cytoplasm ([Ca(2)+](cyt)) were interpreted as representing changes in [Ca(2)+](cyt)-sensitive exocytosis and endocytosis. A continuous increase in [Ca(2)+](cyt) resulted in a sigmoidal increase in C(m) values with a half-maximal concentration at approximately 1 microM. The steep increase in C(m) values was followed by a variable slow phase in changing C(m) values. When [Ca(2)+](cyt) increased at a rate of 0.6 micromol L(-)(1) sec(-)(1), the initial steep increase in C(m) values lasted approximately 5 to 10 sec. During this time, protoplasts increased in surface area by approximately 2.5%. The biphasic dynamics of [Ca(2)+](cyt)-stimulated increases in C(m) values can be described by a kinetic model containing two pools of vesicles with two [Ca(2)+](cyt)-sensitive steps in the exocytotic pathway.

Ca2+-sensitive and Ca2+-insensitive exocytosis in maize coleoptile protoplasts

Ca2+-sensitive and Ca2+-insensitive exocytosis in maize coleoptile protoplasts

Ca 2+ -Stimulated Exocytosis in Maize Coleoptile Cells

Ca 2+ -Stimulated Exocytosis in Maize Coleoptile Cells

Endo-1,3;1,4-β-glucanase from coleoptiles of rice and maize: role in the regulation of plant growth

The Matrix Polymer Hydrolysis Model for regulation of growth in plants is based on the simultaneous hydrolysis and incorporation of new glucans into the cell wall observed in growing plant tissues. The inhibition of growth in rice coleoptile tissues treated with glucanase antibodies confirms similar results observed previously in maize coleoptiles and provides direct evidence for a role of glucanase in control of plant growth. Analysis of two-maize coleoptile endo-glucanase ESTs shows that these sequences are not related to any other previously known family of glycosyl hydrolase. Thus, the coleoptile endo-glucanase enzyme should be classified as a new enzyme group (E.C. 3.2.1.xx). These discoveries enable new initiatives for further investigation of the glucanase role in control of plant growth.

Quantitative Changes in Maize Membrane Proteins Induced by Aluminium

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for monitoring Al-induced changes in polypeptide composition of membrane proteins isolated from 3-d-old maize seedlings subjected to aluminium stress. Analysis of peripheral membrane proteins isolated from maize root showed an Al-induced increase in accumulation of 14 polypeptides with apparent molecular mass from 10 to 135 kDa. Qualitative differences were found between peripheral membrane proteins isolated from root tip (increased accumulation of 4 polypeptides with Mr 42 000 - 135 000) and from root base (increased accumulation of 10 polypeptides with Mr 10 000 - 59 000). On the other hand, no Al-induced changes were observed in peripheral membrane proteins isolated from maize coleoptile and integral membrane proteins isolated either from root or coleoptile. These results indicate that peripheral membrane proteins undergo considerable changes during 24-h Al treatment while integral membrane proteins pattern is stable.