Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Genome-wide identification,expression and chromosomal location of the genes encoding chitinolytic enzymes in <Emphasis Type="Italic">Zea mays</Emphasis>

Chitinolytic enzymes are important pathogenesis and stress related proteins. We identified 27 putative genes encoding endochitinases in the maize genome via in silico techniques and four exochitinases. Only seven of the endochitinases and segments of the exochitinases were heretofore known. The endochitinases included members of family 19 chitinases (classes I-IV of PR3, II of PR4) and members of family 18 chitinases (class III of PR8). Some similar enzymes were detected on adjacent regions of the same chromosome, and seem to result from duplication events. Most of the genes expressed were identified from EST libraries from plants exposed to biotic or abiotic stresses but also from libraries from tissues not exposed to stresses. We isolated proteins from seedlings of maize in the presence or absence of the symbiotic root colonizing fungus Trichoderma harzianum strain T22, and analyzed the activity of chitinolytic enzymes using an in-gel activity assay. The activity bands were identified by LC/MS/MS using the database from our in silico study. The identities of the enzymes changed depending on whether or not T22 was present. One activity band of about 95 kDa appeared to be a heterodimer between an exochitinase and any of several different endochitinases. The identity of the endochitinase component appeared to be dependent upon treatment.     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

Water stress and indol-3yl-acetic acid content of maize roots

Water-stress conditions were applied to the apical 12 mm of intact or excised roots ofZea mays L. (cv. LG 11) using mannitol solutions (0 to 0.66 M) and changes in weight, water content, growth and IAA level of these roots were investigated. With increasing stress a decrease in growth, correlated with an increased IAA level, was observed. The largest increase in IAA (about 2.7-fold) was found in the apical 5 mm of the root and was obtained under a stress corresponding to an osmotic potential of −1.39 MPa in the solution. This stress led to an isotonic state in the cells after 1 h. When the duration of water stress (−1.09 MPa) was increased to 2 or 3 h, no further increase in the IAA content was observed in the root segments. This indicated that there was no correlation between a hypothetical passive penetration of mannitol in the cells and IAA content. Indol-3yl-acetic acid rose to the same level in excised as in intact roots. In both cases, IAA accumulation was apparently independent of the hydrolysis of the conjugated form. The caryopsis and shoot seem not to be necessary to induce the increase of the IAA level in the roots during water stress (−1.09 MPa). Therefore, there seems to be a high rate of IAA biosynthesis in excised maize roots under water-stress conditions. Exodiffusion of IAA was observed during an immersion in either buffer or stress (−1.09 MPa) solution. In both cases, this IAA efflux into the medium represented about 50% of the endogenous level. Considering the present results, IAA appears to play an important part in the regulation of maize root metabolism and growth under water deficiency.     

Development of the Casparian strip in primary roots of maize under salt stress

The Casparian strip in the endodermis of vascular plant roots appears to play an important role in preventing the influx of salts into the stele through the apoplast under salt stress. The effects of salinity on the development and morphology of the Casparian strip in primary roots of maize (Zea mays L.) were studied. Compared to the controls, the strip matured closer to the root tip with increase in the ambient concentration of NaCl. During growth in 200 mM NaCl, the number and the length of the endodermal cells in the region between the root tip and the lowest position of the endodermal strip decreased, as did the apparent rate of production of cells in single files of endodermal cells (the rate of cell formation being equal to the rate at which cells are lost from the meristem). The estimated time required for an individual cell to complete the formation of the strip after generation of the cell in the presence of 200 mM NaCl was not very different from that required in controls. Thus, salinity did not substantially affect the actual process of formation of the strip in individual cells. The radial width of the Casparian strip, a morphological parameter that should be related to the effectiveness of the strip as a barrier, increased in the presence of 200 mM NaCl. The mean width of the lignified region was 0.92 m in distilled water and 1.33 m in 200 mM NaCl at the lowest position of the strip. The mean width of the strip relative to that of the radial wall at this position was significantly greater after growth in the presence of 200 mM NaCl than in the controls, namely, 20.5% in distilled water and 33.9% in 200 mM NaCl. These observations suggest that the function of the strip is enhanced under salt stress.     

Comparison of Site I auxin binding and a 22-kilodalton auxin-binding protein in maize

Several properties of a 43-kilodalton (kDa) auxin-binding protein (ABP) having 22-kDa subunits are shared by a class of auxin binding designated Site I. The spatial distribution of the ABP in the maize (Zea mays L.) mesocotyl corresponds with the distribution of growth induced by naphthalene-1-acetic acid and with the distribution of Site I binding as previously shown by J.D. Walton and P.M. Ray (1981, Plant Physiol. 68, 1334–1338). The greatest abundance of both ABP and Site I activity is at the apical region of the mesocotyl. The ABP and Site I activity co-migrate in isopycnic centrifugation with the endoplasmic-reticulum marker, cytochrome-c reductase. Red light, at low and high fluence, far-red and white light were used to alter the elongation rate of apical 1-cm sections of etiolated maize mesocotyls, the amount of auxin binding, and the abundance of the ABP. Relative changes in auxin binding and the ABP were correlated, but the growth rate was not always correlated with the abundance of the ABP.Abbreviations ABP auxin-binding protein - ER endoplasmic reticulum - FR far-red light - kDa kilodalton - NAA naphthalene-1-acetic acid - PM plasma membrane - R red light - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Monoclonal antibodies detect an auxin-induced conformational change in the maize auxin-binding protein

The monoclonal antibody MAC 256 precipitates specifically the auxin-binding protein (ABP) of maize membranes. Auxin-binding activity was recovered from the immunoprecipitate and MAC 256 can, therefore, bind undenatured, native ABP. A sandwich enzyme-linked immunosorbent assay was used to present native ABP to MAC 256 and under these conditions auxins inhibit antibody binding. Millimolar naphthalene-1-acetic acid completely blocks MAC 256 binding and the characteristics of monoclonal antibody MAC 259 are similar. The ability of a range of auxins and related compounds to displace MAC 256 correlates with the known structure-activity relationships of these compounds in vivo and in binding assays. The results are interpreted in terms of an auxin-induced conformational change in ABP, auxin binding leading to a change in, or concealment of, the epitope of the antibody. The epitope for MAC 256 and 259 lies close to the carboxy terminus of the protein, implying that the part of ABP containing the sequence of amino acids responsible for retention within the endoplasmic reticulum is conformationally active.Abbreviations ABP auxin-binding protein - ELISA enzyme-linked immunosorbent assay - IAA indole-3-acetic acid - Mab monoclonal antibody - NAA naphthalene-1-acetic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TIBA 2,3,5-triiodobenzoic acid - 2,4,5-T, 2,4,6-T 2,4,5-trichloro- and 2,4,6-trichlorophenoxyacetic acid, respectively We are grateful to Neville Huskisson and Pat Baker of the Microchemical Facility, AFRC IAPGR, Babraham, UK for the aminoacid sequencing and to the staff at the AFRC Monoclonal Antibody Centre, Babraham where the Mabs were produced. This work was partially funded by the Biotechnology Action Programme of the European Economic Community.To whom correspondence should be addressed.     

In-vitro auxin transport in membrane vesicles from maize coleoptiles

When membrane vesicles from maize (Zea mays L.) coleoptiles are extracted at high buffer strength, a pH-driven, saturable association of [¹⁴C] indole-3-acetic acid is found, similar to the in-vitro auxin-transport system previously described for Cucurbita hypocotyls. The phytotropins naphthylphthalamic acid and pyrenoylbenzoic acid increase net uptake, pressumably by inhibiting the auxin-efflux carrier.Abbreviations IAA indole-3-acetic acid - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA, 2-NAA 1-, 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid     

Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP auxin-binding protein - DEAE diethylaminoethyl - Ig immunoglobulin - kDa kilodalton - NAA naphthalene-1-acetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis

MicroRNA398 targets two Cu/Zn superoxide dismutases (CSD1 and CSD2) in higher plants. Previous investigations revealed both decreased miR398 expression during high Cu²⁺ or paraquat stress and increased expression under low Cu²⁺ or high sucrose in the growth medium. Here, we show that additional abiotic stresses such as ozone and salinity also affect miR398 levels. Ozone fumigation decreased miR398 levels that were gradually restored to normal levels after relieved from the stress. Furthermore, miR398 levels decreased in Arabidopsis leaves infiltrated with avirulent strains of Pseudomonas syringae pv. tomato, Pst DC3000 (avrRpm1 or avrRpt2) but not the virulent strain Pst DC3000. To our knowledge, miR398 is the first miRNA shown to be down-regulated in response to biotic stress (P. syringae). CSD1, but not CSD2, mRNA levels were negatively correlated with miR398 levels during ozone, salinity and biotic stress, suggesting that CSD2 regulation is not strictly under miR398 control during diverse stresses. Overall, this study further establishes a link between oxidative stress and miR398 in Arabidopsis.     

Carotenoid synthesis and pleiotropic effects in carotenoid-deficient seedlings of maize

Plastid-envelope membranes from seedlings ofZea mays L. made carotenoid-deficient by either norflurazon treatment or mutation lack an activity permitting conversion of phytoene to -carotene. This activity in membrane fractions was measured by coincubation in vitro with a soluble system from spinach chloroplasts capable of converting [¹⁴C]isopentenyl pyrophosphate into phytoene. When grown in light, the carotenoid-deficient seedlings lack many soluble chloroplast proteins, including NADP-dependent malic enzyme (EC 1.1.1.40), pyruvate phosphate dikinase (EC 2.7.9.1), and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), but apparently still contain the soluble activities permitting synthesis of phytoene.Abbreviations IPP isopentenyl pyrophosphate - LHCP light-harvesting chlorophylla/b-binding protein - norflurazon 4-chloro-5(methylamine)-2-(,,-trifluoro-m-tolyl)-3-(2H)-pyrazinone - TLC thin-layer chromatography - Tris 3-amino-2-(dihydroxymethyl)-1,3-propanediol     

Two forms of NADP-dependent malic enzyme in expanding maize leaves

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a K_m (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a K_m (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar K_m (NADP⁺), and the typical increase of K_m for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min^-1 mg protein^-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.Abbreviation MES 2 (N-morpholino)ethane sulfonic acid Work supported by grants from the Consiglio Nazionale delle Ricerche for years 1975 and 1976     

Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (52 residues out of a total of 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.Abbreviations A absorbance - Bis N,N-methylene bisacrylamide - IEF isoelectric focusing - 2-ME 2-meroaptoethanol - mol wt molecular weight - 62 opaque-2 - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - PAS periodic acid-Schiff stain - SDS sodium dodecyl sulphate - ICA trichloroacetic acid - TEMED N,N,N,N-tetramethyl ethylene diamine - Z₁ zein extracted with 55% isopropanol - Z₂ zein extracted with 55% isopropanol and 0.6% 2-ME - Z 9.6 zein of mol wt 9600 - Z 13.5 zein of mol wt 13,500 - Z 21 zein of mol wt 21,000 - Z 23 zein of mol wt 23,000     

Patterns of polypeptide synthesis in various maize organs under anaerobiosis

The pattern of protein synthesis was compared in several organs of maize (Zea mays L.) under aerobic and anaerobic conditions. Protein synthesis was measured by [³⁵S]methionine incorporation and analysis by two-dimensional native-SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis and fluorography. The aerobic protein-synthesis profiles were very different for root, endosperm, scutellum and anther wall. However, except for some characteristic qualitative and quantitative differences, the patterns of protein synthesis during anaerobiosis were remarkably similar for these diverse organs and also for mesocotyl and coleoptile. The proteins synthesized were the anaerobic polypeptides (ANPs) which have been previously described in anaerobic roots of seedlings. Leaves exhibited no detectable protein synthesis under anaerobic conditions, and died after a short anaerobic treatment. Evidence is presented that the ANPs are not a generalized response to stress. This indicates that the ANPs are synthesized as a specific response to anaerobic conditions such as flooding.Abbreviations ADH alcohol dehydrogenase - ANP anaerobic polypeptide - SDS sodium lauryl sulfate     

Cytochemical identification of arabinogalactan protein in the outer epidermal wall of maize coleoptiles

Artificial carbohydrate antigen (Yariv reagent), fluorescence-labeled -l-fucose-binding lectin, and -D-galactose-binding lectin were used to localize arabinogalactan protein in sections of maize (Zea mays L.) coleoptiles. All three probes bind to cell walls of vascular tissue and the outer epidermis. Intense staining is obtained at the outer and inner faces of the growth-controlling outer epidermal wall. At the inner face of this wall the auxin-inducible osmiophilic particles, hitherto observed only by electron microscope (Kutschera et al. 1987, Planta 170, 168–180), are strongly stained by all three probes and can therefore be identified as deposits of arabinogalactan protein. It is proposed that this proteoglycan acts as an epidermal wallloosening factor in auxin-mediated coleoptile growth.Abbreviation AGP arabinogalactan protein I thank Dr. R. Bergfeld for the electron micrograph of Fig. 13. This work was supported by the Deutsche Forschungsgemeinschaft.