Cytokinins from Zea mays

A number of adenine derivatives with cytokinin activity were isolated from immature sweet corn (Zea mays) kernels. The following structures were assigned: 9-β-d-ribofuranosylzeatin, 9-β-d-ribofuranosylzeatin 5′-monophosphate, 6-(1-carboxy-2-hydroxypropylamino)-9-ribofuranosylpurine, 6-(2,3,4-trihydroxy-3-methylbutylamino)purine, 2-hydroxy-6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine, 6-(3,4-dihydroxy-3-methylbutylamino)purine, a 9-glycoside of zeatin(identity of sugar moiety not established), and 6-(1,2-dicarboxyethylamino)-9-β-d-ribofuranosylpurine.     

Purification and characterization of a galactose-specific lectin from corn (Zea mays) coleoptyle

We purified and characterized a lectin from the corn coleoptyle (Zea mays). The lectin (CCL) was purified by affinity chromatography on a Lactosyl–Sepharose 4B column. It is a glycoprotein of 88.7 kDa, composed mainly by glutamic, aspartic, glycine, and Ser residues; in a minor proportion, it contained methionine and cysteine residues. Carbohydrates that constituted 12% of the total weight comprised galactose, mannose, and N-acetyl-D-glucosamine. The lectin contained the blocked amino-terminus. Analysis of the lectin, determined from peptides obtained after trypsin digestion by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight), indicated that CCL has 18% homology with a putative calcium-dependent Ser/Thr protein kinase, from Arabidopsis thaliana, and 39% homology with a NADPH-dependent reductase from Z. mays. The lectin showed hemagglutinating activity toward several erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that the lectin interacts specifically with the OH on C4 from galactose residues. OH- on C1 plays a relevant role in the interaction with CCL, since β-galactose residues are better recognized than those from the anomeric α-galactose. Lack of lectin activity was observed in corn extracts; the highest specific activity was obtained from coleoptyle obtained at the 7th day after seeding.     

Flavonoids from Zea mays pollen

The flavonol glycosides of quercetin, isorhamnetin and kaempferol were isolated from Zea mays pollen. The most prominent flavonols were diglycosides of quercetin and isorhamnetin. Flavonol 3-O-glucosides of quercetin, isorhamnetin and kaempferol, and triglucosides of quercetin and isorhamnetin, were minor components. The flavonoid pattern of maize pollen is characterized by the accumulation of quercetin and isorhamnetin diglycosides and by the absence of flavones, which are common in other maize tissues.     

Spreading synaptonemal complexes from Zea mays

Four different inversion heterozygotes of maize were examined for the occurrence of synaptic adjustment. Three substages of pachytene were identified in synaptonemal complex (SC) spreads using side-by-side comparisons of chromosome squashes with two-dimensional spreads of SCs. In SC spreads, inversion loop frequency did not change substantially from early through late pachytene for any of the four inversion heterozygotes examined. In addition, the position and size of the inversion loops remained essentially constant throughout pachytene. These results indicate that synaptic adjustment of inversion loops does not occur during pachytene in Zea mays.     

Pollen specific cDNA clones from Zea mays.

We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-ripening polygalacturonase and to a pollen-specific polygalacturonase from Oenothera. The other three clones have no significant homologies to any reported sequence.     

Structure of the Threonine-Rich Extensin from Zea mays

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the `P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF-solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot.     

Somatic embryogenesis from cultured leaf segments of Zea mays

Basal leaf segments of 3 to 4 week old maize (Zea mays L.) seedlings plated on SH medium with 30 M dicamba produced embryogenic callus and/or somatic embryos. Histological evidence showed that some of the embryos arose directly from the explant. When leaf segments with embryos were transferred to MS medium with 1.0 M NAA, 1.0 M IAA, 2.0 M 2iP, and 60 g/l sucrose, the embryos germinated and the resulting seedlings could be established in culture tubes. These responses were obtained from three inbred lines, CHI31, S615, and S7.Abbreviations SH Schenk and Hildebrandt (1972) medium - MS Murashige and Skoog (1962) medium - dicamba 3,6-dichloro-o-anisic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2iP 2-isopentyladenine     

Growth inhibitor from the root cap of Zea mays

Summary The downward lateral transport of at least one inhibitor produced in or released by the cap of a maize root partly explains differences in growth of the upper and lower sides of roots placed in the horizontal position.The relative activity or level of such a regulator increases with the increase in root length.     

Site-specific recombination in Zea mays

The elimination of marker genes after selection is recommended for the commercial use of genetically modified plants. We compared the applicability of the two site-specific recombination systems Cre/lox and Flp/FRT for marker gene elimination in maize plants. The selection marker gene pat surrounded by two identically directed lox or FRT sites was introduced into maize. Sexual crossing with plants harboring the corresponding constitutively expressed recombinase led to the precise and complete excision of the lox-flanked marker gene in the F₁ progeny, whereas Flp-mediated recombination of FRT sequences occurred rarely. Further examination of site-specific integration was done by biolistic bombardment of immature embryos harboring only one lox site with a lox.uidA sequence with results indicating directed integration.     

An electron microscopic study of the mature megagametophyte in Zea mays

With light microscopy maize megagametophytes stained with Alcian blue-periodic acid-Schiff (AB-PAS) reveal acid or neutral polysaccharides in various cell walls. Comparative fine structural studies were made of permanganate- or OsO₄-fixed material. Organelle distribution is random in the vacuolate and multinucleate antipodal cells; organelles are abundant; starch is scarce. Antipodal cell walls have large openings forming several syncytia. Some walls are papillate. In the central cell (primary endosperm cell) a thin peripheral layer of cytoplasm surrounds the large vacuole; organelle number is moderate; starch is abundant. The central cell wall is also papillate adjacent to the antipodals and around the egg apparatus. In the synergids organelle distribution is non-random; nuclei and numerous organelles occupy the micropylar cytoplasm of each synergid; vacuoles dominate the chalazal cytoplasm of these cells. The filiform apparatus stains with AB-PAS and is composed of both lightly and darkly stained amorphous material. In the egg, organelle distribution is perinuclear with vacuoles proximal to the micropyle; mitochondria are large, abundant and polymorphic; starch is abundant. Nucleolar diameter is five times greater in the central cell and egg than in the antipodal cells and ten times greater than in the synergids. Plasmodesmata occur in all cell walls within the gametophyte, but none appear in the gametophyte wall itself. It is suggested that the antipodals and synergids might be secretory, the latter probably being involved in pollen tube attraction, and that stored metabolites in the central cell and egg cytoplasm support rapid increase in metabolism following fertilization.     

A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein

Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize cell suspension cultures yield a new extensin rich in histidine (HHRGP) that also has characteristics of arabinogalactan proteins (AGPs). Thus, chymotryptic peptide maps of anhydrous hydrogen fluoride (HF)-deglycosylated HHRGP showed repetitive motifs related to both extensins and AGPs as follows. HHRGP contains Ala-Hyp₃ and Ala-Hyp₄ repeats that may be related to the classical dicot Ser-Hyp₄ extensin motif by the single T → G (Ser → Ala) base change. Furthermore, HHRGP also contains the repetitive motif Ala-Hyp-Hyp-Hyp-His-Phe-Pro-Ser-Hyp-Hyp related to the Ser-Hyp₄-Ser-Hyp-Ser-Hyp₄ motif of P3-type dicot extensin. However, HHRGP also has AGP characteristics, notably an elevated alanine content, near sequence identity with the known Lolium AGP peptide Ser-Hyp-Hyp-Ala-Pro-Ala-Pro, the putative presence of glucuronoarabinogalactan, and precipitation by Yariv antigen, but β-elimination of arabinogalactan indicates its O-linkage to serine rather than the characteristic O-hydroxyproline link of other AGPs. Although HHRGP might be a “chimera” of two different proteins, i.e. an extensin and an AGP, this is unlikely because one can account for the apparent chimera by the codon relationships of the five common hydroxyproline-rich glycoprotein amino acid residues, Ser, Pro, Thr, Ala (TCx, CCx, ACx, GCx) and histidine (CAT or CAC), which facilitate interconversion of major motifs by single point mutations. Thus, we propose that the extensin family of wall proteins consists of a highly diversified phylogenetic series ranging from basic minimally glycosylated repetitive pro-rich proteins to the highly glycosylated acidic AGPs. To relate this diversity of form and function at the molecular level, we identified putative functional domains hypothetically involved in properties such as reptation, recognition, adhesion, intermolecular cross-linkage, and self-assembly. Not previously noted, peptide palindromes feature prominently in HHRGP: Hyp-Hyp-Ala-Ala-Asn-Ala-Ala-Hyp-Hyp and Hyp-Hyp-Hyp-His-His-His-Hyp-Hyp-Hyp; in P3: Hyp₄-Ser-Hyp-Ser-Hyp₄, and in other extensins. Such palindromes would enhance glycoprotein stereoregularity, thereby possibly promoting quasicrystalline interactions between wall components.     

A kinetic investigation of phosphoenolpyruvate carboxylase from Zea mays.

The reaction catalyzed by phosphoenolpyruvate carboxylase from Zea mays has been studied kinetically. Results of initial velocity patterns and inhibition studies indicate that phosphoenolpyruvate carboxylase has a random sequential mechanism in which there is a high level of synergism in the binding of substrates. The preferred order of addition of reactants is Mg2+, phosphoenolpyruvate, and bicarbonate. The binding of Mg2+ is at equilibrium. Values for the various kinetic parameters are KiMg = 2.3 +/- 0.4 mM, KPEP = 3.6 +/- 0.6 mM, KiPEP = 0.2 +/- 0.07 mM, and Kbicarbonate = 0.18 +/- 0.04 mM. In addition, double inhibition experiments have been performed to examine the nature of the active site interactions with the putative intermediates, carboxy phosphate and the enolate of pyruvate. Highly synergistic inhibition of phosphoenolpyruvate carboxylase was observed in the presence of oxalate and carbamyl phosphate (alpha = 0.0013). However, an antisynergistic relationship exists between oxalate and phosphonoformate (alpha = 2.75).     

A cDNA clone from Zea mays endosperm sucrose synthetase mRNA

A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.     

Somatic embryogenesis from three commercially important inbreds of Zea mays

Zea mays (maize) genotypes B73, Mo17 and LH38 were evaluated for their capacity to undergo somatic embryogenesis. Over 1500 immature embryos (ie's) of B73, 2900 ie's of LH38 and 400 ie's of Mo17 were excised 10–17 days after pollination and plated on six different media. Overall response, reported as a percentage of the ie's plated that developed embryogenic callus, was 2.1%, 1.6% and 26% for LH38, B73 and Mo17, respectively. Best response on a given medium for each of these genotypes was 9.2% (LH38), 4.4% (B73) and 100% (Mo17). Other parameters examined for their effects on production of embryogenic callus included self vs. sib pollination, ear ranking (1st, 2nd or 3rd ear), and temperature shock, all of which had no significant effect. Plantlets regenerated from selected treatments of B73 have been grown to maturity, selfed or sibbed and seed collected for field evaluation.Abbreviations i.e. immature embryo - 2,4-D 2,4-di-chlorophenoxyacetic acid     

Assessment of the mutagenicity of fractions from s-triazine-treated Zea mays

A water-soluble extract from maize plants exposed to 3 s-triazine herbicides (atrazine, simazine and cyanazine) has been shown to be mutagenic in strain TA100 of Salmonella. No mutagenic activity was observed in any control plant extracts using either water or a variety of organic solvents. Gel permeation studies of the extracts suggest that the mutagen(s) are small molecules (less than 1000 MW). HPLC fractionation suggests that the mutagens formed from each of the 3 herbicides are similar in polarity and water solubility, eluting in a 50/50 water:methanol fraction. Approximately 89% of 14C-labeled HPLC chromatographable metabolites of atrazine were also associated with this fraction, suggesting a close chemical link between a labeled but unidentified metabolite and the mutagenic activity.