The rea (red embryonic axis) phenotype describes a new mutation affecting the response of maize embryos to abscisic acid and osmotic stress

During a screen for mutants with defective germination, a newphenotype was observed consisting of red pigmentation of theembryonic axis in the dormant seed. Segregation ratios, as determinedin F₂ and back-crossed progeny, indicate that the phenotypeis due to a recessive single gene mutation that has been symbolizedrea to denote red embryonic axis. A closer inspection of therea phenotype revealed that the mutant is occasionally viviparous,indicating a defect in abscisic acid (ABA) metabolism. The mutationprobably affects ABA sensitivity since no difference in ABAcontent was detected in mutant versus normal tissues. Moreover,when immature mutant and wild-type embryos were incubated onmedia containing 10 M ABA, only the mutants germinated. ABA-regulatedgene expression in rea embryos differed from that of embryosof the viviparous mutant vp1 which does not respond to the inhibitoryaction of ABA at the level of immature embryo germination. Theseresults, therefore, indicate that the two genes exert a differentrole in the control of embryogenesis. Key words: Zea mays L, embryo dormancy, ABA     

Is pathogenicity of Ustilago maydis (huitlacoche) strains on maize related to in vitro production of indole-3-acetic acid?

Corn smut caused by Ustilago maydis (DC) Corda on maize (Zea mays L.) is characterized by gall (tumour) formation on aerial parts of the plant. Young galls on immature corn ears are called huitlacoche and are highly appreciated as a food delicacy. Several reports have suggested that production of the auxin indole-3-acetic acid (IAA) by U. maydis might be involved in tumour formation. Because strains showing defects in IAA biosynthesis (Iaa^– phenotype) would be valuable in elucidating the role of IAA in pathogenicity, in a previous study we isolated and analysed several such mutants. In the present work, we have crossed a null Iaa^– auxotrophic mutant with compatible wild-type strains. The main objective of the present work was to evaluate the pathogenicity of crosses involving wild-type and Iaa^– strains, in order to examine its relation to IAA production. Significant differences were found in growth, IAA production and ability to survive in water suspension among wild-type and mutant progeny strains. In general, high levels of pathogenicity were associated to high levels of IAA production by the strains, which supports the hypothesis that U. maydis strains require the ability to produce IAA in order to induce tumours in the host.     

The Nuclear Endoreduplication Cycle in Metaxylem Cells of Primary Roots of Zea mays L.

The nuclear DNA content of metaxylem cells in roots of Zea mayscv. Golden Bantam reaches 16C or 32C by successive rounds ofDNA endoreduplication. Each phase of endoreduplication (endo-S)is separated by a non-DNA synthetic phase (endo-G). These phasesseem to occur in zones at fixed distances from the root tip.The duration of the phases in two of the endoreduplication cycles(4C–8C, 8C–16C) has been estimated in two ways.The first makes use of the rate of movement of cells throughthe positions along the root where the different phases of thecycle are occurring, the second uses labelling with methyl-[³H]thymidineand autoradiography. Both methods indicate that the endo-S phaseswhich cause the nuclear DNA content to rise from 4C to 8C andfrom 8C to 16C last 8–10 h, and that the intervening endo-Gphase lasts 8–12 h. DNA endoreduplication keeps pace withthe increase of nuclear volume; cell volume increases at a morerapid rate, however. Comparison of the endoreduplication cyclein the metaxylem with the mitotic cycle in the adjoining filesof parenchyma cells shows that the mitotic cells complete theircycle more slowly. DNA synthesis, endoreduplication cycle, mitotic cycle, root apex, Zea mays     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Role of Auxin in Maize Endosperm Development (Timing of Nuclear DNA Endoreduplication,Zein Expression,and Cytokinin)

The timing of developmental events and regulatory roles of auxin were examined in maize (Zea mays L.) endosperms. Zeatin, zeatin riboside, and indole-3-acetic acid (IAA) levels were determined by enzyme-linked immunosorbent (ELISA). Zeatin and zeatin riboside increased to maximal concentrations at an early stage (9 d after pollination [DAP]), corresponding to the stage when cell division rate was maximal. In contrast, IAA concentration was low at 9 DAP and abruptly increased from 9 to 11 DAP, thus creating a sharp decline in the cytokinin to auxin ratio. Coincident with the increase in IAA was an increase in DNA content per nucleus, attributed to postmitotic DNA replication via endoreduplication. Exogenous application of 2,4-dichlorophenoxyacetic acid (2,4-D) at 5 or 7 DAP hastened the time course of DNA accumulation per nucleus and increased the average nuclear diameter, whereas 2-(para-chlorophenoxy)isobutyric acid delayed such development. Exogenously applied 2,4-D hastened the accumulation of the zein polypeptides of apparent molecular masses of 12, 14, and 16 kD and the expression of mRNA hybridizing with a zein DNA probe. We conclude that an abrupt increase in auxin induces cellular differentiation events in endosperm, including endoredupliction and expression of particular zein storage proteins.     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.     

Auxin-Cytokinin Interactions in Wild-Type and Transgenic Tobacco

Cytokinins and auxins are important regulators of plant growthand development, but there is incomplete and conflicting evidencethat auxins affect cytokinin metabolism and vice versa. We haveinvestigated these interactions in Nicotiana tabacum L. by separatein planta manipulation of levels of the hormones followed byanalysis of the induced changes in the metabolism of the otherhormone. Cytokinin-overproducing plants (expressing the Agrobacte-riumtumefaciens ipt gene) had lower than wild-type levels of freeIAA, and reduced rates of IAA synthesis and turnover, but therewere no differences in the profiles of metabolites they producedfrom fed IAA. Similarly, auxin-overproducing plants (expressingthe A.tumefaciens iaaM and iaaH genes), had lower levels ofthe major cytokinins than wild-type plants and lower cytokininoxidase activity, but there were no differences in the profilesof metabolites they produced from fed cytokinins. The data demonstratethat cytokinin or auxin overproduction decreases the contentof the other hormone, apparently by decreasing its rate of synthesisand/or transport, rather than by increasing rates of turnoveror conjugation. Implications for the importance of cytokinin: auxin ratios in plant development are considered. (Received September 24, 1996; Accepted December 4, 1996)     

Ontogenetic Changes in the Transport of Indol-3yl-acetic Acid into Maize Roots from the Shoot and Caryopsis

The quantities of endogenous indol-3yl-acetic acid (IAA) in endosperms and scutella of 6-day-old maize seedlings (Zea mays L. cv Giant White Horsetooth) were determined by a fluorimetric method. Endosperms were found to contain 33.4 nanograms IAA per plant, and scutella 7.5 nanograms IAA per plant. [5-³H]IAA applied to endosperms of 6-day-old seedlings moved into the roots and radioactivity accumulated at the apex of the primary root within 8 hours. Two to 7-day-old seedlings were treated simultaneously with [5-³H]IAA in the endosperm and [2-¹⁴C] IAA on the shoot apex. The patterns of transport into the root were found to change during ontogeny: in successively older plants, transport from the shoot into the roots increased relative to transport from the endosperm into the roots. The auxin required for the growth of maize roots could, therefore, partially be contributed by the shoot and endosperm. Ontogenetic changes in the relative importance of these two supplies could be of significance for the integration of growth and development between shoot and root.     

A Maize Mutant with an Altered Vascular Pattern

A recessive maize mutant with disrupted seedling developmentwas isolated following transpositional mutagenesis with Mutator.This mutant, initially identified during germination on thebasis of abnormal growth of the scutellar node, was designatedlsn1 (l arge s cutellar n ode). The mutant seedling exhibitsan enlarged primary root with a longitudinal groove and multipleseparate root tips. The mutant root is shorter than normal,because of defective cell elongation, and lacks lateral roots.The mutant plant shows defective leaves and reduced internodeelongation. Histological analyses on primary root, shoot, scutellarnode and juvenile leaves revealed a series of defects, all relatedto an irregular differentiation of vascular elements. In addition,in situ hybridization of mutant leaves demonstrates an abnormalpattern of expression of Knotted-1, a marker of meristem function.The presence of multiple roots fused together can be interpretedas suppression of the negative control responsible for the differentiationof only one root primordium. Therefore, the data obtained onseedlings of lsn1 point to a relationship between meristem activity,vascular differentiation and auxin polar transport, and mayallow the identification of a gene which is active during embryogenesis.Copyright2000 Annals of Botany Company Auxin, defective seedling, maize, meristem activity, vascular differentiation, Zea mays.     

Zinc Deficiency Affects the Levels of Endogenous Gibberellins in Zea mays L.

Zinc deficiency in Zea mays L. markedly reduced the level ofGA₁, but not GA₂₀, suggesting blockage of 3rß-hy-droxylation.The level of IAA was also decreased although not as markedly.Castasterone was affected less than IAA by zinc deficiency. (Received February 24, 1997; Accepted June 24, 1997)     

Identification and localisation of auxin in primary roots of Zea mays by mass spectrometry

Summary Roots of 3.5-day-old seedlings of Zea mays L. var. Giant White Horsetooth contain an extractable auxin which has been identified unequivocally as IAA. A mass spectrometric technique has been used to determine quantitatively the levels of IAA in the cortical, stelar and apical regions of the roots. The IAA is predominantly located in the steles.     

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.     

Free and bound indole-acetic acid is low in the endosperm of the maize mutantdefective endosperm-B18

Summary The maize mutant defective endosperm-B18 (de ^*-B18), which is recessive to its wildtype, accumulates substantially less dry matter in the endosperm than its normal counterpart. Both free and bound indole-acetic acid (IAA) content has been measured at 5 different developmental stages. In endosperm tissue, the level of IAA is at least 15 times lower in the mutantde ^* -B18 than in the wildtype. The situation found in the diploid tissues is somewhat different: in the mature embryo the level of total IAA is lower in the mutant than in the wildtype, while in 4-day old seedlings the level of total IAA is, to some degree, similar in both genotypes. Naphthalene-acetic acid (NAA), a stable synthetic auxin which mimics IAA in its biochemical effects, is able to normalize the seed weight of the mutant when applied to developing grains. The results favor the conclusion that in maize endosperm the mutationde ^* -B18 is involved in IAA metabolism.     

Further characterization of the spontaneous growth response in Zea coleoptile segments

Excised segments of corn (Zea mays L., Bear Hybrid WF 9?38)coleoptiles show a strong "spontaneous" increase in growth rateabout 4.0 hr after excision. The response can be delayed about2 hr using a brief (10 min) exposure to IAA during the latentperiod. An established spontaneous growth response can be suppressedby a 30 to 60 min exposure to auxin and does not reappear untilabout 2.5 hr after withdrawal of the hormone. During the 3 hrperiod following withdrawal of exogenous auxin there is a two-foldincrease in magnitude and a three-fold decrease in latent periodof a growth response to a sub-optimal level of auxin. The dataare consistent with the hypothesis that the spontaneous growthresponse is caused by a time-dependent change in sensitivityof isolated tissue to auxin and/or a change in the endogenouslevel of auxin. Apical sections of Zea coleoptiles with the tip intact do notgrow at the rapid rate one might expect of tissue with an endogenousauxin supply. Instead they grow very poorly and exhibit botha weak spontaneous growth response and a poor response to exogenouslysupplied auxin. Indirect evidence suggests that this is dueto the production of a growth inhibitor by the tip. (Received August 3, 1976; )     

Cytological characterization of the embryo-lethal mutantdek-1 of maize

Summary The recessive embryo-lethal mutantdek-1 of maize, showing arrest of embryo development at the proembryo stage, lack of carotenoids and anthocyanins and absence in the endosperm of the aleurone layer, was characterized at a cytological level. Cytofluorimetric analysis excluded endoreduplication or polyploidization events in mutant embryonic cells, in spite of an evident increase in nucleolus and nucleus diameters.The data seem to point to an involvement ofDek-1 in the progression of the embryo toward specific developmental steps and in the differentiation of the aleurone layer in the endosperm. Cellular proliferation is not affected by the mutation, as is shown by DNA replication even after the arrest in development and by the possibility of inducing callus from mutant embryos.Abbreviation DAP days after pollination     

Effects of Morphactin on Indol-3yl-acetic Acid Transport, Growth, and Geotropic Response in Cereal Coleoptiles

The effects of the morphactin 2-ehloro-9-hydroxyfluorene-9-carboxylicacid methyl ester [CFM] on growth, geotropic curvature and transportand metabolism of indol-3yl-acetic acid [IAA-5-³H] in the coleoptilesof Zea mays and A vena saliva have been investigated. A strongcorrelation has been found to exist between the inhibition ofthe geotropic response and the inhibition of auxin transport.CFM supplied at concentrations sufficient to abolish auxin transporthas been shown to promote the elongation of Zea, but not ofAvena, coleoptile segments. CFM does not change the patternof metabolism of IAA in Zea coleoptile segments. In these segmentsIAA is metabolized when its concentration is high, but the radioactivitytransported basipetally, or laterally in geotropically stimulatedcoleoptiles, is virtually confined to the IAA molecule. Radioactivityexported into the basal receiver blocks is wholly confined toIAA. It is concluded that CFM inhibits the geotropic responsein coleoptiles by suppression of the longitudinal and lateralauxin transport mechanisms. The growth-promoting propertiesof this substance cannot be linked with its effects on eitherauxin metabolism or transport.