Endorsperm Development of Maize Defective Kernel (dek) Mutants. Auxin and Cytokinin Levels

To provide insight into the regulatory roles of auxin and cytokininin endosperm development, defective kernel (dek) mutants ofmaize (Zea mays L.) were examined. The mutants dek6, dek18 anddek 26 had substantially lower indole-3-acetic acid (IAA) levelsthan wild-type counterparts while dek8, dek12 dek24 had higherIAA levels than wild-type counterparts. The mutant dek6 hada somewhat lower zeatin level than its wild-type counterpart.The mutation-induced changes in IAA levels and effects of exogenously-appliedauxin were not consistently related to changes in numbers ofnuclei per endorsperm; however, there was a correspondence betweenauxin level in endosperm and nuclear diameter, which is proportionalto the extent of post-mitotic DNA synthesis (endoreduplication).We conclude that auxin may be involved in stimulating endoreduplication.Copyright1993, 1999 Academic Press Zea mays L., maize, defective kernel mutant, dek, auxin, cytokinin, hormone, ELISA, cell number, cell division, endopolyploidy, endoreduplication, DNA amplification     

Gametophytic expression of genes controlling endosperm development in maize

Summary An indirect approach was adopted to select viable mutants affecting the male gametophytic generation in maize. This approach consists of a selection of endosperm defective mutants followed by a test for gametophytic gene expression, based on the distortion from mendelian segregation and on the measurement of pollen size and pollen sterility. The material used consisted of 34 endosperm defective viable mutants introgressed in B37 genetic background. Complementation tests indicated that the mutation in the collection of mutants affected different genes controlling endosperm development. The study of the segregation in F2 revealed four classes of de (defective endosperm) mutants: (1) mutants in which the mutation does not affect either gametophytic development or function; (2) mutants in which the effect on the gametophyte affects pollen development processes; (3) mutants showing effects on both pollen development and function, and (4) mutants where only pollen tube growth rate is affected. Positive and negative interactions between pollen and style were detected by means of mixed pollination (pollen produced by de/de plants and pollen from an inbred line used as a standard and carrying genes for colored aleurone), on de/de and de/ + plants. Positive interactions were interpreted as methabolic complementation between defective pollen and normal styles.     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

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.     

Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Movement of C-compounds from Maternal Tissue into Maize Seeds Grown in Vitro

Uptake from nutrient media into the cob and translocation of various ¹⁴C-compounds from maternal tissue (cob) into developing maize seeds was examined by using caryopsis cultures. Based on relative ¹⁴C concentrations in the cob and the endosperm, it was concluded that the relative efficiencies of movement of amino acids (leucine, phenylalanine, proline), vitamins (thiamine HCl, nicotinic acid), and nucleic acid bases (adenine, thymine) from the cob to the endosperm were 11 to 250 times lower than that of sucrose. Thiamine was unique in that it was concentrated in the embryo at a level that was almost 10 times higher than in the endosperm. The absence of auxotrophic mutants requiring an organic supplement in higher plants (other than thiamine auxotrophs) may be explained by inadequate translocation of these essential metabolites into the mutant zygotes (embryos) to enable their development to mature seeds.     

Translocation and Metabolism of Endosperm-Applied [2-C] Indoleacetic Acid in Etiolated Avena sativa L. Seedlings

The role of free indole-3-acetic acid (IAA) in the endosperm of Avena sativa L. seedlings was investigated to determine its contribution to free IAA in the shoot. [2-¹⁴C]IAA was injected into the endosperm of darkgrown seedlings and the transport and metabolism of the [¹⁴C]-labeled compounds determined. It was concluded that translocation of free IAA directly from the endosperm is probably not a significant source of free IAA in the shoot, mainly because even small amounts of [¹⁴C]IAA introduced into the endosperm were rapidly metabolized. This suggested that, in Avena, free IAA does not normally exist in the liquid endosperm.     

Construction of IAA-Deficient Mutants of Pseudomonas moraviensis and Their Comparative Effects with Wild Type Strains as Bio-inoculant on Wheat in Saline Sodic Soil

Tryptophan role in microbial biosynthesis of Indole Acetic Acid (IAA) is very distinct. In present study IAA producing bacteria Pseudomonas moraviensis was applied on wheat for improving growth and physiology; in the presence or absence of L-tryptophan in saline sodic field. Aqueous solution of tryptophan was added to the rhizosphere soil at 10?mg L^?1 with irrigated water. The survival efficiency of P. moraviensis measured in the presence of NaCl and mixture of salts. P. moraviensis increased P, NO₃–N and K contents in soil by 18–35% and further 12–15% increase was recorded in the presence of tryptophan. There were 40–80% increases in indole acetic acid (IAA), abscisic acid (ABA) and gibberellic acid (GA) contents of rhizosphere soil, and 40–45% increase in leaves when tryptophan was added with P. moraviensis. In the second phase, IAA deficient mutants of P. moraviensis were constructed and tested for the conversion of tryptophan to IAA. In transposon mutagenesis, 1800 trans-conjugants were generated and tested for tryptophan conversion. Among these, 11 mutants were selected and inoculated into wheat to compare their growth responses to the wild type. P. moraviensis wild type served as PGPR under salinity, but IAA- deficient mutants of P. moraviensis were unable to produce IAA and halted plant growth.     

Genetic relationship between o6 and pro-1 mutants in maize

Summary This paper reports that the opaque-6 (o6) mutation of maize, which causes seedling lethality and interferes in the endosperm with the synthesis of zeins and b-32 protein, is a proline requiring mutant functionally allelic to proline-1 (pro-1). Furthermore, immunological studies on the b-32 content of ten independently originated o6 and pro-1 alleles demonstrated that four alleles contain an apparently normal b-32 protein while the others are either devoid of it or contain trace amounts of cross-reacting proteins of lower molecular weight.     

Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electrophoresis, it is resolved into four fractions of different molecular weight: 10, 14, 20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelectric pH. For example, the 20 kd fraction contains at least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we present evidence that the structural genes coding for the 20 kd proteins are clustered on the short arm of chromosome 7, a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De^*-B30)]. The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated duplication and divergence of chromosome segments.     

The Effect of Epibrassinolide on Arabidopsis Seedling Morphogenesis and Hormonal Balance under Green Light

We studied the effect of 24-epibrassinolide (EB) on the levels of endogenous hormones and photomorphogenesis of Arabidopsis thaliana (L.) Heynh wild-type (Ler) and mutant (hy4) seedlings. This mutant is deficient in the cryptochrome 1 (CRY1) synthesis. CRY1, which is a product of the HY4 gene, is a blue light photoreceptor in wild-type plants, but is sensitive to green light as well. In dark-grown seven-day-old mutant seedlings, the ABA/zeatin ratio differed from this ratio in wild-type seedlings. Thehy4 mutant exhibited a lower zeatin and higher free-ABA contents, which could retard its hypocotyl growth in darkness. EB retarded the growth of hypocotyls in etiolated hy4 seedlings and enlarged their cotyledons more efficiently than in wild-type seedlings. Green light (GL) did not affect the growth of hypocotyls but enlarged cotyledons of hy4 seedlings, which might be associated with some increase in the level of free IAA and a considerable decrease in free ABA and also with a decrease in the cytokinin level in seedlings. The hy4 cotyledon response to GL depended evidently on photoreceptors other than CRY1. GL enhanced the effects of EB on the morphogenesis of both Ler and hy4 seedlings, which was coupled with changes in the balance of endogenous IAA, ABA, and cytokinins. We may suppose that EB is involved in the control of photomorphogenesis by interaction with endogenous hormones, which are involved in the transduction of a light signal absorbed by the GL photoreceptors.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

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.     

A comparison between 3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoic acid. II. Effects on uptake and efflux of IAA in maize roots

An explanation is sought for the inhibition of maize root growth and gravireaction brought about by treatment with 3,5-diiodo-4-hydroxybenzoic acid (DIHB). The effects of DIHB and 2,3,5-triiodobenzoic acid (TIBA) on the uptake and efflux of [³H]-indol-3yl-acetic acid (IAA) were tested using segments prepared from the elongation zone (2 to 7 mm region) of maize (Zea mays L. cv. LG11) roots. The uptake of [³H]-IAA (21 nM) by root segments incubated in buffered solutions (pH 5.0) was measured over a 5-min time-course. No significant effect of DIHB at 100 μM was observed, whereas TIBA at 10 μM slightly stimulated the uptake of [³H]-IAA. This experiment was repeated with the addition of non-radioactive IAA (total IAA concentration 1.0 μM). Up to 3 min DIHB (100 μM) had no significant effect, but thereafter a slight stimulation of IAA net uptake was observed. Treatment with TIBA (10 μM) stimulated the accumulation of IAA in the segments. The effects of DIHB (10, 50, 100 μM) and TIBA (10 and 50 μM) on the efflux of [³H]-IAA from segments that had been pretreated in [³H]-IAA (22 nM) were then tested. Treatment with DIHB or TIBA at pH 5.0 inhibited IAA efflux; the inhibition by TIBA was more marked than that produced by DIHB. This experiment was repeated using DIHB (10, 50, 100 μM) buffered at pH 6.0, and an inhibition of IAA efflux was again observed. Both DIHB (10 μM) and TIBA (10 μM) inhibited the binding of [³H]-NPA to a 5000–48000 g membrane fraction prepared from whole maize roots. The effects of the two substances were similar: 40% inhibition of specific binding by DIHB and 41% inhibition by TIBA. This indicates that DIHB, like TIBA, binds to the N-1-naphthyl-phthalamic acid-sensitive carrier for IAA efflux. It is concluded that DIHB, like TIBA, inhibits IAA transport at the level of efflux. The similarity between DIHB and TIBA as regards chemical structure and their inhibitory effects on IAA efflux and NPA binding strongly suggest that they act on the same carrier for IAA efflux across the plasmalemma.     

Regulation of programmed cell death in maize endosperm by abscisic acid

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.     

Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants

Summary Symbiotic mutants of Rhizobium meliloti were isolated following Tn5 mutagenesis. Besides four nodulation mutants (Nod^-) unable to induce nodule formation on alfalfa, five infection mutants (Inf^-), which induce the formation of root nodules without detectable infection threads or bacteroids, were obtained. The Inf^- mutants were subdivided into two classes. One class contains mutants which fail to synthesize acidic exopolysaccharide (EPS^-). The other class is comprised of mutants which produce excess amounts of acidic exopolysaccharide (EPS^*). ¹³C nuclear magnetic resonance spectroscopy of the exopolysaccharide isolated from one of the latter type of Inf^- mutant, 101.45, revealed that the side chain of the repeating octosaccharide unit lacks the terminal pyruvate residue. Complementing cosmids were isolated for all Inf^- mutants. In the case of the Inf^- EPS^- mutants the complementing cosmids contain DNA segments which overlap and are part of megaplasmid 2. For two mutants the mutations were found to map on a 7.8 kb EcoRI fragment. In the case of the Inf^- EPS^* mutants the complementing cosmids carry chromosomal DNA. The mutations of two Inf^- EPS^* mutants were localized on a 6.4 kb EcoRI fragment. Coinoculation of alfalfa plants with Nod^- and Inf^- EPS^- mutants resulted in effective symbiosis. The nodules appeared wild type and fixed nitrogen. In constrast, coinoculations with Nod^- mutants and the Inf^- EPS^* mutant 101.45 did not result in the formation of effective nodules.