A pleiotropic mutation results in cross-resistance to auxin, abscisic acid and paclobutrazol

Summary Mutant lines of Nicotiana plumbaginifolia resistant to the synthetic auxins 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) were isolated as germinating seedlings on selective medium. In each case, resistance was conferred by a single recessive nuclear mutation at one of 3 loci designated iba1, iba2 and iba3. Labelling studies with ¹⁴C NAA suggest that resistance was not due to changes in the uptake or metabolism of NAA. Plants homozygous for the iba1 mutation exhibit a syndrome of atypical germination and growth suggestive of a defect in the biosynthesis, metabolism or localization of abscisic acid. Wild-type seeds treated with gibberellin exhibit the same syndrome, including resistance to NAA and IBA. On the basis of these observations, we propose that auxin toxicity in seeds may be mediated by a block in gibberellin biosynthesis.Abbreviations ABA abscisic acid - GA gibberellic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - p-cell protoplast-derived cell - 2,4-D 2,4-dichlorophenoxyacetic acid     

Interaction of jasmonic acid with some plant growth regulators in the control of apple (Malus domestica) embryo germination

Embryos isolated from dormant apple seeds were treated with jasmonic acid (JA), gibberellin A₃ (GA₃), abscisic acid (ABA) and hydrogen cyanide in darkness and in light. The chemicals were present in the culture medium continuously and simultaneously or applied for 2 days and in different sequences. All treatments stimulated embryo germination except ABA, which was strongly inhibitory. Additive effects of JA with light and with GA₃ on embryo germination were observed, whereas ABA interacted synergically with JA, HCN and light. ABA and GA₃ were most effective when applied early during embryo incubation, but the late JA treatment was more stimulatory. It is concluded that JA does not act on the regulatory pathway that is initiated by light and which leads to embryo germination through gibberellin accumulation and alkaline lipase activation. ABA and HCN appear to be involved in the control of this pathway. JA and ABA may be involved in the control of alkaline lipase activity, independently of this regulatory chain.Abbreviations ABA abscisic acid - GA₃ gibberellin A₃ - JA jasmonic acid     

Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.     

Evidence for the accumulation of a germination inhibitor during progressive thermoinhibition of seeds of celery (Apium graveolens L.)

The germination of seeds of celery (Apium graveolens L.) becomes progressively thermoinhibited on incubation in the dark at high temperatures, the inhibitory temperature being dependent on the cultivar used. In two high-dormancy cultivars of celery, the production of germination inhibitors in seeds incubated in the dark at 26°C gradually increased over a 7-day period. Inhibitor production was measured by incubating seeds of the low-dormancy cultivar Florida 683 in homogenates of the thermoinhibited seeds of the high-dormancy cultivars and recording germination either in the light or with the gibberellins A₄ and A₇ (GA_4/7) in the dark. Most Florida 683 seeds which failed to germinate in the homogenates after 15 days were induced to germinate by addition of N⁶-benzyladenine (BA). The presence of BA in addition to GA_4/7 throughout incubation in the dark completely overcame the inhibitory effects of homogenates. This indicates that thermoinhibition of celery seeds is associated with the accumulation of a germination inhibitor which interacts with cytokinins. This does not appear to be abscisic acid (ABA) since ABA levels in thermoinhibited seeds were lower than in untreated seeds and did not increase with duration of high temperature treatment.Abbreviations ABA Abscisic acid - BA N⁶-benzyladenine - GA_4/7 a mixture of the gibberellins A₄ and A₇ - HTP high-temperature pretreatment     

Growth regulators differentially affect photosensitivity induced by low temperature and osmotic stresses in germinating white clover seeds

Negative photoblastism induced in white clover seeds at 5°C or by lowered water potential (–0.3 MPa, polyethylene glycol) was affected by ethrel, gibberellin A₃, benzylaminopurine and kinetin treatments. The effects were different for water and temperature stressed seeds. The observed synergistic and additive effects of light and growth regulators confirm the earlier suggestion that there are two different mechanisms involved in the light inhibition of white clover seed germination induced by various adverse environmental conditions.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellin A₃ - PEG polyethylene glycol     

The influence of the genes An1, An2, and An4 on the activity of the enzyme UDP-glucose:Flavonoid 3-O-glucosyltransferase in flowers of Petunia hybrida

A relation between gene dosage and UDP-glucose:flavonoid 3-O-glucosyl-transferase (UFGT) activity was found in homozygous dominant and recessive parental lines and their F₁ progeny for both of the genes An1 and An2. In both F₂ crosses, progeny plants could be classified as belonging to groups showing either a low or a medium to high UFGT activity. Test crosses showed that heterozygous and homozygous dominant plants were present throughout the medium- to high-active group. The dosage relation in F₂ plants is most probably confounded by the segregation of modifiers. Thermal inactivation experiments indicated that structurally different UFGT enzymes are formed in homozygous dominant lines as well as in lines homozygous recessive for either An1 or An2. Lines homozygous recessive for the gene An4 contain a UFGT with a half-life time at 55° C of less than 8 min, whereas UFGTs from lines homozygous dominant for An4 show a half-life time of 25 min or above, with one exception. This relation was confirmed in the F₂ progeny; heterozygotes for An4 showed an intermediate half-life time. It is concluded that An4 might be the structural gene for the enzyme; An1 and An2 are both regulatory genes. UFGT activity in flowerbuds of An4/An4 plants seems to be lower than in an4/an4 plants. Anthers of flowers of an4/an4 lines, however, are virtually devoid of UFGT activity.     

Somatic embryogenesis in Cucurbitaceae

Strategies based on the application of biotechnologies to crop improvement programmes generally require regeneration of whole plants from cells or tissues cultivated in vitro. In Cucurbitaceae, regeneration can occur either through a caulogenic or an embryogenic developmental pathway. Reports of somatic embryogenesis have dealt with the main cultivated crops, i.e. cucumber, melon, squash and watermelon. Somatic embryogenesis and plant recovery are obtained from numerous sources including protoplasts, but the best results are observed with explants coming from seedlings, especially cotyledons and hypocotyls. The genetic constitution of mother plants also seems to play a key role in the success of embryogenesis, but few systematic studies on genotype effect have been published. Somatic embryos can exhibit developmental abnormalities, particularly when they arise from protoplast-derived cultures. Generally, data concerning embryo yield, rate of germination and plant development and characteristics of regenerated plants and their progeny, has not been provided in previous reports. The potential use of somatic embryogenesis in cucurbit breeding programmes is stressed in this review.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - B₅ Gamborg et al. (1968) - CH casein hydrolysate - CW coconut water - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellin A₃ - GA₄ gibberellin A₄ - H Heller (1953) - IAA indole 3 acetic acid - IBA indole 3 butyric acid - 2ip 2 isopentenyladenine - KIN kinetin - MS Murashige & Skoog (1962) - N Nitsch (1951) - N6 Chu et al. (1975) - NAA 1 naphthalene acetic acid - TDZ thidiazuron - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Interactions of Abscisic Acid, Cytokinin and Gibberellin in the Control of Betacyanin Synthesis in Seedlings of Amaranthus caudatus

In de-rooted seedlings of Amaranthus caudatus L., betacyanin synthesis induced by white light or cytokinin was inhibited by abscisic acid (ABA) or a mixture of gibberellins A₄ and A₇ (GA_4/7). The GA_4/7 and ABA effects were additive. Thus ABA inhibited the cytokinin action but had no effect on the gibberellin response.     

Changes in phytohormone contents in chickpea seeds germinating under lead or zinc stress

The present work describes the changes that take place in phytohormone contents in germinating chickpea (Cicer arietinum cv. Aziziye-94) seeds in response to heavy metal stress. For this aim, endogenous abscisic acid (ABA), gibberellic acid (GA₃), zeatin (Z) and zeatin riboside (ZR) contents were followed for 24, 48 and 72 h in chickpea seeds germinating at the concentrations of 0.1, 1.0 and 5.0 mM Pb or 0.1, 1.0 and 10 mM Zn. The results showed that Pb and Zn significantly delayed and impeded the germination of chickpea seeds. The negative effect of Pb on germination was higher than that of Zn. Further, Pb increased ABA and Z contents while decreased GA₃ content in the germinating seeds. The high concentrations of Zn (1.0 and 10 mM) decreased contents of Z, ZR and GA₃ while 0.1 mM Zn increased the content of the same hormones. The ABA content was enhanced by Zn in all concentrations used.     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Cloning,characterization and expression of<Emphasis Type="Italic"> OsGLN2</Emphasis>, a rice endo-1,3-β-glucanase gene regulated developmentally in flowers and hormonally in germinating seeds

We report here the isolation and characterization of a new endo-1,3--glucanase (1,3--GLU) cDNA, OsGLN2, that is expressed both in flowers and in germinating seeds of rice (Oryza sativa L.). The isolated OsGLN2 gene encoded a protein which displayed 72%, 93% and 92% identity at the amino acid level with those encoded by barley GII, rice Gns4 and glu1 1,3--GLU genes, respectively. A GST-OsGLN2 recombinant protein expressed in Escherichia coli preferentially hydrolyzed Laminaria digitata 1,3;1,6--glucan and liberated only oligosaccharides, suggesting that the enzyme can be classified as a 1,3--GLU. Northern analysis with a 3-UTR gene-specific probe revealed that OsGLN2 is expressed exclusively in the paleae and lemmas during flowering, and no expression of OsGLN2 was detected in other tissues such as leaf blades, leaf sheaths, stems, nodes and roots in mature rice plants. The OsGLN2 gene is also expressed in germinating seeds, where its expression is predominant in endosperms rather than embryos. In de-embryonated rice half-seeds, addition of gibberellin A₃ (GA) greatly enhanced expression of the OsGLN2 gene, while the GA-induced gene expression was suppressed strongly by abscisic acid (ABA). This is the first report, to our knowledge, that OsGLN2 encodes a 1,3--GLU and is expressed specifically in paleae and lemmas during flowering and in germinating seeds, where its expression is enhanced by GA and suppressed by ABA.Abbreviations ABA Abscisic acid - GA Gibberellin A₃ - 1,3--GLU Endo-1,3--glucanase - GST Glutathione S-transferase - IPTG Isopropyl-1-thio--galactopyranoside - 3-UTR 3-Untranslated region     

Implication of Ethylene in the Release of Secondary Dormancy in Amaranthus caudatus L. Seeds by Gibberellins or Cytokinin

Ethephon (Eth), gibberellin A₃, A_{4 + 7} (GA₃, GA_{4 + 7}), and 6-benzyladenine (BA) removed secondary dormancy of Amaranthus caudatus seeds. The GAs and BA potentiated the effect of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene biosynthesis precursor, in terms of the rate or final percent of germination. Aminoethoxyvinylglycine (AVG), an ACC synthase activity inhibitor, was observed to simultaneously inhibit the release from dormancy effected by GA₃ or BA as well as the ethylene production stimulated by these regulators. Breaking of secondary dormancy by GA₃, GA_{4 + 7} or BA was prevented by 2,5-norbornadiene (NBD), an inhibitor of ethylene binding. Ethylene completely or markedly reversed the inhibitory effect of NBD. We thus conclude that the removal of secondary dormancy in Amaranthus caudatus seeds by gibberellin or benzyladenine involves ethylene biosynthesis and action.     

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development

Gibberellins A₁, A₈, A₂₀ and A₂₉ were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A₁ within these ovary types was correlated with pod size. Gibberellin A₁, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A₂₀. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A₃. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A₂₀ and A₂₉ in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A₁, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A₂₀ and A₂₉, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.Abbreviations GA_(a) gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFK perfluorokerosene - PVP polyvinylpyrrolidone     

The identification of gibberellins in immature seeds of Vicia faba,and some chemotaxonomic considerations

GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄ and 13-hydroxy-GA₁₂, now named GA₅₃, were identified by GC-MS in immature seeds of Vicia faba (broad bean). Also identified were a GA catabolite, two polyhydroxykauranoic acids, and abscisic, phaseic and dihydrophaseic acids. The GAs of Vicia are hydroxylated at C-13, in common with those of other legumes. However the GAs of Vicia are not hydroxylated at C-3, nor do they appear to be readily conjugated. In these respects Vicia resembles Pisum, another member of the tribe Viciae. Vicia differs from Phaseolus and Vigna, of the tribe Phaseoleae, in both these respects.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA_n gibberellin A_n - GC gas chromatography - GC-MS gas chromatography mass spectrometry - KA kauranoic acid - PA phaseic acid - TLC thin layer chromatography     

A simple system for determining the phloem mobility of compounds using excised pods of lupin (Lupinus albus L.)

Lupin pods were isolated from the plant and the pedicels placed in solutions of either radiolabeled sucrose, glucose, abscisic acid, or gibberellin A₉ (GA₉). Phloem exudate was collected from the cut stylar ends of the pods and both quantitative and qualitative data were obtained on the activity in the sap. Fed sucrose, abscisic acid, and GA₉ were found in phloem exudate, but labeled glucose was immobile as the monosaccharide and activity in exudate appeared in sucrose.Abbreviations GA gibberellic acid - ABA abscisic acid - TLC thin layer chromatography     

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A₄ (GA₄) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A₄ is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA₄ in the presence of an exogenous gibberellin, such as GA₁, which is not a biosynthetic precursor for GA₄. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA₁ is indirect and appears to be mediated by GA₄. Embryos form predominantly GA₁; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography     

Enzymatic control of anthocyanin expression in the flowers of pea (Pisum sativum) mutants

Using enzymological and immunological methods we have investigated the relationship between chalcone synthase and the A locus, a major gene involved in the control of anthocyanin expression in pea (Pisum sativum L.) flowers. Pea plants containing the dominant allele A usually synthesize anthocyanins in the petal tissue, whereas plants homozygous for the a allele do not produce anthocyanins. We sought to determine whether or not the A locus also controlled the presence or absence of chalcone synthase, the first enzyme of the flavonoid pathway in the flowers of three genetic lines (A, purple-violet flowers; A,am, white flowers with sometimes pink edges; and a, white flowers). Chalcone synthase was found to be present in all three genetic lines by enzyme activity measurement, indirect enzyme-linked immunosorbent assay (ELISA), and Western blotting. Spectroscopic investigations showed that only the genetic lines A and A,am contained anthocyanins and flavonol glycosides, respectively, in the flowers; line a accumulated p-coumaric acid or its derivatives. These data suggest that the A locus in Pisum is not the structural gene for chalcone synthase and it does not appear to regulate the expression of this enzyme.This work was supported by a grant from the Cornell University Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation and a consortium of industries.     

Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana

SAP1-1 and SAP1-2 were isolated from the male reproductive buds of willow (Salix discolor, clone S365). SAP1-1 differs from SAP1-2 based on a few nucleotide substitutions, but the sizes of their full-length cDNAs are identical. The deduced amino acid sequences of SAP1-1 and SAP1-2 were 98% similar and contain the same C-terminal amino acid motif “GYGA” like that of PTAP1-2 from Populus trichocarpa. The expression patterns of SAP1 in various parts of the male reproductive buds of S. discolor implicate this gene in the formation of the inflorescence meristems, bracts, and floral meristems. To characterize the functions of SAP1, we assessed Arabidopsis thaliana transformed with 35S∷SAP1-1. A total of 52 transgenic T₁ lines were obtained, and a 3:1 segregation ratio was obtained in the T₂ generation of each line. In the T₃ generation, five homozygous transgenic lines were obtained, which were used for further analysis. Screening of transgenic lines was greatly facilitated by the detection of GFP expression starting with germinating seeds. Phenotypes of the homozygous transgenic lines included early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, and formation of flowers with greater number of petals, stamens, and pistils. Northern analysis showed similar expression levels in all five lines. This study provides the first functional analysis of an APETALA1 (AP1)/SQUAMOSA (SQUA) homolog from a dioecious species and suggests that SAP1 is a homolog of the AP1/SQUA gene.     

An in vitro method for screening for the presence of thepat-2 gene in tomatoes (Lycopersicon esculentum Mill.)

Under field conditions,pat-2, the gene which conditions parthenocarpy in tomatoes, is recessive. A simple method has been devised for distinguishing the heterozygote from the two homozygotes using tissue culture. Ovaries of plants segregating for thepat-2 gene were excised and cultured on a medium containing 100 ppm gibberellic acid. After three weeks in culture, three distinct ovary sizes could be seen. It was shown, using F ₃ progeny tests, that the largest ovaries corresponded to those plants homozygous for thepat-2 gene, the smallest ovaries corresponded to those plants homozygous for the wild type allele, and the intermediate sized ovaries were the heterozygotes. The ability to identify the heterozygote would greatly simplify a backcross breeding program aimed at incorporating thepat-2 gene into commercial cultivars by eliminating the need for an F ₃ progeny test to determine the genotype of a plant.Abbreviations GA ₃ gibberellic acid - IAA indole acetic acid - ppm parts per million