Gibberellins and the procera mutant of tomato

The procera mutant of tomato (Lycopersicon esculentum L.) has a phenotype which is remarkably similar to that of normal tomatoes treated with exogenous gibberellin (GA), indicating that it might be a GA over-producer. However, analysis of endogenous GAs by gas chromatography-mass spectrometry showed that Procera actually has lower levels of GA₂₀ and GA₁ than normal. The reason for these anomalously low GA levels is not clear, as there was no difference between procera and normal plants in their ability to metabolize [³H]GA₂₀. The procera mutant responded to exogenous gibberellic acid with increased extension growth, but the proportional response for a given dose of GA was the same in procera and normal plants. It therefore appears that the procera mutation does not directly affect either the GA status of the plant, or its ability to respond to GA.Abbreviations GA gibberellin - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MeTMSi methyl trimethylsilyl - SIM selected ion monitoring     

Somatic instability of carotenoid biosynthesis in the tomato ghost mutant and its effect on plastid development

The tomato (Lycopersicon esculentum (L.) Mill.) ghost plant is a mutant of the San Marzano cultivar affected in carotenoid biosynthesis. ghost plants exhibit a variable pattern of pigment biosynthesis during development. Cotyledons are green but true leaves are white. Green sectors, which appear to be clonal in origin, are frequently observed in the white tissue. Because of the lack of photosynthesis ghost plants have a very low viability in soil. We have developed a strategy for propagating ghost plants that employs organ culture to generate variegated green-white plants which, supported by the photosynthetic green areas, develop in soil to almost wild-type size. These plants were used to analyze the pigment content of the different tissues observed during development and plastid ultrastructure. Cotyledons and green leaves contain both colored carotenoids and chlorophyll but only the colorless carotenoid phytoene accumulates in white leaves. the plastids in the white tissue of ghost leaves lack internal membrane structures but normal chloroplasts can be observed in the green areas. The chromoplasts of white fruits are also impaired in their ability to form thylakoid membranes.     

Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Immunoassay and ultrastructural localization of isopentenyladenine and related cytokinins using monoclonal antibodies

Two hybridoma cell lines, J40-IV-A1 and J40-IV-C4 were obtained from a fusion of spleen cells of Balb/c mice immunized against an isopentenyladenosine-bovine serum albumin conjugate with X63. Ag 8.653 myeloma cells. These hybrids secrete monoclonal antibodies of the immunoglobulin G (IgG) class and share high affinities and specificities to isopentenyladenine and isopentenyladenosine suitable for the detection of femtomole amounts of these cytokinins in plant extracts by enzyme-linked immunosorbent assay (ELISA). One of the monoclonal antibodies (J40-IV-C4) has been employed to localize isopentenyladenine immunoreactivity in a cytokinin-over-producing mutant of the moss, Physcomitrella patens. After fixation and embedding at low temperature, immunoreactivity was visualized in protonemal filaments of the moss mutant by the use of indirect immunogold labelling. In the mutant, the labelling was predominantly in the wall of the protonemal cells. Neither the wild-type nor control treatments showed any labelling. The signficance of these observations is discussed with respect to the applicability of immunocytochemical techniques for the localization of low-molecular-weight compounds in plant tissue.Abbreviations ELISA enzyme linked immunosorbent assay - HPLC high-performance liquid chromatography - IP isopentenyladenine - IPA isopentenyladenosine - mAB monoclonal antibody - OVE cytokinin-over-producing mutant - RIA radioimmunoassay     

Radioimmunoassays for trans-zeatin and related cytokinins

Radioimmunoassays for the quantitation of trans-zeatin and related cytokinins have been developed. Antisera produced against bovine serum albumin conjugates of trans-zeatinriboside have a high affinity (K_a=2.4.10^-11 M) for zeatinriboside and for zeatin, but show a negligible cross reaction to isopentenyladenosine (0.1%) and cis-zeatinriboside (0.4%), only a slight cross reaction to dihydrozeatin (1.7%), and no cross reaction at all to other purines, such as adenosine and related, compounds, was observed. The assays are sensitive and measuring ranges extend from 0.06–30 pmol (0.02–10 ng) of zeatinriboside. This has been achieved by employing as tracers immunoreactive zeatin derivatives with high-specific activity, (tritiated zeatinriboside-dialcohol: 8.37.10¹¹ Bq mmol^-1 and zeatinribosyl-[¹²⁵I]tyramine: ca. 1.9.10¹³ Bq mmol^-1. The detection limit is 40 fmol (15pg) for the assay employing the tritiated tracer, and assay reproducibility is high (variation coefficients of triplicates less than 5%). Several hundred assays can be completed in one day, and, due to the high specificity of this assay, crude extracts may be used for analysis. The course of zeatin levels in developing fruits of Lycopersicon esculentum cv. Moneymaker is given.Abbreviations GC gas chromatography - LC liquid chromatography - MS mass spectroscopy - RIA radioimmunoassay - TLC thin layer chromatography - UB unspecific binding Part 12 in the Series: Use of Immunoassay in Plant Science     

Accumulation of an ABA analogue in the wilty tomato mutant,flacca

A new abscisic acid (ABA) analogue has been isolated from tomato plants. High levels of the compound are found in flacca mutants compared with normal isogenic controls. The analogue also accumulates in response to water stress. Three alternative structures, consistent with the mass spectrum, have been proposed. The possibility that the compound may be a biosynthetic precursor of ABA is considered.We are grateful for the financial support of the ARC.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe cytokinin-like phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially empty leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Synthesis of polygalacturonase during tomato fruit ripening

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in Never ripe mutants and the lack of activity in ripening inhibitor mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.Abbreviations PG polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

Gibberellins regulate seed germination in tomato by endosperm weakening: a study with gibberellin-deficient mutants

The germination of seeds of tomato [Lycopersicon esculentum (L.) Mill.] cv. Moneymaker has been compared with that of seeds of the gibberellin-deficient dwarf-mutant line ga-1, induced in the same genetic background. Germination of tomato seeds was absolutely dependent on the presence of either endogenous or exogenous gibberellins (GAs). Gibberellin A₄₊₇ was 1000-fold more active than commercial gibberellic acid in inducing germination of the ga-1 seeds. Red light, a preincubation at 2°C, and ethylene did not stimulate germination of ga-1 seeds in the absence of GA₄₊₇; however, fusicoccin did stimulate germination independently. Removal of the endosperm and testa layers opposite the radicle tip caused germination of ga-1 seeds in water. The seedlings and plants that develop from the detipped ga-1 seeds exhibited the extreme dwarfy phenotype that is normal to this genotype. Measurements of the mechanical resistance of the surrounding layers showed that the major action of GAs was directed to the weakening of the endosperm cells around the radicle tip. In wild-type seeds this weakening occurred in water before radicle protrusion. In ga-1 seeds a similar event was dependent on GA₄₊₇, while fusicoccin also had some activity. Simultaneous incubation of de-embryonated endosperms and isolated axes showed that wild-type embryos contain and endosperm-weakening factor that is absent in ga-1 axes and is probably a GA. Thus, an endogenous GA facilitates germination in tomato seeds by weakening the mechanical restraint of the endosperm cells to permit radicle protrusion.Abbreviations GA(s) gibberellin(s) - GA₃ gibberellic acid     

Cellular basis of the effects of gibberellin and the pro gene on stem growth in tomato

Tomato (Lycopersicon esculentum Mill.) plants homozygous for the mutant pro gene, exhibiting the distinctive procera phenotype, appeared virtually identical to gibberellic acid (GA₃)-treated isogenic normal plants. The pro gene and GA₃ caused analogous increases in internode length, and in the length and number of cells in the outer cell layers of each internode. Internode number was also increased by pro and GA₃ over the period of the experiment. Despite their greater length, the internodes of GA₃-treated and pro plants reached their final size within a time period similar to that of internodes of untreated normal plants. The pro mutant itself was responsive to GA₃, especially in the seedling stage, but the proportional increase in height seen in the later stages of growth was less than that of normal plants.Abbreviations GA gibberellin - GA₃ gibberellic acid - LSD least significant difference     

Characterisation of a temperature-sensitive gametophore over-producing mutant of the moss, Physcomitrella patens

Summary A mutant of the moss, Physcomitrella patens, was isolated which was temperature-sensitive for the production of gametophores. At 17° C this mutant, designated ove 409, produced normal leafy shoots. At 24° C ove 409 produced many abnormal buds characteristic of bud-over-producing (ove) mutants. ove 409 produced an intermediate phenotype at 21° C. The cytokinin levels in the culture medium of this mutant, the wild-type and a cytokinin overproducing mutant, oveA78, were measured by combined gas chromatography mass spectrometry at the permissive and nonpermissive temperatures. Production of cytokinin was found to be affected by temperature in all strains; the change in phenotype of ove 409 correlated with the production of N⁶-(²-isopentenyl) adenine. Complementation analysis was performed using this mutant by protoplast fusion. ove 409 was found to be in the same complementation group as a previously isolated ove mutant, oveA78.     

Abscisic-acid metabolism in a wilty mutant of Nicotiana plumbaginifolia

A mutant of Nicotiana plumbaginifolia, CKR1, isolated on the basis of its enhanced resistance to cytokinins was found to have a greater tendency to wilt than the wild type (Blonstein et al., 1991, Planta 183, 244–250). Further characterisation has shown that the wiltiness in the mutant is not caused by an insensitivity to abscisic acid (ABA) because the external application of ABA leads to stomatal closure and phenotypic reversion. The basal ABA level in the mutant is < 20% of that in the wild type. Following stress, the ABA level in wild-type leaves increases by approx 9-to 10-fold while the mutant shows only a slight increase. This deficiency in ABA is unlikely to be the consequence of accelerated catabolism as the levels of two major metabolites of ABA, phaseic and dihydrophaseic acid, are also much reduced in the mutant. The qualitative and quantitative distributions of carotenoids, the presumed presursors of ABA, are the same for the leaves of both wild type and mutant. Biosynthesis of ABA at the C15 level was investigated by feeding xanthoxin (Xan) to detached leaves. Wild-type leaves convert between 9–19% of applied Xan to ABA while the mutant converts less than 1%. The basal level of trans-ABA-alcohol (t-ABA-alc) is 3-to 10-fold greater in the mutant and increases by a further 2.5-to 6.0-fold after stress. This indicates that the lesion in the wilty mutant of N. plumbaginifolia affects the conversion of ABA-aldehyde to ABA, as in the flacca and sitiens mutants of tomato and the droopy mutant of potato (Taylor et al., 1988, Plant Cell Environ. 11, 739–745; Duckham et al., 1989, J. Exp. Bot. 217, 901–905). Wild-type tomato and N. plumbaginifolia leaves can convert trans-Xan into t-ABA-alc, and Xan into ABA, while those of flacca and the wilty N. plumbaginifolia mutant convert both Xan and t-Xan to t-ABA-alc.     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate     

Immunocytochemical localization of histamine in flatworms

Summary Specific antibodies against histamine were used to demonstrate the occurrence and cellular distribution of histamine-like immunoreactivity in three species of flatworms (phylum Platyhelminthes). In the parasitic cestode Diphyllobothrium dendriticum, histamine-reactivity was found in neurons of the main nerve cords, and in cells lining the central and peripheral excretory ducts. In the free-living microturbellarian Microstomum lineare and in the planarian Polycelis nigra, histamine-immuno-reactivity was restricted to cells and fibres of the nervous system. The occurrence of histamine or a related substance in the nervous system of flatworms, which represent primary bilateria, indicates the importance of this neuroactive substance in the animal kingdom.     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

On the biogenesis of cytokinins in roots of Phaseolus vulgaris

Roots of intact bean plants were supplied with [¹⁴C]adenine by pulse-chase experiments. The rate of incorporation of radioactivity into tRNA and oligonucleotides of roots as well as the content of radioactive labeled cytokinin nucleotides in these RNA fractions were determined. On the average, 1/70 of the radioactivity incorporated into tRNA was localized in N⁶(²isopentenyl)adenosine. The half life of tRNA was estimated to be 65–70 h. Shortly after the pulse period, oligonucleotides contained zeatin riboside at a ratio of 1:800, on the basis of radioactivity. The half life of these oligonucleotides was determined to be about 8 h. The main free radioactive cytokinin of roots and leaves was zeatin. Comparing the rate of degradation of ¹⁴C-labeled tRNA and the oligonucleotides of roots and the rate of appearance of radioactive cytokinins in roots and leaves, we found strong indications for their dependency. The results contradict the hypothesis of de novo synthesis of cytokinins in roots of intact bean plants.Abbreviations AMP adenosine monophosphate - IPA N⁶(²isopentenyl)adenosine - IPAde N⁶(²isopentenyl)adenosine - Z zeatin - ZR zeatinriboside - TLC thin-layer chromatography - HPLC high performance liquid chromatography Part of the doctoral thesis, Bonn 1980     

Photoisomerization of lycopene during carotenogenesis in mutant C-6D of Scenedesmus obliquus

Mutant C-6D of the unicellular green alga Scenedesmus obliquus has lost the ability to form cyclic carotenoids during heterotrophic growth in the dark. In the dark it accumulates acyclic intermediates, i.e. lycopene, neurosporene and ζ-carotene. The lycopene and two neurosporene forms were identified to be cis-isomers. Upon transfer to light, intermediates decrease and a normal set of carotenoids is synthesized. Inhibition of the cyclization reaction by nicotine reveals a lightinduced isomerization of cis-lycopene to trans-lycopene. Since the spectral characteristics of these two isomers differ drastically the isomerization can be followed in vivo by measuring a light-induced absorbance change. This absorbance change has its maximum at 520 nm and shows fast kinetics under high light intensities reaching a saturation level after about 2 min. Fluence-response curves for this absorbance change were performed for different wavelengths of actinic light. From the linear parts of these curves an action spectrum was caculated showing maxima at 670, 630 and 440 nm originating from chlorophyll and a maximum at shorter wavelengths (400–510 nm) which is interpreted to derive from ζ-carotene. A model for the light regulation of carotenogenesis in mutant C-6D is presented and the relation to the so-called 520-change observed in many plants is discussed.