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     

Feeding responses to solanaceous allelochemicals by larvae of the tobacco hornworm, Manduca sexta

Feeding responses of the oligophagous tobacco hornworm to allelochemicals prevalent in their host plants were determined in food choice-tests using filter paper discs laced with a test solution or water (control). Six solanaceous alkaloids, tomatine, tomatidine, solanine, solanocapsine, atropine and nicotine, were tested and only tomatine and solanocapsine were found to influence preference behavior. Solanocapsine (5 mM) deters feeding whereas tomatine (1 mM) stimulates feeding slightly. No synergistic effect of either tomatine or tomatidine with sucrose was found.The responses to tomatine are affected by previous feeding experience. Tomatine slightly stimulates feeding in larvae reared on tomato (Lycopersicon esculentum), but slightly deters feeding in larvae reared on Jerusalem cherry (Solanum pseudocapsicum). Such induced preference is absent for the other alkaloids tested, which indicates that these alkaloids do not by themselves induce preferences for the plants containing them.The non-alkaloid allelochemicals, chlorogenic acid, rutin, and 2-tridecanone also influenced food choice behavior. Chlorogenic acid is slightly stimulatory at its natural concentration (1mM), but strongly deterrent at higher concentrations. Rutin stimulates feeding in a concentration-dependent manner. Its activity must be due to the glycosylated structure, because both the aglycone (quercetin) and the sugar moiety (rutinose) are neutral. Removal of the glucose part of rutin, as in quercitrin, results in feeding deterrent activity. 2-Tridecanone is neutral at its concentration in cultivated tomato (1 mM), but strongly deterrent and toxic at higher concentrations. Preference behavior is not affected by solanesol, GABA, and a mixture of host plant compounds stimulatory for anothe solanaceous-specific feeder, the Colorado potato beetle (Leptinotarsa decemlineata).We conclude that the prevalent solanaceous alkaloids and other allelochemicals tested do not play important roles in food selection of the tobacco hornworm, although some may make small contributions.

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Résumé Des experiences de choix de chenilles oligophages de M. sexta ont été réalisees avec des disques de papier filtre imbiles d'eau ou de solutions des substances allélochimiques dominantes dans les plantes consommées. Sur les six alcaloïdes de solanées examinés: tomatine, tomatidine, solanine, solanocapsine, atropine et nicotine, seuls la tomatine et la solanocapsine ont influé sur le choix; la solanocapsine (5 mM) empêche la prise de nourriture, tandis que la tomatine (1 mM) la stimule légèrement. Aucun effet synergique de la tomatine ou de la tomatidine n'a été observé en présence de sucrose.La réponse à la tomatine est modifiée par la prise de nourriture antérieure. Elle stimule légèrement l'alimentation de chenilles élevées sur tomates (Lycopersicon esculentum), mais dissuade légèrement les chenilles élevées sur Solanum pseudocapsicum. II n'y a pas d'action induite semblable avec les autres alcalïdes examinés, ce qui indique que ces alcaloïdes ne peuvent pas induire par eux-mêmes de préférences pour les plantes qui les contiennent.Des substances allélochimiques non-alcaloïdes: acide chlorogénique, rutine, et 2-tridécanone, influent aussi sur le comportement de choix alimentaire. L'acide chlorogénique est légèrement stimulant à sa concentration naturelle (1 mM), mais fortement dissuasif aux concentrations supérieures. La rutine stimule la prise de nourriture en fonction de sa concentration. Son activité doit être due à sa structure glucosylate, puisqu'aussi bien l'aglycone (quercitine) que la moiteé sucrée (rutinose) sont neutres. La suppression de la partie glucose de la rutine, comme dans le cas de la quercitine, a un effet dissuasif. A sa concentration dans la tomate cultivée (1 mM), le 2-tridécanone est neutre, mais il est fortement dissuasif et toxique à des concentrations supérieures.Le comportement de choix n'est pas modifié par le solanésol, le GABA, et par un mélange de composés végétaux stimulant un consommateur spécifique de solanées, comme le doryphore (Leptinotarsa decemlineata).Nous pouvons conclure que les principaux alcaloïdes et autres substances allélochimiques des solanées que nous avons examinés n'interviennent pas d'une façon importante, mais peuvent avoir une influence secondaire, dans les choix alimentaires de Manduca sexta.

     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.     

Requirement for the action of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus

The role of endogenous ethylene during germination of non-dormant seeds of Amaranthus caudatus L. was investigated. The seeds readily germinated in water and darkness at 24°C. Application of ethylene or of its precursor I-aminocyclopropane-I-carboxylic acid (ACC) slightly increased the rate of germination. Both compounds effectively antagonized osmotic inhibition by polyethyleneglycol. Application of aminoethoxyvinylglycine (AVG) reduced ethylene production by 90% but did not inhibit germination. However, germination was inhibited by 2,5-norbornadiene, a competitive inhibitor of ethylene action. This inhibition was counteracted by ethylene, ethephon or ACC and enforced by AVG. It is concluded that the action of endogenous ethylene is an indispensable factor during germination of non-dormant seeds of A. caudatus. Ethylene action is required from the start of imbibition on. In water, low levels of endogenous ethylene are sufficient for this action. PEG increased the ethylene requirement considerably.     

Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh.

Mutant lines of Arabidopsis thaliana (L.) Heynh., which are characterized by symptoms of withering and the absence of seed dormancy, showed much lower levels of endogenous abscisic acid (ABA) in developing seeds and fruits (siliquae) than the wild type. Reciprocal crosses of wild type and ABA-deficient mutants showed a dual origin of ABA in developing seeds. The genotype of the mother plant regulated a sharp rise in ABA content half-way seed development (maternal ABA). The genotype of the embryo and endosperm was responsible for a second ABA fraction (embryonic ABA), which reached much lower levels, but persisted for some time after the maximum in maternal ABA. The onset of dormancy correlated well with the presence of the embryonic ABA fraction and not with the maternal ABA. Dormancy developed in both the absence and presence of maternal ABA in the seeds. In this respect maternal ABA resembled exogenously applied ABA which did not induce dormancy in ABA-deficient seeds. However, both maternal and applied ABA stimulated the formation of a mucilage layer around the testa, which could be observed during imbibition of the mature seeds. In the mature state, ABA-deficient seeds germinated in the siliquae on the plant, but only when the atmosphere surrounding the plant was kept at high relative humidity. In younger stages germination in siliquae occurred after isolation from the plants and incubation on wet filter paper. Therefore, it seems that limited access to water is the primary trigger for the developmental arrest in these seeds.     

Continuous monitoring of lactate during exercise in humans using subcutaneous and transcutaneous microdialysis

We have evaluated the possibility of monitoring the plasma lactate concentration in human volunteers during cycle ergometer exercise using subcutaneous and transcutaneous microdialysis. In transcutaneous microdialysis, the relative increase in dialysate lactate concentration exceeded that of plasma lactate concentration by a factor of 6 during exercise due to exercise-induced lactate secretion in sweat. During exercise the subcutaneous microdialysis dialysate lactate concentration underestimated the plasma lactate concentration possibly due to diffusion limitation or adipose tissue lactate production. While it was demonstrated that microdialysis can be used for on-line lactate monitoring, neither subcutaneous nor transcutaneous dialysate lactate concentration were linearly related to the plasma lactate concentration during exercise, and it was found therefore that it was not possible to monitor directly plasma lactate concentration during exercise.     

Nuclear replication activities during imbibition of abscisic acid- and gibberellin-deficient tomato (Lycopersicon esculentum Mill.) seeds

The role of cis-abscisic acid (ABA) and gibberellins (GAs) in the induction of cell-cycle activities has been studied during imbibition and subsequent germination of tomato seeds. Using flow cytometry, nuclear replication activity was investigated in embryo root tips isolated from seeds of the ABA-deficient mutant sit ^w , the GA-deficient mutant gib-1, and the wild-type (MM) tomato (Lycopersicon esculentum Mill. cv. Moneymaker) upon imbibition in water, 10 μM GA₄₊₇, 5 μM ABA or 5 μM ABA+10 μM GA₄₊₇. The nuclei of fully matured dry MM, sit ^w and gib-1 seeds predominantly showed 2C DNA signals, indicating that the cell-cycle activity of most root-tip cells had been arrested at the G₁ phase of nuclear division. However, ABA-deficient sit ^w seeds contained a significantly higher amount of G₂ cells (4C DNA) compared with the other genotypes, suggesting that, during maturation, cell-cycle activity in sit ^w seeds is less efficiently arrested in G₁. Upon imbibition in water, an induction of the 4C signal, indicating nuclear replication, was observed in the root tip cells of both MM and sit ^w embroys. The augmentation in the 4C signal occurred before visible germination. Gib-1 seeds did not show cell-cycle activity and did not germinate in water. Upon imbibition in GA₄₊₇, both cell-cycle activity and subsequent germination were enhanced in MM and sit ^w seeds, and were induced in gib-1. In ABA, the germination of MM and sit ^w seeds was inhibited while nuclear replication of these seeds was not affected. It is concluded that GA influences germination by acting upon processes that precede cell-cycle activation, while ABA affects growth by acting upon processes that follow cell-cycle activation.     

Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria

Summary The accuracy and reproducibility of the gas-chromatographic method for the analysis of PHB and PHA in whole cells of Alcaligenes eutrophus H16 and Pseudomonas putida KT2442 were determined. It was found that for analysis of PHA the methanolysis time in the assay had to be increased to 4 h. Accuracy of the PHB and PHA assay were 0.018 mg and 0.304 mg respectively, based on estimation of the measurement error.