Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde

Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [²H]-ent-kaurenoic acid (KA) and [¹⁴C]-gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [²H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [²H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A₉ (GA₉) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA₉. In contrast, thermo- and noninduced leaves metabolized exogenous [²H]-KA into GA₂₀ equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [¹⁴C]-GA₁₂-aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA₁₂-aldehyde.     

Localization of the Site of Perception of Thermoinductive Temperatures in Thlaspi arvense L

This paper describes attempts to localize the site of perception of low temperatures (0-10°C) during thermoinduction in Thlaspi arvense L. Reproductive development (stem elongation and flower formation) was observed when shoots were cooled to 4°C for 4 weeks and then returned to 21°C while maintaining the roots constant 21°C. However, chilling the roots was ineffective for initiating reproductive development. The apparent site of perception of thermoinductive temperatures was further localized to the shoot tip (apex and immature leaves) by controlling the temperature of the shoot tip independently of the rest of the plant. Furthermore, excised apices regenerated flowering plants in organ culture only if they were subjected to a 4 week cold treatment. Grafting experiments also support the notion that the shoot tip or the apex is the site of perception of thermoinductive temperatures: noninduced shoot tips grafted onto bolting donors remained as vegetative rosettes. Paradoxically, it was found that the cells of the shoot tip are not the only ones capable of being thermoinduced. Shoots regenerated from leaf cuttings excised from thermoinduced plants exhibited all signs of reproductive development, while regenerated shoots from control leaves developed into vegetative rosettes. It is suggested that many cell types are capable of being thermoinduced and that the shoot tip may appear to be the site of perception of thermoinductive temperatures because structures associated with reproductive development originate from this tissue.     

Regulation of Gibberellin Biosynthesis in Gibberella fujikuroi

Gibberellin production by Gibberella fujikuroi started only after the nitrogen source was depleted and ceased upon its renewal. Nitrogen repression of gibberellin biosynthesis is not an indirect effect of the growth arrest that follows the depletion of an essential nutrient because gibberellins were not produced upon depletion of phosphate. Mycelia produced gibberellins when suspended in a glucose solution. Production ceased some time after depletion of glucose and resumed upon its readdition. Under certain conditions, the gibberellin production rate was inversely proportional to the glucose concentrations. The specific regulation of gibberellin biosynthesis by the nitrogen source imposes a revision of the concept that gibberellins are secondary metabolites whose production is triggered by imbalance or cessation of growth.     

Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR₁) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA₃, was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA₃. Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress.     

Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA₁ or its immediate precursors. The primary site of synthesis of GA₂₀ from GA₁₉ was immature leaflets and tendrils, and the synthesis of bioactive GA₁ and its inactive catabolite GA₈ occurred predominantly in stem tissue. GA₂₉, the inactive catabolite of GA₂₀, was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA₁, GA₁₉, or even GA₂₀. During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [³H]GA₁ (and [³H]GA₂₉) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA₂₀ 3β-hydroxylase protein.     

Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L

Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA_{1,9,12,15,19,20,24,29,44,51,53}. Traces of GA₈ and GA₂₅ were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA₁₉ and GA₄₄ were most abundant, while GA_{12,15,20,24,29,53} occurred in lesser amounts. Only small amounts of GA_1,9,51 were present. The levels of GA₈ and GA₂₅ were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA_{1,8,19,20,29,44,53}) and another whose members are either nonhydroxylated (GA_{9,12,15,24,25}) or lack a C-13 hydroxyl group (GA₅₁). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA₁₂: an early C-13 hydroxylation pathway, leading ultimately to GA₁ and GA₈ and a C-13 deoxy pathway culminating in the formation of GA₉ and GA₅₁.     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation II. Tryptophan Dehydrogenase Activity in Plants.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutáček died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990     

Determination of the Cellular Mechanisms Regulating Thermo-Induced Stem Growth in Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a species with a cold requirement for the initiation of reproductive development (thermoinduction). Work in this laboratory has been focused on elucidating the biochemical and molecular mechanisms underlying the bolting or rapid stem elongation response that is an intricate part of reproductive development in this species. In the present paper the cellular basis for thermo-induced stem growth was determined. Evidence is presented indicating that bolting results from the production of new cells that elongate to their original length before thermoinduction. This increase in cell division occurs in the pith and cortex approximately 0.5 to 5.0 millimeters below the stem apex. For at least the early stages of thermo-induced stem growth, enhanced cell elongation does not appear to be a factor because average lengths of pith cells from stems of thermo-induced plants were similar or less than noninduced controls. In addition, both the amount of increase in the production of new pith cells and stem growth were positively correlated with the length of the cold treatment. Two other lines of evidence are presented corroborating previous assertions (JD Metzger [1985] Plant Physiol 78: 8-13) that gibberellins mediate thermo-induced stem growth in field pennycress. First, treatment of noninduced plants with gibberellin A₃ completely mimicked the effects of a 4-week cold treatment on mitotic activity in the pith and cortex. Second, very little increase in the production of new cells was observed in the pith and cortex of thermo-induced plants of a gibberellin-deficient dwarf mutant of field pennycress. It is also shown that the influence of photoperiod on stem growth is mediated by an effect on the final length that cells ultimately attain.     

Light Regulation of Sink Metabolism in Tomato Fruit : II. Carbohydrate Metabolizing Enzymes

Effects of light on carbohydrate levels and certain carbon metabolizing enzyme activities were studied during the early development of tomato (Lycopersicon esculentum) fruit. Sucrose levels were low and continued to decline during development and were unaffected by light. Starch was significantly greater in light. Invertase activity was similar in both light- and dark-grown fruit. Sucrose synthase activity was much lower than invertase and showed a slight decrease in light-grown fruit between days 21 and 28. Light-grown fruit also had higher ADP glucose pyrophosphorylase activity than dark-grown fruit, which was correlated with higher starch levels. The rapidly decreasing activity of ADP glucose pyrophosphorylase during early fruit development in the dark in conjunction with reduced starch levels and rates of accumulation indicates that ADP glucose pyrophosphorylase is crucial for carbon import and storage in tomato. The differential stimulation of ADP glucose pyrophosphorylase activity from light- and dark-grown tissue by 3-phosphoglycerate suggests that this enzyme may be allosterically altered by light.     

CO(2) Metabolism in Corn Roots. II. Intracellular Distribution of Enzymes

Three enzymes assumed to mediate CO₂ metabolism in corn root tips, P-enolpyruvate carboxylase, malic dehydrogenase, and the malic enzyme, were extracted to determine their relative specific activities and their partitioning between soluble and particulate fractions. The data indicated that the intracellular location of these 3 enzymes is nonparticulate and thus these enzymatic reactions of CO₂ metabolism are apparently nonparticulate. The soluble malic dehydrogenase fraction differed from the particulate fraction in several kinetic properties, viz., response to the thionicotinamide analog of nicotinamide-adenine dinucleotide, oxaloacetate substrate inhibition at pH 8.3, and Km's for nicotinamide-adenine dinucleotide and l-malate. It was concluded that the soluble-malic dehydrogenase differed from the particulate forms in both structure and function. The soluble malic dehydrogenase is apparently involved in CO₂ metabolism.     

Role of Gibberellins in the Environmental Control of Stem Growth in Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual that requires a cold treatment for the induction of stem elongation. An inbred line was selected in which no stem elongation was observed in plants grown for 6 months at 21°C regardless of the prevailing photoperiod. Increased exposure time of plants grown initially at 21°C to cold (2°C) induced a greater rate of stem elongation when the plants were returned to 21°C. Moreover, longer cold treatments resulted in a greater maximum stem height and reduced the lag period for the onset of measurable internode elongation. The optimal temperature range for thermoinduced stem growth was broad: rates of stem growth in plants maintained for 4 weeks at either 2° or 10°C were virtually identical. However, a 4-week thermoinductive treatment at 15°C resulted in a greater lag period for the initiation of stem elongation and a decreased growth rate. The rate of cold-induced stem elongation was greater in plants subjected to long days than for plants subjected to short days following the cold treatment. Thus, photoperiod does not control the induction of stem elongation, but does regulate stem elongation in progress. Exogenous gibberellin A₃ (GA₃) was able to substitute for the cold requirement, but elicited a greater response in plants maintained under long days than short days. This indicates that photoperiod influences the plant's sensitivity to GAs. The GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride, inhibited low temperature-induced stem elongation, and this inhibition was completely reversed by exogenous GA₃. These results suggest that cold-induced stem elongation in field pennycress is mediated by some change in the endogenous GA status.     

Plasticity of reproductive components at different stages of development in the annual plant Thlaspi arvense L.

Summary This study examines the effect of different densities and the removal of all neighbours at different stages of development on all components of reproduction in the inbreeding annual Thlaspi arvense L. A 64-fold increase in density significantly reduced all repooductive components. The number of flower buds per plant was decreased most strongly; the order of decreasing plasticity among the other components was number of capsules per flower, individual seed weight, ovule number per capsule, flowers per flower bud and seeds per ovule. Removing neighbours at all stages of development increased seed yield of plants in comparison to the control without density reduction, but patterns of plasticity depended strongly on time of treatment. The main effect of the removal of neighbours at the vegetative stage was to increase the number of flowers per plant, but the number of ovules per capsule and seed weight increased also, and abortion of capsules decreased. Removing neighbours at the onset of flowering initially failed to affect flower number per plant, instead it resulted in a strong reduction of capsule abortion and an increase in seed weight. However, several weeks after flowering had initially ceased, fresh lateral inflorescences were produced, resulting in a second flush of reproduction. Removing neighbours at the stage of fruit ripening resulted at first only in an increase in seed witht, but later a second reproductive phase occurred. Fresh lateral branches were produced, but the apical meristem was also reactivated. The overall pattern of plasticity among all reproductive components in response to a removal of neighbours was the same as in response to density. The position of a capsule along the inflorescence influenced its number of ovules, the rate of seed abortion and the mean weight of seeds, with the type of effect depending on the developmental stage at which neighbours were removed. Significant negative correlations were found between the mean weight of seeds and the number of seeds in a capsule under all treatments.     

Cyclic Purine Mononucleotides: Induction of Gibberellin Biosynthesis in Barley Endosperm

The de novo synthesis of α-amylase in barley endosperm and isolated aleurone layers is induced by 3′,5′-cyclic purine mononucleotides and gibberellic acid. The induction of α-amylase by cyclic purine mononucleotides is prevented by 2,4-DNP, inhibitors of RNA and protein syntheses, CCC, AMO-1618 and phosfon. The induction of α-amylase formation by 3′,5′-cyclic purine mononucleotides, but not by gibberellic acid, is also blocked by inhibitors of DNA synthesis. Extracts from cyclic AMP-treated endosperm halves exhibit a characteristic gibberellin-like activity which is detectable within 12 hours from the addition of the cyclic AMP. On paper chromatograms this gibberellin-like activity is located at the Rf typical for GA₃. Its formation is prevented by inhibitors of DNA synthesis, CCC and AMO-1618. Glucose inhibits the formation of α-amylase induced by gibberellic acid. Glucose has no effect on the cAMP-induced gibberellin biosynthesis. The evidence shows that the cyclic purine mononucleotides induce DNA synthesis, which results in gibberellin biosynthesis, which in turn activates the synthesis of α-amylase.     

Auxin-induced Fruit Set in Capsicum annuum L. Requires Downstream Gibberellin Biosynthesis

A hierarchical scheme for the central role of the plant hormones auxin and gibberellins in fruit set and development has been established for the model plants Arabidopsis and tomato. In the fruit crop Capsicum annuum, the importance of auxin as an early signal in fruit set has also been recognized; however, the effect of gibberellins and their interaction with auxin has not yet been studied. The aim of this study was to determine the role of gibberellin and the hierarchy between auxin and gibberellin. We applied gibberellin alone or in combination with auxin or with the gibberellin biosynthesis inhibitor paclobutrazol on stigmas of emasculated flowers. Gibberellin application enhanced fruit set, whereas application of paclobutrazol reduced fruit set. The effect of paclobutrazol treatment could be counteracted by coapplication of gibberellin but not by auxin_. These results indicate that in C. annuum, like in Arabidopsis and tomato, auxin is the major inducer of fruit set that acts in part by inducing gibberellin biosynthesis. Interestingly, gibberellin does not significantly contribute to the final fruit size but seems to play an important role in preventing flower and fruit abscission, a major determinant of production loss in C. annuum. At the same time, gibberellin together with auxin seems to balance cell division and cell expansion during fruit growth.     

Herbicide Chlorsulfuron Decreases Assimilate Transport Out of Treated Leaves of Field Pennycress (Thlaspi arvense L.) Seedlings

Treatment of field pennycress (Thlaspi arvense L.) leaves with the herbicide chlorsulfuron resulted in a decrease in the export of assimilate. Twelve hours after a spot application of 1 microgram, assimilate translocation was 70% of that in control leaves. In excised leaves treated with chlorsulfuron the total amounts of sugars and free amino acids were 150 and 170%, respectively, of the amounts in control leaves, 30 hours after herbicide treatment. The amount of sucrose was 247% of that in control leaves. The increase in the concentration of sucrose in the chlorsulfuron-treated leaves, combined with the absence of an effect of chlorsulfuron on carbon dioxide fixation, suggests that the decrease in assimilate transport is not due to an effect on the synthesis of assimilates, but rather to an effect on their movement out of the leaves. Supplying branched-chain amino acids to the field pennycress seedlings prior to the application of chlorsulfuron prevented the occurrence of the effects described.     

Genetic Regulation of Development in Sorghum bicolor (IX. The ma3R Allele Disrupts Diurnal Control of Gibberellin Biosynthesis)

The diurnal regulation of gibberellin (GA) concentrations in Sorghum bicolor was studied in a mutant lacking a light-stable 123-kD phytochrome (ma3Rma3R), wild-type (ma3ma3,Ma3Ma3), and heterozygous (ma3ma3R) cultivars. GAs were determined in shoots of 14-d-old plants by gas chromatography-selected ion-monitoring-mass spectrometry. GA12 levels fluctuated rhythmically in Ma3Ma3, ma3ma3, and,ma3Rma3R; Peak levels occured 3 to 9 h after lights-on. In some experiments, GA53 levels followed a similar pattern. There was no rhythmicity in levels of GA19 and GA8 in any genotype. In ma3ma3 and Ma3Ma3, GA20 levels increased at lights-on, peaked in the afternoon, and decreased to minimum levels in darkness. In ma3Rma3R, peak GA20 levels occured at lights-on, 9 h earlier than in the wild-type genotypes. The pattern for GA1 levels closely followed GA20 levels in all cultivars. One copy of ma3 restored near wild-type regulation of GA20 levels. GA rhythms persisted in 25-d-old ma3ma3 plants. Since absence of the 123-kD phytochrome disrupted diurnal regulation of the GA19 -> GA20 step, the ma3Rma3R genotype may be viewed as being phase shifted in the rhythmic levels of GA20 and GA1 rather than as simply overproducing them.     

Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L

Metabolism of [¹⁴C]gibberellin (GA) A₁₂ (GA₁₂) and [¹⁴C]gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [¹⁴C]GA₁₂ was slower in seed coats than in cotyledons. [¹⁴C]GA₁₂-aldehyde was also metabolized to conjugates in seed coats. Seed coat [¹⁴C]-metabolites produced from [¹⁴C]GA₁₂-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [¹⁴C]GA₅₃-aldehyde, [¹⁴C]GA₁₂-aldehyde conjugate, and [¹⁴C]GA₅₃-aldehyde conjugate were major metabolites produced from [¹⁴C]GA₁₂-aldehyde by seed coats aged 20-22 days or older. The dilution of ¹⁴C in these compounds by ¹²C, as compared to the supplied [¹⁴C]GA₁₂-aldehyde, indicated that they are endogenous. Feeding [¹⁴C]GA₅₃-aldehyde led to the production of [¹⁴C]GA₅₃-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [¹⁴C]GA₁₂, [¹⁴C]GA₁₂-aldehyde, or [³H]GA₉, were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA₁₅, GA₂₄, GA₉, GA₅₁, GA₅₁-catabolite, GA₂₀, GA₂₉, and GA₂₉-catabolite were detected in seed coats, whereas GA₉, GA₅₃, GA₄₄, GA₁₉, GA₂₀, and GA₂₉ were found in cotyledons. The highest GA levels were for GA₂₀ and GA₂₉ in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA₂₉ and GA₂₉-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).