Specific oxidative cleavage of carotenoids by VP14 of maize.

The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C25 apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.

Induction of ABA 8'-hydroxylase by (+)-S-, (-)-R- and 8'-8'-8'-trifluoro-S-abscisic acid in suspension cultures of potato and Arabidopsis.

Suspension cultures of potato and Arabidopsis were incubated with 50 microM of (+)-ABA and (-)-ABA for 3 hr. These pretreatments were found to increase the rate, by two- to seven-fold, of formation of [2H6] phaseic acid (PA) from [2H6] ABA, applied in a subsequent incubation. Pretreatment with trifluoro-ABA had a higher efficacy, increasing the rate of conversion 15-fold. Suspension cell cultures that had been dehydrated and then rehydrated in the presence of [2H6] ABA displayed a much lower enhancement of PA formation. We conclude that ABA induces its own oxidative catabolism in suspension cultures.

Synthesis and confirmation of structure for a new gibberellin, 2 beta-hydroxy-GA12 (GA110), from spinach and oil palm.

The identity of a new gibberellin (GA) in spinach and oil palm sap has been confirmed as 2 beta-hydroxy-GA12 (GA110) by comparisons of GC-mass spectral data obtained for the trimethylsilyl ether methyl ester derivatives with those of a synthetic sample prepared by means of a 24 step sequence from gibberellic acid; 2 beta-hydroxy-GA24 was also prepared. Experimental details for the latter part of the syntheses are described.

Identification of three C20-gibberellins: GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19).

Three new C20-gibberellins, GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19), have all been isolated from spinach, GA97 also from tomato root cultures and pea pods, and GA98 from maize pollen. The structures of these compounds were established by GC-mass spectrometric comparisons of the trimethylsilylated methyl esters with authentic samples prepared from gibberellic acid (GA3).

Sites of Abscisic Acid Synthesis and Metabolism in Ricinus communis L.

The sites of abscisic acid (ABA) synthesis and metabolism in Ricinus communis L. were investigated by analyzing the levels of ABA and its two metabolites phaseic acid (PA) and dihydrophaseic acid (DPA) in the shoot tips, mature leaves, and phloem sap of stressed and nonstressed plants.Water stress increased the concentration of ABA, PA, and DPA in phloem exudate and also increased the levels of all three compounds in mature leaves and in shoot tips. The latter had a very high DPA content (18.7 mug/g fresh weight) even in plants not subjected to water stress. When young and mature leaves were excised and allowed to wilt, the level of ABA increased in both, demonstrating that leaves at an early stage of development have the capacity to produce ABA.These results have been interpreted to mean that in mature leaves of nonstressed Ricinus plants, ABA is synthesized and metabolized, and that ABA itself, as well as its metabolites, are translocated in the phloem to the shoot tips (sinks). Since DPA, but not ABA, accumulates in the shoot tips, it follows that ABA is metabolized rapidly in the apical region. To what extent ABA present in young leaves of nonstressed plants is the consequence of synthesis in situ and of import from older leaves remains to be determined.

Levels of (+/-) Abscisic Acid and Xanthoxin in Spinach under Different Environmental Conditions.

The levels of the growth inhibitors(+)-abscisic acid and xanthoxin were determined in the long day plant spinach (Spinacia oleracea L. cv. Savoy Hybrid 612) grown under different environmental conditions. When plants were transferred from light to darkness, the (+)-abscisic acid level always decreased, whereas the xanthoxin content did not change. The (+)-abscisic acid content was higher in plants grown under low than under high relative humidity.Xanthoxin levels were not affected by photoperiod, whereas the (+)-abscisic acid content increased 2 to 3 times upon transferring plants from short day to long day. Shoot tips with young leaves and mature leaves of the same plants analyzed separately did not differ in their inhibitor content when expressed per unit dry weight. No increase in xanthoxin level was observed in wilting plants. In general, the xanthoxin levels of spinach were much less affected by changes in the environment than were those of (+)-abscisic acid. In conclusion, there is no correlation between xanthoxin and (+)-abscisic acid levels in spinach on the one hand, and growth and flowering responses on the other.

Gibberellin A20 content of Bryophyllum daigremontianum under different photoperiodic conditions as determined by gas-liquid chromatography.

The gibberellin A20 content of the long-short-day plant Bryophyllum daigremontianum (R. Hamet and Perr.) Berg. under different photoperiodic conditions was determined by gas-liquid chromatography. Purified extracts from ten plants were adequate for quantitative analysis by this method. The level of GA20 increased following transfer from long-day (LD) to short-day (SD) conditions until after 38 SD it was three times higher on a dry weight basis than in comparable plants under continuous LD. No GA20 could be detected in extracts of plants under permanent SD. These results are in agreement with earlier data obtained by assaying Bryophyllum extracts with the d-5 mutant of corn.

Changes in the Levels of Abscisic Acid and Its Metabolites in Excised Leaf Blades of Xanthium strumarium during and after Water Stress.

The time course of abscisic acid (ABA) accumulation during water stress and of degradation following rehydration was investigated by analyzing the levels of ABA and its metabolites phaseic acid (PA) and alkalihydrolyzable conjugated ABA in excised leaf blades of Xanthium strumarium. Initial purification was by reverse-phase, preparative, high performance liquid chromatography (HPLC) which did not require prior partitioning. ABA and PA were purified further by analytical HPLC with a muBondapak-NH(2) column, and quantified by GLC with an electron capture detector.The ABA content of stressed leaves increased for 4 to 5 hours and then leveled off due to a balance between synthesis and degradation. Since PA accumulated at a constant rate throughout the wilting period, it was concluded that the rate of ABA synthesis decreased after the first 4 to 5 hours stress. Conjugated ABA increased at a low rate during stress. This is interpreted to indicate that free ABA was converted to the conjugated form, rather than the reverse.Following rehydration of wilted leaves, the ABA level immediately ceased increasing; it remained constant for 1 hour and then declined rapidly to the prestress level over a 2- to 3-hour period with a concomitant rise in the PA level. In contrast to the rapid disappearance of ABA after relief of stress, the high PA content of rehydrated leaves declined only slowly. The level of conjugated ABA did not change following rehydration, indicating that conjugation of ABA was irreversible.Detached Xanthium leaves that were subjected to a wilting-recovery-rewilting cycle in darkness, responded to the second wilting period by formation of the same amount of ABA as accumulated after the first stress period.

Metabolism of Abscisic Acid and Its Regulation in Xanthium Leaves during and after Water Stress.

Metabolism of abscisic acid was compared in stressed and in rehydrated leaf blades of Xanthium strumarium L. Chicago strain that were either detached or left intact on the plant. Under all conditions, phaseic acid was the major metabolite. The high level of phaseic acid that was observed in intact plants 1 day after recovery from stress declined slowly and had not yet reached the prestress level 1 week later. The glucosyl ester of abscisic acid, beta-d-glucopyranosyl abscisate, accumulated at a low rate during periods of prolonged stress. Repeated stress-recovery cycles resulted in a gradual increase in the level of the glucosyl ester, which did not decline following relief of stress for at least 34 days. The level of the glucosyl ester of abscisic acid may therefore serve as a cumulative indicator of the water stresses to which a particular leaf has been exposed.Darkness stimulated abscisic acid metabolism in both detached and attached leaves. Treatment of Xanthium leaves in light with ethylene or chemicals that release ethylene also resulted in a faster breakdown of abscisic acid. Inasmuch as darkness is known to stimulate ethylene production, it is proposed that enhancement of abscisic acid metabolism in darkness is mediated by ethylene.

Inhibition of stem growth and gibberellin production in Agrostemma githago L. by the growth retardant tetcyclacis.

The effects of the new growth retardant tetcyclacis (TCY) on stem growth and endogenous gibberellin (GA) levels were investigated in the long-day rosette plant Agrostemma githago. Application of TCY (10 ml of a 5·10(-5)M solution daily) to the soil suppressed stem elongation in Agrostemma grown under long-day conditions. A total of 10 µg GA1 (1 µg applied on alternate days) per plant overcame the growth retardation caused by TCY.Control plants and plants treated with TCY were analyzed for endogenous GAs after exposure to nine long days. The acidic extracts were fractionated by high-performance liquid chromatography. Part of each fraction was tested in the d-5 maize bioassay, while the remainder was analyzed by combined gas chromatography-selected ion monitoring. The bioassay results indicated that the GA content of plants treated with TCY was much lower than that of untreated plants. The data obtained by gas chromatography-selected ion monitoring confirmed that the levels of seven GAs present in Agrostemma were much reduced in TCY-treated plants when compared with the levels in control plants: GA53 (13%), GA44 (0%), GA19 (1%), GA17 (33%), GA20 (15%), GA1 (4%), and epi-GA1 (13%). These results provide evidence that TCY inhibits stem growth in Agrostemma by blocking GA biosynthesis and thus lowering the levels of endogenous GAs.

Flower formation in the short-day plant Kalanchoë by grafting with a long-day and a short-long-day Echeveria.

Flower formation was induced in the shortday plant Kalanchoë blossfeldiana Poellnitz under long-day conditions by grafting with flowering shoots of the short-long-day plant Echeveria harmsii (Rose) MacBr. and of the long-day plant Echeveria 'pulvoliver' (E. pulvinata Rose x E. harmsii). Vegetative shoots from induced Echeveria plants failed to cause a flowering response in Kalanchoë. The presence of flowering and vegetative shoots side by side on induced Echeveria plants provides evidence for physiological chimeras in this genus.

Reduction of the Gibberellin Content of Pharbitis Seeds by CCC and After-Effects in the Progeny.

Plants of Pharbitis nil were treated with the growth retardant (2-chloroethyl) trimethylammonium chloride (CCC) shortly before and after anthesis. Fresh and dry weight of immature seeds were not affected by the CCC treatment.The level of gibberellin-like activity in Pharbitis seeds as compared to control seeds was strongly reduced by CCC application. The progenies of the treated plants also had a much reduced GA content in the seedling stage. These results are interpreted to indicate that CCC blocks gibberellin biosynthesis in higher plants, as it does in the fungus Fusarium.CCC applied via the roots accumulated in the immature seeds and was carried over to the following generation. Consequently, growth of CCC progenies was dwarfed and flower formation inhibited. Both phenomena were overcome by application of gibberellin A(3).Three gibberellin-like substances (called fractions I, II, and III) were present in Pharbitis seeds and could be separated by thin-layer chromatography. All 3 fractions were also present in seeds treated with CCC. Fractions II and III were present in much higher quantities than fraction I. Both fractions II and III promoted growth of d5 corn but only fraction II was active in dwarf peas grown under red light.

Inhibition of stem growth and flower formation in Pharbitis nil with N, N-dimethylaminosuccinamic acid (B 995).

In the short-day plant Pharbitis nil, strain "Violet", flower formation is inhibited by application of the growth retardant N,N-dimethylaminosuccinamic acid (B 995) via the roots for a period of 24 hours prior to one inductive long night. Terminal flower bud formation is suppressed by a B 995 concentration of 100 mg/l, but for complete suppression of all axillary flower buds 2000 mg/l is required. Inhibition of flower formation is also caused by B 995 application via plumules or cotyledons, even if made at the end of the inductive night. B 995 treatment always results in short, thick internodes and dark-green leaves.Transport of (14)C-labeled B 995 from cotyledons to plumules and roots takes place during a 16-hour dark period. However, very little label moves from a treated to an untreated cotyledon. Application of B 995 to one of the two cotyledons results in flower inhibition, although the untreated cotyledon produces sufficient flower hormone to induce optimal flower formation. It is concluded therefore that in the short-day plant Pharbitis B 995 does not affect flower hormone production, but rather inhibits floral initiation by interfering with the action of the hormone in the shoot apex.Inhibition of flower formation by B 995 can be completely overcome by application of gibbrellin A3 to the plumulus before the long nigh. A dose of 0.01 µg GA3/apex is sufficient to re-establish flowering, but much more GA3 is required to restore internode length equal to that of the control. Indole-3-acetic acid and naphthalene acetic acid are totally inactive in overcoming B 995 inhibition of flower formation and growth.The growth rate of Pharbitis plants treated with B 995 and continuously grown in long-day conditions is initially low, but reaches the same level as in untreated plants approximately 25 days after treatment. (14)C-labeled B 995 applied to cotyledons accumulates to a high degree in roots and in the basal part of the shoots. (14)C-B 995 is metabolized very slowly and persists therefore in Pharbitis plants for prolonged periods of time.

Lack of evidence for distinguishing florigen and flower hormone in Perilla.

The minimal duration of short-day (SD) treatment necessary to cause flower formation in the SD plant Perilla is 9 days, whereas leaves exposed to at least 12 SD can function as donors in grafting experiments. Zhdanova's report that SD given consecutively to different individual leaves for 2 or 3 days resulted in flowering, could not be confirmed.

Effects of photoperiod on growth rate and endogenous gibberellins in the long-day rosette plant spinach.

The earliest visible responses of spinach plants (Spinacia oleracea L., cv. Savoy Hybrid 612) transferred from short to long days (8 hours of high intensity light supplemented with 16 hours of low intensity illumination from incandescent lamps) were upright leaf orientation and increased elongation of the petioles. The effect of long days on growth rate was direct; i.e., there was no after-effect if the plants were transferred to short days. Gibberellin A(3) applied to plants under short days had an effect similar to that of long days, whereas application of the growth retardant AMO-1618 [2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidinel-carboxylate] under long days caused a growth habit typical of short-day conditions. Gibberellin A(3) caused more stem growth in plants under long days in which the endogenous gibberellin content had been reduced by AMO-1618 than in plants under short days not treated with the growth retardant.Three gibberellin-like substances, called I, II, and III in order of increasing R(F) value, were present in acidic extracts of spinach under short days. After transfer to long days, II increased, whereas I and III decreased, the latter below the level of detection in the d5 corn assay. Following application of AMO-1618 the gibberellin content of plants under long days fell off more rapidly than in those under short days, indicating that gibberellin turnover was markedly higher under long days. This increased rate of gibberellin metabolism was established after 2 long days. When plants were returned to short days, the turnover of gibberellins declined. It is suggested that a higher rate of gibberellin biosynthesis combined with increased sensitivity to gibberellin is responsible for the observed growth responses in spinach under long days.

(+)-abscisic Acid content of spinach in relation to photoperiod and water stress.

Levels of (+)-abscisic acid present in the long-day plant spinach (Spinacia oleracea L., cv. Savoy Hybrid 612) grown under different photoperiodic regimes were measured in purified extracts by optical rotary dispersion. When plants were transferred from short to long days, the abscisic acid content increased 2- to 3-fold. This rise in the level of abscisic acid took place during the 1st long day. Abscisic acid levels of plants under short days as well as under long-day conditions were higher at the end of the 8-hour high intensity light period than at its beginning.The growth retardant AMO-1618 [2'-isopropyl-4'-(trimethyl-ammonium chloride)-5'-methylphenyl piperidine-1-carboxylate], which strongly reduces the gibberellin content of spinach under long days, did not affect the abscisic acid content.When water was withheld from plants until wilting symptoms appeared, the abscisic acid content increased more than 10-fold over that of turgid plants. There was no evidence that the sudden rise of abscisic acid level during wilting was due to release from a water-soluble bound form.Bioassays of crude acidic extracts in the wheat coleoptile section test did not indicate the presence of other specific growth inhibitors besides abscisic acid. It is concluded that abscisic acid does not function as an endogenous regulator of stem growth and flower formation in the long-day plant spinach.

The leaf as the site of gibberellin action in flower formation in Bryophyllum daigremontianum.

The long-short-day plant Bryophyllum daigremontianum can be induced to flower by transfer from long to short days (LD→SD), or by gibberellin (GA) application under SD. Application of GA to mature leaves of intact or partially defoliated plants induces flowering more effectively than when applications are made to the youngest leaf pair and the shoot tip.Mature leaves on de-budded plants in SD are induced to produce floral stimulus by GA application, as demonstrated by grafting LD receptor scions onto the debudded plants, or by grafting SD leaves treated with GA onto receptor stocks in LD. This shows that GA applied to Bryophyllum in SD exerts its flower-promoting effect in the leaves.The minimal number of SD necessary for flower formation in Bryophyllum is approximately 15, both in case of photoinduction by the shift LD→SD, and after GA treatment in SD. It is concluded that the LD part of photinduction establishes a high level of endogenous GAs in the leaves which is a prerequisite for production of floral stimulus under subsequent SD.

Gibberellin-like substances in Bryophyllum daigremontianum and the distribution and persistence of applied gibberellin A3.

Acidic extracts of the long-short-day plant Bryophyllum daigremontianum contain two gibberellin (GA)-like substances called fractions I and II. In plants under permanent short-day (SD) conditions the levels of both I and II are very low. In continuous long days (LD) the total GA content is approximately 20 times higher than in SD, mainly due to an increased level of II. Extracts of plants induced to flower by the shift LD→SD show a further increase in the level of II. Application of GA3 to plants in SD causes normal flower formation, but the level of fraction II remains as low as in vegetative plants in permanent SD.Approximately 10% of the GA3 applied could still be recovered from leaves and inflorescences after 45 days, indicating that GA3 is very stable in Bryophyllum, Most of the GA3 recovered was still associated with the treated leaves, but small amounts could be detected in other leaves and in inflorescences. Results of grafting experiments indicate that these low levels of GA3 are adequate to induce production of the floral stimulus.

ent-kaurene biosynthesis is enhanced by long photoperiods in the long-day plants Spinacia oleracea L. and Agrostemma githago L.

The effect of photoperiod on ent-kaurene biosynthesis was determined in the long-day (LD) plants spinach (Spinacia oleracea L.) and Agrostemma githago L. Further metabolism of ent-kaurene was blocked by application of the growth retardant tetcyclacis, and ent-kaurene accumulation was measured by isotopic dilution using gas chromatography-selected ion monitoring (GC-SIM) (E. Grosselindemann, J.E. Graebe, D. Stöckl, P. Hedden [1991] Plant Physiol 96: 1099-1104). In spinach, the rate of ent-kaurene accumulation in shoots grown under LD conditions was 3 times higher than in shoots grown under short-day (SD) conditions. ent-Kaurene also accumulated in fully expanded leaves, but at a lower rate than in shoots (15 and 55 pmol g-1 dry weight h-1, respectively). In Agrostemma, ent-kaurene accumulated at a rate 2.5 times higher in plants grown under LD conditions than in those grown under SD conditions. In spinach, enhanced ent-kaurene accumulation was detectable after 1 long day, and with exposure to additional long days, the rate of ent-kaurene accumulation increased further. Conversely, when plants were exposed to LD conditions and then returned to SD conditions, the rate of ent-kaurene accumulation decreased. Following tetcyclacis application, ent-kaurene accumulation was observed in all parts of spinach that were analyzed, but there were large quantitative differences between organs of different ages. As the leaves matured, ent-kaurene biosynthesis declined. Petioles accumulated more ent-kaurene than the corresponding leaf blades. It is concluded that stimulation of ent-kaurene biosynthesis by LD conditions leads to a higher rate of gibberellin biosynthesis, which is essential for stem elongation in rosette plants.

Phenotypic expression of wild-type tomato and three wilty mutants in relation to abscisic Acid accumulation in roots and leaflets of reciprocal grafts.

Lycopersicon esculentum Mill. cv Rheinlands Ruhm (RR) and cv Moneymaker and the three wilty mutants flacca (flc), sitiens (sit), and sitiens(w) (sit(w)), together with most reciprocal grafts, were grown in pots and in solution culture. Detached leaflets, and control and steam-girdled intact plants, were left turgid or were wilted in air. Detached leaflets and the leaflets and roots of the intact plants were analyzed for their abscisic acid (ABA) content. Turgid RR leaflets contained about 2.9 ng ABA per milligram dry weight. On average, the flc and sit leaflets contained 33 and 11% of this amount, respectively. The lack of ABA approximately correlated with the severity of the mutant phenotype. Mutant roots also contained less ABA than wild-type roots. Wild-type scions on mutant stocks (wild type/mutant) maintained the normal phenotype of ungrafted plants. Mutant scions grafted onto wild-type stocks reverted to a near wild-type phenotype. After the wild-type leaves were excised from solution culture-grown mutant/wild-type plants, the revertive morphology of the mutant scions was maintained, although endogenous ABA levels in the leaflets fell to typical mutant levels and the leaflets became wilty again. When stressed in air, both leaflets and roots of RR plants produced stress-induced ABA, but the mutant leaflets and roots did not. The roots and leaflets of the grafted plants behaved according to their own genotype, with the notable exception of mutant roots grown with wild-type scions. Roots of flc and sit(w) recovered the ability to accumulate stress-induced ABA when grafted with RR scions before the stress was imposed.