Gibberellins are required for embryo growth and seed development in pea

The gibberellin (GA) biosynthesis mutants lh-1 and lh-2 have been used to examine the physiological role of GAs in pea seed development. The LH protein is required for the three-step oxidation of ent -kaurene to ent -kaurenoic acid early in the GA biosynthesis pathway. The allele-specific interaction of lh-1 and lh-2 with chemical inhibitors of these three steps suggests that LH encodes the multi-functional GA biosynthesis enzyme ent -kaurene oxidase. Unlike the lh-2 mutation which reduces seed weight and decreases seed survival by ∼50% compared with wild-type plants, the lh-1 allele has a transient effect on embryo and seed growth and only slightly increases seed abortion. These seed phenotypes parallel the effects of the two mutant alleles on GA levels in young seeds. Detailed examination of the growth of lh-1 seeds reveals homeostatic regulation of GA-promoted embryo and seed growth. Although GA-deficient seeds grow more slowly than WT seeds, decreased assimilate availability to the developing seeds is not the primary reason for the altered seed development. Instead, GAs act to promote some process(es) required for embryo and seed growth and only indirectly influence the distribution of assimilates. How GA deficiency causes seed abortion is not known but it may simply be a consequence of reduced seed or embryo growth rate. These results demonstrate that even relatively small changes in the levels of GAs in young seeds can alter seed development and suggest that the available GA-related mutants may represent only a subset of all possible mutants with reduced GA levels or GA signalling.     

Effects of the triazole plant growth retardant BAS 111¨W on gibberellin levels in oilseed rape, Brassica napus

Three-week-old shoots of the spring oilseed rape cv. Petranova ( Brassica napus L. ssp. napus ) were found by combined gas chromatography-mass spectrometry to contain GA₁, GA₈, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, 3-epi-GA₁ and a previously uncharacterised C₁₉ dicarboxylic acid that is probably structurally related to GA₂₄. Shoots of the winter cultivar Belinda, harvested at the early flowering stage, contained the same GAs with the exception of the C₁₉ dicarboxylic acid and, in addition, GA₃₄ and GA₅₁ were identified. All material contained higher levels of GA₂₀ than of GA₁; the ratio of GA₁ to GA₂₀ was highest in shoots containing the largest proportion of young immature tissues. Soil treatment of cv. Petranova seedlings with the growth retardant BAS 111¨W [1-phenoxy-5,5-dimethyl-3-(1,2,4-triazol-1-yl)-hexan-4-ol] caused 80% reduction in height 18 days after treatment and the levels of all GAs were 20% or less that of control plants. Foliar treatment at the same dosage reduced height by 50% and caused an 85% or greater reduction in the concentrations of the GA₁ precursors GA₂₀, GA₁₉ and GA₄₄. However, the levels of GA₁, GA₈ and GA₂₉ were affected to a much smaller extent. Foliar application of BAS 111¨W to cv. Belinda 1 month after sowing resulted in only a 20% height reduction at flowering, but no uniform decrease in the concentrations of endogenous GAs at this stage.     

Identification and quantitation of gibberellins in fruits of Lycopersicon esculentum, and their relationship to fruit size in L. esculentum and L. pimpinellfolium

GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were identified by combined gas chromatography-mass spectrometgry (GC-MS) in immature seeds and pericarp of Lycopersicon esculentum Mill. (tomato). Higher levels of these GAs were present in the seeds than in the pericarp; seeds in addition contained GA₁₅, GA₂₄, GA₂₅, and GA₄₄. Fruits of the Lycopersicon pimpinellifolium Mill. mutant I were smaller and contained lower GA₁ concentrations, but higher GA₂₀ concentrations, than those of mutants III and IV. In contrast, differences in fruit size in L. esculentum due to position on the truss did not correlate with GA₁ concentration in either the pericarp or seeds.     

Germination of annual celery (Apium graveolens) seeds: Inhibition by paclobutrazol and its reversal by gibberellins and benzyladenine

Plants of annual celery ( Apium graveolens L.) were treated with paclobutrazol during anthesis. Seeds collected from the treated plants showed a marked reduction in germination in light and failed to germinate in the dark. Application of GA_4/7 to the imbibition solution reversed the inhibitory effect of paclobutrazol while gibberellic acid (GA₃) was ineffective. Benzyladenine (BA) interaction with GA_4/7 was light and concentration-dependent. At relatively low concentrations in the dark there was a synergistic effect, but at higher concentrations, especially in the light, BA, antagonized the GA_4/7 effect. Seedlings emerging from the seeds from paclobutrazol-treated plants were only slightly shortened. It is suggested that paclobutrazol applied to the mother plants inhibited the biosynthesis of endogenous GAs, which normally enable the germination of annual seeds under unfavorable conditions. Exogenously applied GA_4/7 fulfills the function of the absent endogenous GAs.     

Gibberellins in relation to flowering in Polianthes tuberosa

Endogenous gibberellins (GAs) in corms of Polianthes tuberosa L. (cv. Double) were isolated and identified by high performance liquid chromatography, bioassay and combined capillary gas chromatography-mass spectrometry (GC-MS). Gibberellins A₁, A₁₉, A₂₀ and A₅₃ were quantified at the vegetative, early floral initiation and flower development stages. The identification of 13-hydroxylated GAs indicates the presence of the early 13-hydroxylation pathway in P. tuberosa corms. An increase in GA₁ and GA₂₀, and a decrease in GA₁₉ levels, coincided with the transition from the vegetative phase to the stages of early floral initiation and flower development. GA₅₃ stayed at constant levels at the 3 different growth stages. The absence of GA₁ in vegetative corms and its presence in corms at early floral initiation and flower development stages suggest that GA₁ is a causal factor in inducing floral initiation in P. tuberosa . When GA₁, GA₃, GA₄, GA₂₀ and GA₃₂ were applied to corms at the vegetative stage (plants about 5 cm in height), floral initiation was promoted by all of the GAs used, GA₃₂ being the most active. In contrast with the other GAs, GA₃₂ had no effect on stem elongation. Therefore, it is suggested that hydroxylated C-19 GAs play an important role in flower induction in P. tuberosa .     

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3'-nucleotidase activity

Gibberellic acid (GA₃) applied at different times during the growth of wild carrot ( Daucus carota ssp. Carota ) cell suspension cultures inhibited anthocyanin accumulation. Application of 3 × 10^–6 M GA₃ to cultures on day 0 or day 4 gave, respectively, 10 or 35% of anthocyanin accumulation relative to levels occurring when GA₃ was applied at the end of the growth period. Endogenous GAs were separated by high pressure liquid chromatography, and identified and quantified by gas chromatography-selected ion monitoring. Gibberellins GA₁, GA₃ and traces of GA₈. GA₁₉ and GA₂₀ were identified in carrot cell suspension cultures of both high and low anthocyanin-accumulating clones. The concentrations of GA₁. GA₃ and GA₈ in the two clones were similar and were not significantly different after the application of uniconazole which promoted anthocyanin accumulation. This suggests that these endogenous GAs are not the sole factors controlling the accumulation of anthocyanin in these different clones. Exogenous GA₃ and uniconazole had no effect on 3'-nucleotidase and 5'-nucleotidase activity in the carrot cell suspension cultures. Thus 3'-nucleotidase does not appear to play a role in the inhibition of anthocyanin accumulation by exogenous GA₃.     

Internode length in Lathyrus odoratus. The involvement of gibberellins

Evidence was obtained by gas chromatography-mass spectrometry and gas chromatography-selected ion monitoring for the presence of gibberellin A₂₀), GA₁, GA₂₉, GA₈ and 2-epiGA₂₉ in vegetative shoots of tall sweet pea, Lathyrus odoratus L. Both tall (genotype L –) and dwarf (genotype II ) sweet peas elongated markedly in response to exogenous GA₁ attaining similar internode lengths at the highest dose levels. Likewise internode length in both genotypes was reduced by application of the GA biosynthesis inhibitor, PP333. The ratio of leaflet length to width was reduced by application of PP333 to tall plants and this effect was reversed by GA₁. When applied to plants previously treated with PP333, GA₂₀ promoted internode elongation of L – plants as effectively as GA₁, but GA₂₉ was not as effective as GA₁ when applied to II plants. In contrast, GA₂₀ and GA₁ were equally effective when applied to the semidwarf lb mutant but GA-treated lblb plants did not attain the same internode length as comparable GA-treated Lb – plants. The difference in stature between the tall and dwarf types persisted in dark-grown plants. It is concluded that GA₁ may be important for internode elongation and leaf growth in sweet pea. Mutant l may influence GA₁ synthesis by reducing 3β-hydroxylation of GA₂₀ whereas mutant lb appears to affect GA sensitivity.     

Gibberellin substitution for long day secondary induction of flowering in Poa pratensis

Plants of Poa pratensis cv. Holt initiate inflorescence primordia when exposed to short days (SD) and low temperature, but require a secondary induction by at least 4 long days (LD) for further inflorescence development and stem elongation. Single or double applications of 10 µg per plant of gibberellins A₁, A₃, A₅ and 16,17‐dihydro A₅ (DHGA₅) induced inflorescence development in a high proportion of plants in SD, but only if the plants were detillered to a single stem. Exposure to 2 LD cycles did not cause heading and flowering alone but enhanced the effect of exogenous gibberellins (GAs), bringing flowering to 100%. GA₅ and DHGA₅ were less effective than GA₁ and GA₃ in SD, especially with double applications, but were more effective than GA₁ and GA₃ when given together with 2 LD. The GAs had differential effects on vegetative growth and flowering, GA₅ and DHGA₅ causing much less leaf and stem growth than the other two GAs. Marginal induction, whether by LD or GA application, resulted in a high proportion of spikelets with viviparous proliferation. Thus, whereas GAs are inhibitory to the primary induction by SD, they can replace secondary induction by LD when vegetative growth is limited.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

A localised decrease of GA1 in shoot tips of Salix pentandra seedings precedes cessation of shoot elongation under short photoperiod

By application of a recently developed method allowing analysis of gibberellins (GAs) in mg amounts of tissue, the effect of photoperiod on levels of GAs in shoot tips of individual seedlings of the woody species Salix pentandra was studied. In elongating long day-grown seedlings, maximum levels of GA₁ were found 5–20 mm below the apex, approximately twice the levels in other segments. After exposure of plants to 5 or 15 short days, the levels of GA₁ were about 50% lower within this specific region of the stem, as compared with seedlings grown under long days. Short day-induced cessation of shoot elongation also correlated with overall declines in the levels of GA₅₃, GA₁₉, GA₂₀ and GA₈, Within each photoperiodic treatment the levels of these GAs were generally relatively similar throughout the upper 35 mm of stems. No differences in internode lengths or in lengths of pith or epidermal cells were found in plants grown under long days compared with those exposed to 5 short days. In both cases, cells in mitosis were observed in the subapical stem tissues of shoot tips. After 15 short days, stem elongation was completed, and dividing cells were generally not found in the subapical part of the stem. However, short day exposure did not prevent elongation of internodes and cells differentiated before the treatment was started. Thus, the localised decrease in level of GA₁ in shoot tips under short days precedes the morphological and anatomical changes connected with the short day-induced cessation of elongation growth. This supports the hypothesised role for GA₁ in photoperiodic control of shoot elongation in S. pentandra .     

Internode length in Pisum. A new allele at the Lh locus

A new allele at the Lh locus has been identified in Pisum sativum L. and named lhⁱ . This allele results in reduced GA levels in young shoots, and a dwarf phenotype. Gas chromatography-selected ion monitoring (GC-SIM) with dideuterated internal standards has been used to demonstrate a quantitative relationship between the level of endogenous GA₁ and internode length using the three alleles ( Lh, lh and lhⁱ ) at the Lh locus. These results are consistent with previous findings in peas (for alleles at the Le locus) and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis and support the role of GA₁ as the major native GA in peas with biological activity in its own right. However, in contrast to alleles at the Le locus, GA₂₀ levels are also reduced in lh and lhⁱ plants. The lhⁱ allele also has possible pleiotropic effects on seed abortion, leading to a reduction in seed yield compared to plants homozygous for the previously characterised Lh or lh alleles.     

Hormone directed sucrose transport during fruit set induced by gibberellins in Pisum sativum

A new system has been developed to study hormone-directed transport in intact plants during parthenocarpic fruit set induced by gibberellins. Gibberellic acid (GA₃) and gibberellin A₁ (GA₁) applied to unpollinated ovaries of pea ( Pisum sativum L. cv. Alaska) promoted sucrose transport from the leaf to the site of hormone application. In vivo experiments showed an early (30 min) accumulation of [¹⁴C]-sucrose in ovaries of pea stimulated by gibberellins. This activation of sucrose transport appears to be mediated by gibberellins (GA₁, GA₃), increasing both loading of phloem with sucrose in the leaf (source) and sucrose unloading in the ovary (sink). The ability of pea tissue segments to take up sucrose in vitro was not affected by the hormonal treatment.     

Photoperiodic control of endogenous gibberellins in seedlings of Salix pentandra

In the temperate-zone woody species Salix pentandra elongation growth is regulated by the photoperiod. Long days sustain active growth, whereas short days induce cessation of apical growth, which is a prerequisite for winter hardening. It is shown that this is correlated to quantitative changes in levels of endogenous GA₁₉ GA₂₀, and GA₁. Within two short days the amount of the active GA₁ and its immediate precursor GA₂₀, decreased markedly in young leaves us well as in stem tissue. Also, the amount of GA¹⁹, declined, but the decrease was delayed relative to that of GA₁ and GA₂₀. The ability of S. pentandra seedlings to respond to exogenous GA₁₉, decreased with increasing numbers of short days. Observations that support the hypothesis that the level of GA₁ in S. pentandra is regulated by the photoperiod in a quantitative mode with conversion of GA₁₉, to GA₂₀, being one target for control.
Different distribution of GAs in various plant parts was observed. The level of GA was higher in young leaves than in other plant parts, and the amount of GA₁₉ was 5–10 times higher in stem tissue than in leaves and roots. The ratios of GA₈ to GA¹ and GA₂₀, were higher in roots as compared with other parts, as rods contained very low levels of GA₁ and GA₂₀, but amounts of GA₂₀ comparable with other parts.     

Free and conjugated gibberellins in dormancy and germination of apple seeds

Halińska, A. and Lewak, St. 1987. Free and conjugated gibberellins in dormancy and germination of apple seeds.
The presence of gibberellin A₄ (GA₄) was confirmed in partly stratified seeds of apple ( Malus domestica Borb., cv. Antonówka) by mass spectrometry of the methyl ester. Levels of free and conjugated gibberellins A₄₊₇ and A₉ changed during drying of mature seeds, during cold and warm stratification, as well as during germination of dormant and non-dormant embryos. The temporary rise in GA₄₊₇ during cold stratification and during the culture of dormant embryos as well as the lack of it under conditions of warm stratification, allowed us to postulate a role for GA₄₊₇ in the removal of dormancy. In addition, GA₉ was absent in dormant embryos and increased during cold stratification and during the culture of non-dormant embryos. This suggests the involvement of GA₉, in induction of normal development of the seedling. The equivalence between changes in free and conjugated GAs suggests that formation and hydrolysis of conjugates are involved in the control of the physiologically active levels of free GA₄₊₇ and GA₉.     

Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons

Halińska, A., Sińska, I. and Lewak, St. 1987. Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons.
Free and conjugated gibberellins (GAs) A₄₊₇ and A₉ were determined in embryonic axes and in cotyledons of seeds of apple ( Malus domestica Borb., cv. Antonó wka) during breaking of dormancy under cold stratification. In both organs, the maximum level of free GA₄₊₇ was found at day 30 of stratification, but the concentration was 700 times higher in axes than in cotyledons. Comparison of changes in free and conjugated GA₄₊₇ levels during stratification allow us to suggest that the accumulation of free hormone in axes is, at the most, to 40% due to release from conjugates already present in the axis; that maximally 20% is derived from hydrolysis of cotyledonary conjugates translocated to axes; and that at least 40% originate from the novo biosynthesis of the hormone. Free and conjugated GA₉ levels were similarly altered in axes and in cotyledons, markedly increasing at the end of afterripening. Both release of the free hormone from conjugates and biosynthesis of GA₉, appeared to be involved in that increase; no translocation of free or bound GA₉, between axes and cotyledons was demonstrated.     

Gibberellin-biosynthesis and -sensitivity mediated stimulation of seed germination of Sisymbrium officinale by red light and nitrate

Light stimulated seed germination of Sisymbrium officinale (L.) Scop, (hedge mustard) by means of two different mechanisms. Light effect I was absolutely dependent on the simultaneous presence of nitrate. Without nitrate, red (R) irradiated seeds did not escape from the antagonizing action of far-red (FR) irradiation. The data indicated that nitrate acted as a cofactor at the level of the FR absorbing form of phy-tochrome (P_fr). The combined action of R and nitrate could be replaced by addition of the gibberellins 4 and 7 (GA,₄₊₇). This action could be inhibited by the growth re-tardant tetcyclacis, an inhibitor to GA biosynthesis in cell free systems and intact plants. The action of tetcyclacis was fully neutralized by GA₄₊₇. It is concluded that the combination of R and nitrate stimulated GA biosynthesis. Omission of nitrate from the incubation medium enabled the study of light effects apart from G A biosynthesis. In such conditions R stimulated the sensitivity to GA₄₊₇, (light effect II). The two light effects could also be distinguished by their different reactions to the temperature of a pre-treatment in water and darkness. The sensitivity to R and nitrate was subject to breaking and induction of dormancy. Both processes were stimulated at rising temperatures. Due to a different optimum, breaking of dormancy prevailed at lower temperatures and induction of secondary dormancy at more elevated temperatures. The sensitivity to GA₄₊₇ rose and fell in a comparable way during dark incubation at a broad range of temperatures. The capacity of light to stimulate GA₄₊₇, action did not diminish at higher temperatures, it even tended to rise. The study indicated that seed germination is regulated by an increase in both the levels of GAs and the sensitivity to GAs.     

Internode length in Pisum. The lrs mutation reduces gibberellin response and levels

We describe a new mutation, lrs , which reduces internode length in Pisum sativum L. The mutation appears to act by reducing both GA synthesis and the response to GA₁. The levels of the 13‐hydroxylated GAs, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈ in the lrs mutant were greatly reduced compared with the wild‐type. The extent of the reduction in GA₁ content in the apical tissues would, at least in part, account for the dwarf phenotype of the mutant. The reduced GA responsiveness of the new mutant was indicated by the inability of applied GA₁ to remove the difference in elongation between lrs and LRS plants. The lrs mutant appears to be unique amongst internode length genotypes, possessing characteristics of both GA synthesis and GA response mutants.     

Interactions between hydrogen cyanide, gibberellin, abscisic acid and red light in germination of lettuce seeds

Germination of lettuce ( Lactuce sativa L. cy. Grand Rapids) seeds was promoted by red light and by pulse treatments with gibbercllie acid (GA₃) or hydrogen cyanide, whereas it was inhibited by short exposure of seeds to absusic acid (ABA). The eflects of unsalLirating red light and of 10 μ M GA₃ on lettuce germination were completely reversed the effect of ABA (100 μ M ). In contrast, hydrogen cyanide did not reverse the effect of 100 μ M ABA and only partly eliminated the effect of 10μ M ABA, independently of the sequence of treatments. Possible interactions between HCN GA₃, ABA and red light were discussed. It was concluded thai light GA₃ and HCN affect different mechanisms involved in lettuce germination: ABA counteracts the stimulatory action of all these faclors. being the most effective against cvanide Additional key words - Lactuca sativa, pholodormancy, phylohormoncs.