The Distribution of Gibberellins in Vegetative Tissues of Pisum sativum L. : I. Biological and Biochemical Consequences of the le Mutation

The concentrations of endogenous gibberellin (GA) 1, 5, 8, 19, 20, and 29 in the component tissues of maturing tall (Le) and dwarf (le) pea (Pisum sativum) plants have been determined. The following conclusions were drawn from the data obtained: (a) GA₂₀ and its metabolites accumulate only in the growing regions of Le and le plants; (b) the le mutation is biochemically expressed in all immature tissues of the dwarf plants; (c) the quantitative composition of the GA metabolites in the various immature tissues is variable; (d) the total GA concentration in apical buds, unexpanded leaves, and tendrils is considerably higher than in GA₁-responsive stem tissue; and (e) there is very little GA accumulation of the inactive 2β-hydroxylated GAs (GA₈ and GA₂₉) in either the mature vegetative tissues or the roots of pea plants.     

Gibberellin concentration and transport in genetic lines of pea : effects of grafting

Effects of the Na and Le loci on gibberellin (GA) content and transport in pea (Pisum sativum L.) shoots were studied. GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄, GA₈ catabolite, and GA₂₉ catabolite were identified by full-scan gas chromatography-mass spectrometry in extracts of expanding and fully expanded tissues of line C79-338 (Na Le). Quantification of GAs by gas chromatography-single-ion monitoring using deuterated internal standards in lines differing at the Na and Le alleles showed that na reduced the contents of GA₁₉, GA₂₀, and GA₂₉ on average to <3% and of GA₁ and GA₈ to <30% of those in corresponding Na lines. In expanding tissues from Na le lines, GA₁ and GA₈ concentrations were reduced to approximately 10 and 2%, respectively, and GA₂₉ content increased 2- to 3-fold compared with those in Na Le plants. There was a close correlation between stem length and the concentrations of GA₁ or GA₈ in shoot apices in all six genotypes investigated. In na/Na grafts, internode length and GA₁ concentration of nana scions were normalized, the GA₂₀ content increased slightly, but GA₁₉ levels were unaffected. Movement of labeled GAs applied to leaves on Na rootstocks indicated that GA₁₉ was transported poorly to apices of na scions compared with GA₂₀ and GA₁. Our evidence suggests that GA₂₀ is the major transported GA in peas.     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Seed effects on gibberellin metabolism in pea pericarp

Pea fruit (Pisum sativum L.) is a model system for studying the effect of seeds on fruit growth in order to understand coordination of organ development. The metabolism of ¹⁴C-labeled gibberellin A₁₂ (GA₁₂) by pea pericarp was followed using a method that allows access to the seeds while maintaining pericarp growth in situ. Identification and quantitation of GAs in pea pericarp was accomplished by combined gas chromatography-mass spectrometry following extensive purification of the putative GAs. Here we report for the first time that the metabolism of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₉ and [¹⁴C]GA₂₀ occurs in pericarp of seeded pea fruit. Removal of seeds from the pericarp inhibited the conversion of radiolabeled GA₁₉ to GA₂₀ and caused the accumulation of radiolabeled and endogenous GA₁₉. Deseeded pericarp contained no detectable GA₂₀, GA₁, or GA₈, whereas pericarp with seeds contained endogenous and radiolabeled GA₂₀ and endogenous GA₁. These data strongly suggest that seeds are required for normal GA biosynthesis in the pericarp, specifically the conversion of GA₁₉ to GA₂₀.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

The brassinosteroid growth response in pea is not mediated by changes in gibberellin content

The objective of this study was to increase our understanding of the relationship between brassinosteroids (BRs) and gibberellins (GAs) by examining the effects of BR deficiency on the GA biosynthesis pathway in several tissue types of pea (Pisum sativum L.). It was suggested recently that, in Arabidopsis, BRs act as positive regulators of GA 20-oxidation, a key step in GA biosynthesis [Bouquin et al. (2001) Plant Physiol 127:450–458]. However, this may not be the case in pea as GA₂₀ levels were consistently higher in all shoot tissues of BR-deficient (lk and lkb) and BR-response (lka) mutants. The application of brassinolide (BL) to lkb plants reduced GA₂₀ levels, and metabolism studies revealed a reduced conversion of GA₁₉ to GA₂₀ in epi-BL-treated lkb plants. These results indicate that BRs actually negatively regulate GA₂₀ levels in pea. Although GA₂₀ levels are affected by BR levels, this does not result in consistent changes in the level of the bioactive GA, GA₁. Therefore, even though a clear interaction exists between endogenous BR levels and the level of GA₂₀, this interaction may not be biologically significant. In addition to the effect of BRs on GA levels, the effect of altered GA₁ levels on endogenous BR levels was examined. There was no significant difference in BR levels between the GA mutants and the wild type (wt), indicating that altered GA₁ levels have no effect on BR levels in pea. It appears that the BR growth response is not mediated by changes in bioactive GA levels, thus providing further evidence that BRs are important regulators of stem elongation.     

Internode length inPisum: biochemical expression of thele andna mutations in the slender phenotype

Thele andna mutations in pea block GA biosynthesis and normally cause a marked reduction in internode length. However, neither of these genes influences the growth of plants carrying thecry ^s la gene combination. Plants of this genotype have long, thin internodes, pale green foliage, and abnormal flower and fruit development, collectively referred to as the slender phenotype. [¹³C,³H]Gibberellin A₂₀ is metabolized to GA₁, GA₈, and GA₂₉ in slender lines carrying the geneLe but only to GA₂₉ and GA₂₉-catabolite inle lines. Examination of¹²C:¹³C isotopic ratios showed that metabolites were strongly diluted by endogenous [¹²C]GAs inNa lines. However, little if any significant dilution was observed in a line homozygous for thena gene. These results confirm that thele andna mutations are fully expressed at the biochemical level in slender phenotypes of peas and concur with previous reports that internode elongation is entirely independent of GA levels incry ^s la (slender) plants.     

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA₂₀ to bioactive GA₁) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₂₀ to GA₂₉), suggesting a concerted regulation to increase levels of bioactive GA₁ following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₁ to GA₈) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA₁, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [¹⁴C]GA₁₂ to [¹⁴C]GA₁ only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA₁ required for initial fruit set and growth.     

Shoot elongation in Lathyrus odoratus L.: Gibberellin levels in light- and dark-grown tall and dwarf seedlings

The levels of gibberellin A₁ (GA₁), GA₂₀, GA₁₉, GA₈, GA₂₉ and GA₈₁ (2-epiGA₂₉) were measured in tall (L-) and dwarf (ll) sweet-pea plants grown in darkness and in light. In both environments the apical portions of dwarf plants contained less GA₁; GA₈ and GA₁₉, but more GA₂₀, GA₂₉, and GA₈₁ than did those of tall plants. It is concluded that the partial block in 3β-hydroxylation of GA₂₀ to GA₁ is imposed by allele l in darkness as well as in the light. Furthermore, darkness does not appear to enhance elongation in sweet pea by increasing GA₁ levels. The reduction of the pool size of GA₁₉ in dwarf plants supports recent theories on the regulation of GA biosynthesis, formulated on the basis of observations in monocotyledonous species. Darkness results in decreased GA₂₀, GA₂₉, and GA₈₁ levels in the apical portions of tall and dwarf plants and possible reasons for this are discussed.     

Ontogenetic variation in levels of gibberellin A1 in Pisum

The levels of the biologically active gibberellin (GA), GA₁, and of its precursor, GA₂₀, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chromatography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of development the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA₃. Tall plants generally contained 10–18 times more GA₁ and possessed internodes 2–3 times longer than dwarf plants. Further, dwarf plants contained 3–5 times more GA₂₀ than tall plants. No conclusive evidence for the presence of GA₃ or GA₅ could be obtained, even with the aid of [²H₂]GA₃ and [²H₂]GA₅ internal standards. If GA₃ and GA₅ were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA₁, respectively. Comparison of the effects of gene le on GA₁ levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA₁ content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA₃ were present.Abreviations GA_n gibberellin A_n We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuterated GA standards and the Australian Research Council for financial assistance.     

Evidence for Phytochrome Regulation of Gibberellin A(20) 3beta-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L

The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA₂₀), the product (GA₁), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA₁ under red irradiation and in darkness. The lele plants grew in response to GA₂₀ and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA₂₀ and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3β-hydroxylation of GA₂₀ to GA₁ in genotype lele is due to a Pfr-induced blockage in the expression of that activity.     

Further identification of endogenous gibberellins in the shoots of pea, line g2

To interpret the metabolism of radiolabeled gibberellins A₁₂-aldehyde and A₁₂ in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [¹⁴C]GA₁₂ and [¹⁴C]GA₁₂-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). [¹⁴C]GA₁₂ was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [¹⁴C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA₅₃, GA₄₄, GA₁₉ and GA₂₀ was demonstrated and their HPLC peak identity ascertained. The ¹⁴C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA₅₃ to 4% in GA₂₀. Calculated levels of GA₂₀, GA₁₉, GA₄₄, and GA₅₃ were 42, 8, 10, and 0.5 nanograms/gram, respectively.     

Internode length in Pisum

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [¹³C, ³H]GA₂₀. The substrate was metabolised to [¹³C, ³H]GA₁, [¹³C, ³H]GA₈ and [¹³C, ³H]GA₂₉ in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [¹³C, ³H]GA₂₉ and, in one line, [¹³C, ³H]GA₂₉-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3-hydroxylation of GA₂₀ to GA₁. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [¹³C, ³H]GA₂₀ metabolites were considerably diluted by endogenous [¹²C]GAs, implying that the metabolism of [¹³C, ³H]GA₂₀ mirrored that of endogenous [¹²C]GA₂₀. In contrast, the [¹³C, ³H]GA₂₀ metabolites in na lines showed no dilution with [¹²C]GAs, confirming that the na mutation prevents the production of C₁₉-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the ¹²C:¹³C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [¹³C, ³H]GA₂₀ and extraction. No [¹²C]GA₁ and only traces of [¹²C]GA₈ (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

Use of an Acylcyclohexanedione Growth Retardant, LAB 198 999, to Determine Whether Gibberellin A(20) Has Biological Activity per se in Dark-Grown Dwarf (le) Seedlings of Pisum sativum

Dwarf (le⁵⁸³⁹) seedlings of Pisum sativum respond to gibberellin A₂₀ (GA₂₀) in the dark, although the same dosage of GA₂₀ applied to light-grown le⁵⁸³⁹ seedlings elicits no growth response. The acylcyclohexanedione growth retardant, LAB 198 999, which is known to inhibit gibberellin oxidation and in particular 3β-hydroxylation such as the conversion of GA₂₀ to GA₁, also inhibits the growth response of dark-grown dwarf (le⁵⁸³⁹) seedlings to GA₂₀. Thus, the biological activity of GA₂₀ in the dark appears to be a consequence of its conversion to GA₁, even though it is known from studies with light-grown seedlings that the le mutation reduces the conversion of GA₂₀ to GA₁.     

Endogenous gibberellin A1 levels control thermoperiodic stem elongation in Pisum sativum

Gibberellin (GA) is believed to be involved in thermoperiodic stem elongation. With this in mind, we studied the correlation between gibberellin A₁ (GA₁) levels and stem elongation affected by alternating day (DT) and night temperature (NT) in 5 genotypes of Pisum sativum differing in their degree of dwarfism. The endogenous GA content in the tissue of two of the genotypes was determined by combined gas chromatography and mass spectrometry. The wild genotype developed 40 to 50% shorter stems and internodes under a low DT and high NT combination (negative difference [DIF] between DT and NT, DT/NT 15.5/21.5 or 14/24°C) than under the opposite regime of high DT and low NT (positive DIF, DT/NT 22.5/16.5 or 24/14°C). The GA biosynthetic mutants ls and le, and the auxin and brassinosteroid mutant lkb responded in a similar way, but not as strongly as the wild type. The stem length of the GA-insensitive slender mutant (la cry^s) was reduced by only 8% under negative compared to positive DIF. In the wild type endogenous GA levels decreased by 60% from positive to negative DIF in the upper part of the stem. Further, there was a corresponding decrease in the levels of precursors to GA₁, i.e. GA₅₃, GA₄₄, GA₁₉ and GA₂₀, while 2β-hydroxylated GA₂₀ and GA₁, GA₂₉ and GA₈, respectively, were unaffected by DIF. A similar increase in the ratios of GA₂₉ to GA₂₀ and GA₈ to GA₁ from positive to negative DIF was seen in the stem tissue of the le mutant as in the wild type. The temperature regimes affected the levels of GA₁ and its precursors in combined leaf and petiole samples and in the shoot tip in a similar manner as in the stem tissue. However, the different temperature regimes did not affect the ratio of GA₈/GA₁ in the shoot tip. The results indicate that altered stem elongation of the pea plants in response to diurnal temperature alternations may be mediated by changes in endogenous levels of GA₁. The GA₁ levels may be controlled by an effect of DIF on both biosynthetic and inactivation steps.     

The endogenous gibberellins of dwarf mutants of lettuce

The gibberellin (GA) content of E-1, a tall genotype of early flowering lettuce (Lactuca sativa L.), and of three selected GA-responsive dwarfs, dwf1, dwf2, and dwf2¹, has been determined using ¹³C-labeled internal standards and gas chromatographymass spectrometry (GC-MS). In the shoots of the E-1 parent, GA₁, 3-epi-GA₁, GA₃, GA₅, GA₈, GA₁₉, GA₂₀, GA₂₉, and GA₅₃ were identified by full scan GC-MS and Kovats retention indices. Purification by immunoaffinity chromatography selective for 13-hydroxy GAs, was necessary for GA identification. Relative to the parent E-1, the concentrations of GA₁, GA₈, GA₂₀, and GA₂₉ in the shoots of dwf2 plants were reduced to about 10% and in shoots of dwf2¹ plants to less than 50%. In dwf1 the levels of GA₁, GA₈, and GA₂₉ were also reduced to less than 50% of the parent E-1, but the level of GA₂₀ was fivefold higher than in E-1. Plant height was correlated with the endogenous levels of GA₁ and GA₈.