Gibberellins and pea seed development

The gibberellin (GA)-biosynthesis mutations, lh ⁱ , ls and Ie ⁵⁸³⁹ have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh ⁱ possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh ⁱ mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh ⁱ mutation reduces endogenous GA₁ and gibberellic acid (GA₃) levels in the embryo/endosperm a few days after anthesis and fertilizing lh ⁱ plants with wild-type pollen dramatically increases GA₁ and GA₃ levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le ⁵⁸³⁹ mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh ⁱ mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh ⁱ mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA₁ and/or GA₃) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.Abbreviations GA_n gibberellin A_n - DAA days after anthesis - WT wild-type We thank Noel Davies, Katherine McPherson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) for dideuterated GA standards, and the Australian Research Council and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN, Japan), for financial support.     

Seed development in Pisum

The Ih and lh _i alleles have been shown previously to reduce the level of endogenous gibberellin A₁ (GA₁) in shoots of pea (Pisum sativum L.), resulting in a dwarf phenotype compared with the wild type, cv. Torsdag (Lh). In addition, plants homozygous for the lh ⁱ allele have reduced seed yield compared with Lh (tall, wild type) and lh (dwarf) plants. In this paper we show that the lh ⁱ mutation is expressed in developing seeds and pods. Comparison of GA levels in young shoots and developing seeds of genotypes lh and lh ⁱ demonstrates that the relative severity of the two mutations varies in different tissues. Homozygous h ⁱ seeds have reduced GA levels, weigh less, and are less likely to develop to maturity when compared with Lh seeds. However, fertilization of lh ⁱ plants with Lh pollen increases seed GA levels, seed weight and seed survival, indicating that an increase in seed GA levels due to the presence of the Lh allele can restore normal seed growth. Pods developing on self-pollinated lh ⁱ plants are shorter than pods on Lh (wild type) plants, although this may be an indirect effect of the increased seed abortion of lh _i plants. Based on these results we suggest that endogenous GAs play an important role in the development of seeds of P. sativum L.Abbreviations GA_(n) gibberellin A_n We wish to thank Katherine McPherson, Peter Newman, Leigh Johnson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) and Professor B.O. Phinney (UCLA, USA) for labelled GA standards, and the Australian Research Council for financial support.     

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.     

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum

Gibberellins (GAs) A₁₇, A₁₉, A₂₀, A₂₉, A₄₄, 2OH-GA₄₄ (tentative) and GA₂₉-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA₁₉ which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA₁, GA₈, GA₂₀, GA₂₉, GA₈-catabolite and GA₂₉-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA₈, GA₂₀, GA₂₉ and GA₂₉-catabolite, and the presence of GA₁ was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA₁ in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA₂₀, GA₂₉ and GA₂₉-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10²- to 10³-fold less than the levels in developing seeds. The 2-epimer of GA₂₉ is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.Abbreviations cv cultivar - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatographymass spectrometry - HPLC high-pressure liquid chromatography - KRI Kovats retention index - MeTMSi methyl ester trimethylsilyl ether     

Expression of gibberellin mutations in fruits of Pisum sativum L.

The gibberellin (GA) economy of young pea (Pisum sativum L.) fruits was investigated using a range of mutants with altered GA biosynthesis or deactivation. The synthesis mutation lh-2 substantially reduced the content of both GA₄ and GA₁ in young seeds. Among the other synthesis mutations, ls-1, le-1 and le-3, the largest reduction in seed GA₁ content was only 1.7-fold (le-1), while GA₄ was not reduced in these mutants, and in fact accumulated in some experiments (compared with the wild type). Mutation sln appeared to block the step GA₂₀ to GA₂₉ in young pods and seeds, but not as strongly as in older seeds. Mutations ls-1, le-1 and le-3 markedly reduced pod GA₁ levels, but pod elongation was not affected. After feeds of [¹³C,³H]GA₂₀ to leaves, the pods contained ¹³C,³H-labelled GA₂₀, GA₁, GA₂₉ and GA₈₁, and the seeds, [¹³C,³H]GA₂₀ and [¹³C,³H]GA₂₉. These findings are discussed in relation to recent suggestions regarding the role and origin of GA₁ in pea fruits. Received: 6 June 1997 / Accepted: 15 July 1997     

Internode Length in Pisum: Gene na May Block Gibberellin Synthesis between ent-7alpha-Hydroxykaurenoic Acid and Gibberellin A(12)-Aldehyde

The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA₁₂-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7α-hydroxykaurenoic acid and GA₁₂-aldehyde in the na mutant. Feeds of ent-[³H]kaurenoic acid and [²H]GA₁₂-aldehyde to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize [²H]GA₁₂-aldehyde to a number of[²H]C₁₉-GAs including GA₁. However, there was no indication in na genotypes for the metabolism of ent-[³H]kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolised ent-[³H]kaurenoic acid to radioactive compounds that co-chromatographed with GA₁, GA₈, GA₂₀, and GA₂₉. However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-[³H]kaurenoic acid were similar for both Na and na plants and contained ent-16α,17-dihydroxykaurenoic acid and ent-6α,7α,16β,17-tetrahydroxykaurenoic acid (both characterized by GC-MS), suggesting that the metabolites arose from side branches of the main GA-biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7α-hydroxylation.     

The localization,metabolism and biological activity of gibberellins in maturing and germinating seeds of Pisum sativum cv. Progress No. 9

Gibberellin A₂₀ (GA₂₀), GA₂₉ and GA₂₉-catabolite were quantified in cotyledons, embryonic axes, and testas of Pisum sativum cv. Progress No. 9 throughout the final stages of seed maturation and during germination. Stable isotope-labelled GAs were used as internal standards in conjunction with combined gas chromatography-mass spectrometry. Gibberellin A₂₀ and GA₂₉ were mainly located in the cotyledons of maturing seeds, and GA₂₉-catabolite was predominantly located in the testa. Stable isotope- and radio-labelled GA₂₀ and GA₂₉ were fed to both intact seeds developing in vivo, and to isolated seed parts cultured in vitro. The combined results of in-vivo and in-vitro feeds indicated that GA₂₀ is metabolised to GA₂₉ in the cotyledons, that GA₂₉ is transported from the cotyledons to the testa, and that GA₂₉ is metabolised to GA₂₉-catabolite in the testa. Although the metabolism of GA₂₀ in the cotyledons and of GA₂₉ in the testa has been shown definitively, the mobility of GA₂₉ has not yet been demonstrated directly. During seed desiccation and germination GA₂₉-catabolite and products arising from it are transferred from the testa into the embryo. There is no evidence of a physiological function for GA₂₉-catabolite in germination or early seedling growth. Use of a growth retardant indicates that seedling growth, but not germination, is dependent on de-novo GA biosynthesis.     

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).     

The role of gibberellins A1 and A3 in fruit growth of Pisum sativum L. and the identification of gibberellins A4 and A7 in young seeds

Gibberellins A₁ and A₃ are the major physiologically active gibberellins (GAs) present in young fruit of pea (Pisum sativum L.). The relative importance of these GAs in controlling fruit growth and their biosynthetic origins were investigated in cv. Alaska. In addition, the non-13-hydroxylated active GAs, GA₄ and GA₇, were identified for the first time in young seeds harvested 4 d after anthesis, although they are minor components and are not expected to play major physiological roles. The GA₁ content is maximal in seeds and pods at 6 d after anthesis, the time of highest growth-rate of the pod (Garcia-Martinez et al. 1991, Planta 184: 53–60), whereas gibberellic acid (GA₃), which is present at high levels in seeds 4–8 d after anthesis, has very low abundance in pods. Gibberellins A₁₉, A₂₀ and A₂₉ are most concentrated in seeds at, or shortly after, anthesis and their abundance declines rapidly with development, concomitant with the sharp increase in GA₁ and GA₃ content. Application of GA₁ or GA₃ to the leaf subtending an emasculated flower stimulated parthenocarpic fruit development. Measurement of the GA content of the pods at 4 d after anthesis indicated that only 0.002–0.5% of the applied GA was transported to the fruit, depending on dose. There was a linear relationship between GA₁ content and pod weight up to about 2 ng · (g FW)⁻¹, whereas no such correlation existed for GA₃ content. The concentration of endogenous GA₁ in pods from pollinated ovaries is just sufficient to give the maximum growth response. It is concluded that GA₁, but not GA₃, controls pod growth in pea; GA₃ may be involved in early seed development. The distribution of GAs within the seeds at 4 d post anthesis was also investigated. Most of the GA₁, GA₈, GA₁₉, GA₂₀ and GA₂₉ was present in the testa, whereas GA₃ was distributed equally between testa and endosperm and GA₄ was localised mainly in the endosperm. Of the GAs analysed, only GA₃ and GA₂₀ were detected in the embryo. Metabolism experiments with intact tissues and cell-free fractions indicated compartmentation of GA biosynthesis within the seed. Using ¹⁴C-labelled GA₁₂, GA₉, 2,3-didehydroGA₉ and GA₂₀ as substrates, the testa was shown to contain 13-hydroxylase and 20-oxidase activities, the endosperm, 3β-hydroxylase and 20-oxidase activities. Both tissues also produced 16,17-dihydrodiols. However, GA₁ and GA₃ were not obtained as products and it is unlikely that they are formed via the early 13-hydroxylation pathway. [¹⁴C]gibberellin A₁₂, applied to the inside surface of pods in situ, was metabolised to GA₁₉, GA₂₀, GA₂₉, GA₂₉-catabolite, GA₈₁ and GA₉₇, but GA₁ was not detected. Gibberellin A₂₀ was metabolised by this tissue to GA₂₉ and GA₂₉-catabolite. Received: 23 July 1996 / Accepted: 2 September 1996     

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins

We have cloned two genes for gibberellin (GA) 2-oxidase from rice (Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA₁ to GA₈ and GA₂₀ to GA₂₉-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice. Electronic Publication     

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     

Internode length in Pisum sativum L. The kinetics of growth and [3H]gibberellin A20 metabolism in genotype na Le

The relationship between shoot growth and [³H]gibberellin A₂₀ (GA₂₀) metabolism was investigated in the GA-deficient genotype of peas, na Le. [17-¹³C, ³H₂]gibberellin A₂₀ was applied to the shoot apex and its metabolic fate examined by gas chromatographic-mass spectrometric analysis of extracts of the shoot and root tissues. As reported before, [¹³C, ³H₂]GA₁, [¹³C, ³H₂]GA₈ and [¹³C, ³H₂]GA₂₉ constituted the major metabolites of [¹³C, ³H₂]GA₂₀ present in the shoot. None of these GAs showed any dilution by endogenous ¹²C-material. [¹³C, ³H₂]GA₂₉-catabolite was also a prominent metabolite in the shoot tissue but showed pronounced isotope dilution probably due to carry-over of endogenous [¹²C]GA₂₉-catabolite from the mature seed. In marked contrast to the shoot tissue, the two major metabolites present in the roots were identified as [¹³C, ³H₂]GA₈-catabolite and [¹³C, ³H₂]GA₂₉-catabolite. Both of these compounds showed strong dilution by endogenous ¹²C-material. Only low levels of [¹³C, ³H₂]GA₁, [¹³C, ³H₂]GA₈, [¹³C, ³H₂]GA₂₀ and [¹³C, ³H₂]GA₂₉ accumulated in the roots. It is suggested that compartmentation of GA-catabolism may occur in the root tissue in an analogous manner to that shown in the testa of developing seeds. Changes in the levels of [1,3-³H₂]GA₂₀ metabolites over 10 d following application of the substrate to the shoot apex of genotype na Le confirmed the accumulation of [³H]GA-catabolites in the root tissues. No evidence was obtained for catabolic loss of [³H]GA₂₀ by complete oxidation or conversion to a methanol-inextractable form. The results indicate that the root system may play an important role in the regulation of biologically active GA levels in the developing shoot of Na genotypes of peas.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

The identification and metabolism of GA9 and its metabolites in seed coats and shoots of pea,line G2

[³H]gibberellin A₉ was applied to shoots or seed parts of G2 pea to produce radiolabeled metabolites. These were used as markers during purification for the recovery of endogenous GA₉ and its naturally occurring metabolites. GA₉ and its metabolites were purified by HPLC, derivatized and examined by GC-MS. Endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ were identified in pea shoots and seed coats. GA₅₁-catabolite and GA₂₉-catabolite were also detected in seed coats. GA₇₀ was detected in seed coats following the application of 1 g of GA₉. Applied [³H]GA₉ was metabolized through both the 13-hydroxylation and 2-hydroxylation pathways. Labeled metabolites were tentatively identified on the basis of co-chromatography on HPLC with endogenous compounds identified by GC-MS. In shoots [³H]GA₅₁ and [³H]GA₅₁-catabolite were the predominant metabolites after 6 hrs, but by 24 hrs there was little of these metabolites remaining, while [³H]GA₂₉-catabolite and an unidentified metabolite predominated. In seed coats [³H]GA₅₁ was the initial product, later followed by [³H]GA₅₁-catabolite and an unidentified metabolite (different from that in shoots), with lesser amounts of [³H]GA₂₀, [³H]GA₂₉ and [³H]GA₂₉-catabolite. [³H]GA₇₀ was a very minor product in both cases. [³H]GA₉ was not metabolized by pea cotyledons.Edited by T.J. Gianfagna.Author for correspondence     

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     

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.     

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.     

Metabolism of Tritiated Gibberellin A(20) in Immature Seeds of Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₂₀ ([³H]GA₂₀) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of [³H]GA₂₀, the three in highest yield were GA₂₉, an α,β-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA₂₀ → GA₂₉ → compound B → the ketone was indicated. The amount of [³H]GA₂₉ in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [³H]ketone in the seed increased with time to its highest level at maturity. It is suggested that compound B and the ketone represent the major pathway of catabolism of GA₂₉, a 2β-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.