Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.     

The gene encoding tobacco gibberellin 3beta-hydroxylase is expressed at the site of GA action during stem elongation and flower organ development

Gibberellin 3beta-hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3beta-hydroxylase), which encodes a putative GA 3beta-hydroxylase, by RT-PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA 3beta-hydroxylase. A high expression level of Nty was observed in shoot apices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter:GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. It is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3beta-hydroxylase expression in situ.     

Light Alters Cytosolic and Plastidic Phosphorylase Distribution in Pearl Millet Leaves

Treatment of pollinated pea (Pisum sativum L. cv Alaska, line V1) ovaries with 3,5-dioxo-4-butyryl-cyclohexane carboxylic acid ethyl ester (LAB), an acylcyclohexanedione derivative that competitively inhibits 2-oxoglutarate-dependent gibberellin (GA) dioxygenases, caused a reduction of pod elongation proportional to the amount of inhibitor applied. The effect of LAB was counteracted by GA1 and GA3, and partially by GA20. The inhibitor decreased the contents of GA1 and GA3 (the purported active GAs) and GA8, increased those of GA19 and GA20, and did not affect that of GA29 in both the pod and the developing seeds. These results provide evidence that GA1 and/or GA3 control pod development in pea and show that GA20 is not active per se. In contrast to its effect on pollinated ovaries, LAB promoted parthenocarpic development of unpollinated ovaries, which is associated with an increase of GA1 and GA8 content. The inhibitor enhanced the response of unpollinated ovaries to GA1 and GA20, but it did not alter the response to GA3. LAB is proposed to promote parthenocarpic development and enhance the response to exogenous GAs by blocking the 2[beta]-hydroxylation of GA1 more efficiently than 3[beta]-hydroxylation of GA20.     

The gene pat-2, which induces natural parthenocarpy, alters the gibberellin content in unpollinated tomato ovaries

We investigated the role of gibberellins (GAs) in the effect of pat-2, a recessive mutation that induces facultative parthenocarpic fruit development in tomato (Lycopersicon esculentum Mill.) using near-isogenic lines with two different genetic backgrounds. Unpollinated wild-type Madrigal (MA/wt) and Cuarenteno (CU/wt) ovaries degenerated, but GA(3) application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of MA/pat-2 and CU/pat-2 fruits, which occurs in the absence of pollination and hormone application, was not affected by GA(3). Pollinated MA/wt and parthenocarpic MA/pat-2 ovary development was negated by paclobutrazol, and this inhibitory effect was counteracted by GA(3). The main GAs of the early-13-hydroxylation pathway (GA(1), GA(3), GA(8), GA(19), GA(20), GA(29), GA(44), GA(53), and, tentatively, GA(81)) and two GAs of the non-13-hydroxylation pathway (GA(9) and GA(34)) were identified in MA/wt ovaries by gas chromatography-selected ion monitoring. GAs were quantified in unpollinated ovaries at flower bud, pre-anthesis, and anthesis. In unpollinated MA/pat-2 and CU/pat-2 ovaries, the GA(20) content was much higher (up to 160 times higher) and the GA(19) content was lower than in the corresponding non-parthenocarpic ovaries. The application of an inhibitor of 2-oxoglutarate-dependent dioxygenases suggested that GA(20) is not active per se. The pat-2 mutation may increase GA 20-oxidase activity in unpollinated ovaries, leading to a higher synthesis of GA(20), the precursor of an active GA.     

Phytochrome regulation and differential expression of gibberellin 3beta-hydroxylase genes in germinating Arabidopsis seeds.

Despite extensive studies on the roles of phytochrome in photostimulated seed germination, the mechanisms downstream of the photoreceptor that promote germination are largely unknown. Previous studies have indicated that light-induced germination of Arabidopsis seeds is mediated by the hormone gibberellin (GA). Using RNA gel blot analyses, we studied the regulation of two Arabidopsis genes, GA4 and GA4H (for GA4 homolog), both of which encode GA 3beta-hydroxylases that catalyze the final biosynthetic step to produce bioactive GAs. The newly isolated GA4H gene was expressed predominantly during seed germination. We show that expression of both GA4 and GA4H genes in imbibed seeds was induced within 1 hr after a brief red (R) light treatment. In the phytochrome B-deficient phyB-1 mutant, GA4H expression was not induced by R light, but GA4 expression still was, indicating that R light-induced GA4 and GA4H expression is mediated by different phytochromes. In contrast to the GA4 gene, the GA4H gene was not regulated by the feedback inhibition mechanism in germinating seeds. Our data demonstrate that expression of GA 3beta-hydroxylase genes is elevated by R light, which may result in an increase in biosynthesis of active GAs to promote seed germination. Furthermore, our results suggest that each GA 3beta-hydroxylase gene plays a unique physiological role during light-induced seed germination.     

Immunohistochemistry of active gibberellins and gibberellin-inducible alpha-amylase in developing seeds of morning glory

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A(1)-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA(1) and/or GA(3) were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible alpha-amylase in this digestion. We isolated an alpha-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA(3). RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA(1/3). An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA(1/3), was also localized around starch grains in the integument of developing young seeds. The localization of GA(1/3) in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.     

Gibberellin 3-oxidases in developing embryos of the southern wild cucumber, Marah macrocarpus

Immature seeds of the southern wild cucumber, Marah macrocarpus, are a rich source of gibberellins (GAs) and were used in some of the earliest experiments on GA biosynthesis. The main biologically active GAs in developing embryos and endosperm of M. macrocarpus are GA(4) and GA(7), which have been shown previously to be formed from GA(9) in separate pathways, GA(4) being formed directly by 3β-hydroxylation, while GA(7) is produced in two steps via 2,3-didehydroGA(9). In order to identify the enzymes responsible for these conversions, three cDNA clones encoding functionally different GA 3-oxidases, MmGA3ox1, -2 and -3, were obtained from young immature M. macrocarpus embryos. Their biochemical functions were determined by expression of the cDNAs in Escherichia coli and incubation of cell lysates with (14)C-labelled substrates. MmGA3ox1 and MmGA3ox3 converted GA(9) to GA(4) as sole product, while MmGA3ox2 produced several products, including GA(4), 2,3-didehydroGA(9), 2,3-epoxyGA(9), GA(20) and GA(5), these last two products requiring 13-hydroxylation of GA(9) and 2,3-didehydroGA(9), respectively. MmGA3ox1 converted 2,3-didehydroGA(9) to GA(7), while MmGA3ox3 converted this substrate to the 2,3-epoxide, and MmGA3ox2 also formed the epoxide, but also GA(5.) Thus, formation of GA(7) requires the sequential activities of MmGA3ox2 and MmGA3ox1, while MmGA3ox3 is not involved in GA(7) production. The enzymes catalysed similar reactions when incubated with 13-hydroxylated GAs, although with reduced efficiencies. The 13-hydroxylase activity of MmGA3ox2 may be responsible for the production of GA(1) and GA(3), which are present at low levels in developing M. macrocarpus seeds.     

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.     

Expression of the le Mutation in Young Ovaries of Pisum sativum and Its Effect on Fruit Development

The effect of the le mutation on the growth and gibberellin (GA) content of developing fruits was investigated using the near-isogenic lines of Pisum sativum L. 205+ (LeLe) and 205- (lele). Although stem elongation is known to be reduced in 205- plants by approximately 65%, the growth of pods and seeds was unaffected by the le mutation. GA1, GA3, and GA20 stimulated parthenocarpic development of unpollinated ovaries on both 205+ and 205- plants. GA20 was less active on 205- ovaries than on 205+, whereas GA1 had similar, high activity in both lines. The activity of GA3 was even higher than that of GA1 in both lines. Decapitation of 205+ plants induced parthenocarpic development of unpollinated ovaries, but this treatment was much less effective on 205- plants. The contents of GA1 and GA8 in entire ovaries 6 d after anthesis, as well as in the pod and fertilized ovules, were substantially lower in 205- than in 205+ plants, whereas the reverse was true for the levels of GA20 and GA29. These results suggest that 3[beta]-hydroxylation of GA20 to GA1 is reduced in ovaries as well as in vegetative tissues. Thus, the le mutation appears to be expressed in young reproductive organs of the 205- line, even though it does not affect the fruit phenotype. Because the content of GA3 in the ovary was similar in the two lines, one explanation for the normal fruit size in the 205- line is that GA3 is the native regulator of pod growth. Alternatively, sufficient GA1 may still be produced in 205- fruits to maintain normal pod growth.     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.     

Gibberellin biosynthesis: metabolic evidence for three steps in the early 13-hydroxylation pathway of rice

[14C4]GA53, [14C4]GA44, and [2H2/14C4]GA19 were injected separately into seedlings of rice (Oryza sativa) using a dwarf mutant (d35) that has low levels of endogenous gibberellins (GAs). After 8 h incubation, the shoots were extracted and the labeled metabolites were identified by full-scan gas chromatography mass spectrometry (GC-MS) and Kovats retention indices (KRIs). Our results document the metabolic sequence, GA53-->GA44-->GA19-->GA20 and the presence of endogenous GA53, GA44, GA19, GA20 and GA1. Previous metabolic studies have shown the presence of the step, GA20-->GA1 in rice. Taken together, the data establish in vegetative shoots of rice the presence of the early 13-hydroxylation pathway, a pathway that originates from GA12 and leads to bioactive GA1.     

Induced parthenocarpy—a way of changing the levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 1. Extractable hormones

Ethylene, indol-3-acetic acid (IAA), gibberellin-like substances (GAs) and abscisic acid (ABA) were analysed in extracts from normal, seed-containing and parthenocarpic tomato fruits throughout fruit development. Parthenocarpic fruit growth was induced with an auxin (4-CPA), morphactin (CME) or gibberellic acid (GA₃) and compared with that of pollinated control fruits. Fruit growth was only affected by the treatment with GA₃, decreasing size and fresh weight by 60%. The peak sequence of hormones during fruit development was ethylene-GAs-IAA-ABA. Seeded fruits contained the highest levels of IAA and ABA but the lowest levels of GAs. Also, in seeded fruits, a high proportion of IAA and ABA was found in the seeds whereas this was not the case for GAs.Hormone levels of tomato fruits may be successfully, easily and reproducibly altered by inducing parthenocarpic fruit growth and thus eliminating development of seeds which are a major source of hormone synthesis. In spite of markedly changed hormone levels, there was no obvious relationship between fruit growth and extractable hormones per se. However, the results indicate that a high ratio of GAs: auxins is unfavourable for growth of tomato fruits.     

Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis.

The gibberellin (GA) 20-oxidase encoded by the GA5 gene of Arabidopsis directs GA biosynthesis to active GAs, whereas that encoded by the P16 gene of pumpkin endosperm leads to biosynthesis of inactive GAs. Negative feedback regulation of GA5 expression was demonstrated in stems of Arabidopsis by bioactive GAs but not by inactive GA. In transgenic Arabidopsis plants overexpressing P16, there was a severe reduction in the amounts of C20-GA intermediates, accumulation of large amounts of inactive GA25 and GA17, a reduction in GA4 content, and a small increase in GA1. However, due to feedback regulation, expression of GA5 and GA4, the gene coding for the subsequent 3beta-hydroxylase, was greatly increased to compensate for the effects of the P16 transgene. Consequently, stem height was only slightly reduced in the transgenic plants.     

Mendel's stem length gene (Le) encodes a gibberellin 3 beta-hydroxylase.

We describe the isolation of the Le gene of pea, which controls internode elongation and originally was described by Mendel. Heterologous screening of a pea cDNA library yielded a partial clone that was 61% identical to coding regions of the putative Arabidopsis gibberellin 3 beta-hydroxylase gene, GA4. DNA gel blot analysis with this cDNA revealed a HindIII restriction fragment length polymorphism between pea isolines differing at Mendel's Le locus. Genomic clones of the GA4-related gene were isolated from the Le and le isolines. Polymerase chain reaction combined with restriction fragment length polymorphism analysis were used to show that the gene mapped to the Le locus. A cDNA containing a complete open reading frame of the pea GA4-related gene was amplified by polymerase chain reaction from each isoline. Recombinant expression in Escherichia coli demonstrated that the product of the Le cDNA was a gibberellin 3 beta-hydroxylase that is able to convert GA20 to the bioactive GA1. Substantially reduced levels of gibberellin 3 beta-hydroxylase activity were measured, after expression of the le cDNA, by using identical methods. This reduced activity was associated with an alanine-to-threonine substitution in the predicted amino acid sequence of the enzyme near its proposed active site.     

Role of gibberellins in parthenocarpic fruit development induced by the genetic system pat-3/pat-4 in tomato

The role of gibberellins (GAs) in the induction of parthenocarpic fruit-set and growth by the pat-3/pat-4 genetic system in tomato ( Lycopersicon esculentum Mill.) was investigated using wild type (WT; Cuarenteno) and a near-isogenic line derived from the German line RP75/59 (the source of pat-3/pat-4 parthenocarpy). Unpollinated WT ovaries degenerated but GA₃ application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of pat-3/pat-4 fruits, which occurs in the absence of pollination and hormone treatment, was not affected by applied GA₃. Unpollinated pat-3/pat-4 fruit growth was negated by paclobutrazol, an inhibitor of ent -kaurene oxidase, and this inhibitory effect was negated by GA₃. The quantification of the main GAs of the early 13-hydroxylation pathway (GA₁, GA₈, GA₁₉, GA₂₀, GA₂₉ and GA₄₄) in unpollinated ovaries at 3 developmental stages (flower bud, FB; pre-anthesis, PR; and anthesis, AN), by gas chromatography-selected ion monitoring, showed that the concentration of most of them was higher in pat-3/pat-4 than in WT ovaries at PR and AN stages. The concentration of GA₁, suggested previously to be the active GA in tomate, was 2–4 times higher. Unpollinated pat-3/pat-4 ovaries at FB, PR and AN stages also contained relatively high amounts (5–12 ng g⁻¹) of GA₃, a GA found at less than 0.5 ng g⁻¹ in WT ovaries. It is concluded that the mutations pat-3/pat-4 may induce natural facultative parthenocarpy capacity in tomato by increasing the concentration of GA₁ and GA₃ in the ovaries before pollination.     

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.