GA Oxidase Research Articles

PavGA2ox-2L inhibits the plant growth and development interacting with PavDWARF in sweet cherry (Prunus avium L.)

Dwarf dense planting is helpful to improve the yield and quality of sweet cherry, which has enormous market demand. GA2oxs (GA oxidases) affect plant height, dormancy release, flower development, and seed germination by participating in the metabolic regulation and signal transduction of GA (Gibberellin). However, the research on GA2ox in sweet cherry is little and worthy of further investigation. Therefore, we identified the PavGA2ox-2L gene from sweet cherry, close to PynGA2ox-2 from Prunus yedoensis var. Nudiflora. The phylogenetic analysis indicated conserved functions with these evolutionarily closer GA2ox subfamily genes. Subcellular localization forecast analysis indicated that PavGA2ox-2L was localized in the nucleus or cytoplasm. The expression levels of PavGA2ox-2L were higher in winter, indicating that PavGA2ox-2L promoted maintained flower bud dormancy. The expression levels of PavGA2ox-2L were significantly increased after GA4+7 treatment while decreased after GR24 (a synthetic analog of SLs (Strigolactones)) or TIS108 (a triazole-type SL-biosynthesis inhibitor) treatments. Over-expression of PavGA2ox-2L resulted in decreased plant height, delayed flowering time, and low seed germination rate in Arabidopsis thaliana. Furthermore, the interaction between PavGA2ox-2L and PavDWARF was verified by Y2H and BiFC assays. In the current investigation, PavGA2ox-2L functions as a GA metabolic gene that promotes dwarf dense planting, delays flowering time, and inhibits seed germination. In addition, it also participates in regulating plant growth and development through the interaction with the critical negative regulator PavDWARF of Gibberellin. These results will help us better explore the molecular mechanism of GA2ox-mediated dwarf and late-maturing varieties for fruit trees.

Genome-Wide Analysis of the Rice Gibberellin Dioxygenases Family Genes

Gibberellins (GAs), a pivotal plant hormone, play fundamental roles in plant development, growth, and stress response. In rice, gibberellin-dioxygenases (GAoxes) are involved in the biosynthesis and deactivation of gibberellins. However, a comprehensive genome-wide analysis of GA oxidases in rice was not uncovered. Here, a total of 80 candidate OsGAox genes were identified and 19 OsGAox genes were further analyzed. Studies on those 19 OsGAox genes, including phylogenetic tree construction, analysis of gene structure, exploration of conserved motifs and expression patterns, were conducted. Results showed that the GAox genes in Arabidopsis and rice were divided into four subgroups and shared some common features. Analysis of gene structure and conserved motifs revealed that splicing phase and motifs were well conserved during the evolution of GAox genes in Arabidopsis and rice, but some special conserved motifs possessed unknown functions need to be further studied. Exploration of expression profiles from RNA-seq data indicated that each GAox gene had tissue-specific expression patterns, although they varied greatly. The expression patterns of these genes under GA3 treatment revealed that some genes, such as OsGA2ox1, OsGA2ox3, OsGA2ox4, OsGA2ox7, OsGA20ox1, and OsGA20ox4, may play a major role in regulating the level of bioactive GA. Taken together, our study provides a comprehensive analysis of the GAox gene family and will facilitate further studies on their roles in rice growth and development so that these genes can be better exploited.

Genome-Wide Identification and Evolution Analysis of the Gibberellin Oxidase Gene Family in Six Gramineae Crops.

The plant hormones gibberellins (GAs) regulate plant growth and development and are closely related to the yield of cash crops. The GA oxidases (GAoxs), including the GA2ox, GA3ox, and GA20ox subfamilies, play pivotal roles in GAs’ biosynthesis and metabolism, but their classification and evolutionary pattern in Gramineae crops remain unclear. We thus conducted a comparative genomic study of GAox genes in six Gramineae representative crops, namely, Setaria italica (Si), Zea mays (Zm), Sorghum bicolor (Sb), Hordeum vulgare (Hv), Brachypodium distachyon (Bd), and Oryza sativa (Os). A total of 105 GAox genes were identified in these six crop genomes, belonging to the C19-GA2ox, C20-GA2ox, GA3ox, and GA20ox subfamilies. Based on orthogroup (OG) analysis, GAox genes were divided into nine OGs and the number of GAox genes in each of the OGs was similar among all tested crops, which indicated that GAox genes may have completed their family differentiations before the species differentiations of the tested species. The motif composition of GAox proteins showed that motifs 1, 2, 4, and 5, forming the 2OG-FeII_Oxy domain, were conserved in all identified GAox protein sequences, while motifs 11, 14, and 15 existed specifically in the GA20ox, C19-GA2ox, and C20-GA2ox protein sequences. Subsequently, the results of gene duplication events suggested that GAox genes mainly expanded in the form of WGD/SD and underwent purification selection and that maize had more GAox genes than other species due to its recent duplication events. The cis-acting elements analysis indicated that GAox genes may respond to growth and development, stress, hormones, and light signals. Moreover, the expression profiles of rice and maize showed that GAox genes were predominantly expressed in the panicles of the above two plants and the expression of several GAox genes was significantly induced by salt or cold stresses. In conclusion, our results provided further insight into GAox genes’ evolutionary differences among six representative Gramineae and highlighted GAox genes that may play a role in abiotic stress.

Spatiotemporal changes in gibberellin content are required for soybean nodulation.

The plant hormone gibberellin (GA) is required at different stages of legume nodule development, with its spatiotemporal distribution tightly regulated. Transcriptomic and bioinformatic analyses established that several key GA biosynthesis and catabolism enzyme encoding genes are critical to soybean (Glycine max) nodule formation. We examined the expression of several GA oxidase genes and used a Förster resonance energy transfer-based GA biosensor to determine the bioactive GA content of roots inoculated with DsRed-labelled Bradyrhizobium diazoefficiens. We manipulated the level of GA by genetically disrupting the expression of GA oxidase genes. Moreover, exogenous treatment of soybean roots with GA3 induced the expression of key nodulation genes and altered infection thread and nodule phenotypes. GmGA20ox1a, GmGA3ox1a, and GmGA2ox1a are upregulated in soybean roots inoculated with compatible B. diazoefficiens. GmGA20ox1a expression is predominately localized to the transient meristem of soybean nodules and coincides with the spatiotemporal distribution of bioactive GA occurring throughout nodule organogenesis. GmGA2ox1a exhibits a nodule vasculature-specific expression pattern, whereas GmGA3ox1a can be detected throughout the nodule and root. Disruptions in the level of GA resulted in aberrant rhizobia infection and reduced nodule numbers. Collectively, our results establish a central role for GAs in root hair infection by symbiotic rhizobia and in nodule organogenesis.

Analysis of regulatory elements in GA2ox, GA3ox and GA20ox gene families in Arabidopsis thaliana: an important trait

The enzymes that are independently encoded by GA2ox, GA3ox and GA20ox genes control gibberellins (GA) biosynthesis and catabolism. This study analysed the promoter regions, transcription start site (TSS), motifs and CpG islands (CGIs) in 15 GA oxidase genes of Arabidopsis thaliana using 1-kb sequences. Excluding ATGA2ox5 and ATGA3ox4, promoter regions and TSS analysis showed that 60.00% of the genes have more than one TSS in 1000 bp. Relative to the ATG, 80.00% of TSSs with highest score were in the range of −100 bp. Sixty percent of investigated GA genes have TSS in their 5′ untranslated regions (UTRs) with highest prediction score at 0.8 cut-off values. A total of 5 motifs were discovered in 15 GA oxidase genes. More than half (53.33%) of the motifs were distributed throughout promoter regions on both strands. Positional clustering of more than half (55.31%) of motifs was found between +1 and −500 bp. MfIV was detected (100%) in all promoter regions of GA genes. Using the Plant CARE database, various similar motifs were identified in all promoter regions of GA genes. Including two gibberellin-responsive elements (GARE-motif, P-box), numerous motifs such as CAAT-box, TATA-box, ATC-motif, Gap-box and others were distinguished in the promoter regions of GA genes. CGIs were not detected in the promoter sequences of all GA oxidase genes. However, 86.66% of CGIs were discovered in the gene bodies of GA oxidase genes. Analysing the regulatory elements of GA genes is the first step to control crop height, increase yield and resistance to lodging.

Genome-wide identification, characterization and expression profiling of gibberellin metabolism genes in jute

BackgroundGibberellin (GA) is one of the most essential phytohormones that modulate plant growth and development. Jute (Corchorus sp.) is the second most important source of bast fiber. Our result has shown that exogenous GA can positively regulate jute height and related characteristics which mean increasing endogenous GA production will help to get a jute variety with improved characteristics. However, genes involved in jute GA biosynthesis have not been analyzed precisely.ResultsGenome-wide analysis identified twenty-two candidate genes involved in jute GA biosynthesis pathway. Among them, four genes- CoCPS, CoKS, CoKO and CoKAO work in early steps. Seven CoGA20oxs, three CoGA3oxs, and eight GA2oxs genes work in the later steps. These genes were characterized through phylogenetic, motif, gene structure, and promoter region analysis along with chromosomal localization. Spatial gene expression analysis revealed that 11 GA oxidases were actively related to jute GA production and four of them were marked as key regulators based on their expression level. All the biosynthesis genes both early and later steps showed tissue specificity. GA oxidase genes were under feedback regulation whereas early steps genes were not subject to such regulation.ConclusionEnriched knowledge about jute GA biosynthesis pathway and genes will help to increase endogenous GA production in jute by changing the expression level of key regulator genes. CoGA20ox7, CoGA3ox2, CoGA2ox3, and CoGA2ox5 may be the most important genes for GA production.

Tuber-Specific Expression of Two Gibberellin Oxidase Transgenes from Arabidopsis Regulates over Wide Ranges the Potato Tuber Formation

Potato (Solanum tuberosum L.) tuberization is a practically important natural process regulated by various factors including phytohormones. This work was aimed at studying characteristics of in vitro cultivated potato plants transformed with the AtGA20-oxidase gene promoting biosynthesis of bioactive gibberellins (GAs) or with the AtGA2-oxidase gene acting oppositely, i.e. deactivating functional GAs. Both transgenes originated from Arabidopsis and were fused to a sugar-sensitive B33 patatin promoter providing their expression predominantly in tubers. Global phytohormone determination in AtGA20ox-transformants revealed active GA1 at high and moderate concentrations in tubers and shoots, respectively. In control plants, GA1 was virtually absent. Together with GAs, contents of some other phytohormones were altered in transgenic plants. This was especially true for the auxin content which increased ~15-fold in tubers and more than 4-fold in shoots. Also the jasmonic acid content exhibited a tuber-specific increase while the content of abscisic acid decreased both in tubers and shoots. The dynamics of tuberization in transformed and non-transformed potato plants was recorded in in vitro parallel assays. The transgene for GA inactivation enhanced tuber formation while the transgene promoting active GA synthesis reduced potato productivity. Hence, the crucial role of gibberellin in potato productivity was corroborated. These results showed that the manipulation of the local GA level by using the foreign GA oxidase genes and organ-specific promoters is useful not only to investigate the molecular mechanisms governing tuberization, but also as a biotechnological tool for the manipulation of tuber formation without marked impairment of other physiological traits of potatoes.

Comprehensive Analysis of Cucumber Gibberellin Oxidase Family Genes and Functional Characterization of CsGA20ox1 in Root Development in Arabidopsis.

Cucumber (Cucumis sativus L.) is an important vegetable crop worldwide and gibberellins (GAs) play important roles in the regulation of cucumber developmental and growth processes. GA oxidases (GAoxs), which are encoded by different gene subfamilies, are particularly important in regulating bioactive GA levels by catalyzing the later steps in the biosynthetic pathway. Although GAoxs are critical enzymes in GA synthesis pathway, little is known about GAox genes in cucumber, in particular about their evolutionary relationships, expression profiles and biological function. In this study, we identified 17 GAox genes in cucumber genome and classified them into five subfamilies based on a phylogenetic tree, gene structures, and conserved motifs. Synteny analysis indicated that the tandem duplication or segmental duplication events played a minor role in the expansion of cucumber GA2ox, GA3ox and GA7ox gene families. Comparative syntenic analysis combined with phylogenetic analysis provided deep insight into the phylogenetic relationships of CsGAox genes and suggested that protein homology CsGAox are closer to AtGAox than OsGAox. In addition, candidate transcription factors BBR/BPC (BARLEY B RECOMBINANT/BASIC PENTACYSTEINE) and GRAS (GIBBERELLIC ACID-INSENSITIVE, REPRESSOR of GAI, and SCARECROW) which may directly bind promoters of CsGAox genes were predicted. Expression profiles derived from transcriptome data indicated that some CsGAox genes, especially CsGA20ox1, are highly expressed in seedling roots and were down-regulated under GA3 treatment. Ectopic over-expression of CsGA20ox1 in Arabidopsis significantly increased primary root length and lateral root number. Taken together, comprehensive analysis of CsGAoxs would provide a basis for understanding the evolution and function of the CsGAox family.

Genome-wide identification and expression profiling reveal tissue-specific expression and differentially-regulated genes involved in gibberellin metabolism between Williams banana and its dwarf mutant.

BackgroundDwarfism is one of the most valuable traits in banana breeding because semi-dwarf cultivars show good resistance to damage by wind and rain. Moreover, these cultivars present advantages of convenient cultivation, management, and so on. We obtained a dwarf mutant ‘8818-1’ through EMS (ethyl methane sulphonate) mutagenesis of Williams banana 8818 (Musa spp. AAA group). Our research have shown that gibberellins (GAs) content in 8818-1 false stems was significantly lower than that in its parent 8818 and the dwarf type of 8818-1 could be restored by application of exogenous GA3. Although GA exerts important impacts on the 8818-1 dwarf type, our understanding of the regulation of GA metabolism during banana dwarf mutant development remains limited.ResultsGenome-wide screening revealed 36 candidate GA metabolism genes were systematically identified for the first time; these genes included 3 MaCPS, 2 MaKS, 1 MaKO, 2 MaKAO, 10 MaGA20ox, 4 MaGA3ox, and 14 MaGA2ox genes. Phylogenetic tree and conserved protein domain analyses showed sequence conservation and divergence. GA metabolism genes exhibited tissue-specific expression patterns. Early GA biosynthesis genes were constitutively expressed but presented differential regulation in different tissues in Williams banana. GA oxidase family genes were mainly transcribed in young fruits, thus suggesting that young fruits were the most active tissue involved in GA metabolism, followed by leaves, bracts, and finally approximately mature fruits. Expression patterns between 8818 and 8818-1 revealed that MaGA20ox4, MaGA20ox5, and MaGA20ox7 of the MaGA20ox gene family and MaGA2ox7, MaGA2ox12, and MaGA2ox14 of the MaGA2ox gene family exhibited significant differential expression and high-expression levels in false stems. These genes are likely to be responsible for the regulation of GAs content in 8818-1 false stems.ConclusionOverall, phylogenetic evolution, tissue specificity and differential expression analyses of GA metabolism genes can provide a better understanding of GA-regulated development in banana. The present results revealed that MaGA20ox4, MaGA20ox5, MaGA20ox7, MaGA2ox7, MaGA2ox12, and MaGA2ox14 were the main genes regulating GA content difference between 8818 and 8818-1. All of these genes may perform important functions in the developmental processes of banana, but each gene may perform different functions in different tissues or during different developmental stages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0809-1) contains supplementary material, which is available to authorized users.

Divergence and adaptive evolution of the gibberellin oxidase genes in plants.

BackgroundThe important phytohormone gibberellins (GAs) play key roles in various developmental processes. GA oxidases (GAoxs) are critical enzymes in GA synthesis pathway, but their classification, evolutionary history and the forces driving the evolution of plant GAox genes remain poorly understood.ResultsThis study provides the first large-scale evolutionary analysis of GAox genes in plants by using an extensive whole-genome dataset of 41 species, representing green algae, bryophytes, pteridophyte, and seed plants. We defined eight subfamilies under the GAox family, namely C19-GA2ox, C20-GA2ox, GA20ox,GA3ox, GAox-A, GAox-B, GAox-C and GAox-D. Of these, subfamilies GAox-A, GAox-B, GAox-C and GAox-D are described for the first time. On the basis of phylogenetic analyses and characteristic motifs of GAox genes, we demonstrated a rapid expansion and functional divergence of the GAox genes during the diversification of land plants. We also detected the subfamily-specific motifs and potential sites of some GAox genes, which might have evolved under positive selection.ConclusionsGAox genes originated very early—before the divergence of bryophytes and the vascular plants and the diversification of GAox genes is associated with the functional divergence and could be driven by positive selection. Our study not only provides information on the classification of GAox genes, but also facilitates the further functional characterization and analysis of GA oxidases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0490-2) contains supplementary material, which is available to authorized users.

Exogenous GA₃ Application Enhances Xylem Development and Induces the Expression of Secondary Wall Biosynthesis Related Genes in Betula platyphylla.

Gibberellin (GA) is a key signal molecule inducing differentiation of tracheary elements, fibers, and xylogenesis. However the molecular mechanisms underlying the effect of GA on xylem elongation and secondary wall development in tree species remain to be determined. In this study, Betula platyphylla (birch) seeds were treated with 300 ppm GA3 and/or 300 ppm paclobutrazol (PAC), seed germination was recorded, and transverse sections of hypocotyls were stained with toluidine blue; the two-month-old seedlings were treated with 50 μM GA3 and/or 50 μM PAC, transverse sections of seedling stems were stained using phloroglucinol–HCl, and secondary wall biosynthesis related genes expression was analyzed by real-time quantitative PCR. Results indicated that germination percentage, energy and time of seeds, hypocotyl height and seedling fresh weight were enhanced by GA3, and reduced by PAC; the xylem development was wider in GA3-treated plants than in the control; the expression of NAC and MYB transcription factors, CESA, PAL, and GA oxidase was up-regulated during GA3 treatment, suggesting their role in GA3-induced xylem development in the birch. Our results suggest that GA3 induces the expression of secondary wall biosynthesis related genes to trigger xylogenesis in the birch plants.

Transcriptional changes of gibberellin oxidase genes in grapevines with or without gibberellin application during inflorescence development

The concept that gibberellin (GA) application on seeded grapevines induces seedlessness has been known for decades in viticulture. GA was applied to inflorescence clusters of seeded diploid grapevine cultivar 'Tamnara' (Vitis spp.) at 14 days before full bloom (DBF). Morphological and molecular effects of GA application were examined on the induction of parthenocarpic fruit development. With GA application, ovaries were enlarged and pollen tube growth was completely inhibited. Vitis GA oxidase enzymes, key determinants for GA level, were characterized through phylogenetic analysis with Arabidopsis GA oxidase enzymes. Five VvGA 20-oxidase (VvGA20ox), three VvGA 3-oxidase (VvGA3ox), and nine VvGA 2-oxidase (VvGA2ox) family proteins, and one VvGA methyltransferase (VvGAMT) and one Vitis cytochrome P450 714A1 proteins were identified, and their expression patterns were analyzed during inflorescence development from 14 DBF to 5 days after full bloom (DAF). VvGA2ox1, VvGA20ox3, and VvGA3ox2 were the most abundantly expressed genes in each gene family at 7, 5, and 2 DBF, respectively. Following GA application at 14 DBF inducing seedlessness, GA catabolic genes such as VvGAMT2, VvGA2ox3, and VvGA2ox4 were up-regulated at 12 DBF, full bloom, and 5 DAF, respectively. Conversely, most GA biosynthetic genes, VvGA20oxs and VvGA3oxs, were down-regulated at near full bloom, and the timing of their peak expression was changed. These results suggest that GA application at pre-bloom changes the GA biosynthesis into GA catabolic pathway at near full bloom by altering the transcription level and timing of GA oxidase genes during grapevine inflorescence development.

Gibberellin metabolism in Vitis vinifera L. during bloom and fruit-set: functional characterization and evolution of grapevine gibberellin oxidases.

Gibberellins (GAs) are involved in the regulation of flowering and fruit-set in grapes (Vitis vinifera L.), but the molecular mechanisms behind this process are mostly unknown. In this work, the family of grapevine GA oxidases involved in the biosynthesis and deactivation of GAs was characterized. Six putative GA 20-oxidase (GA20ox), three GA 3-oxidase (GA3ox), and eight GA 2-oxidase (GA2ox) proteins, the latter further divided into five C19-GA 2ox and three C20-GA2ox proteins, were identified. Phylogenetic analyses suggest a common origin of the GA3ox and C19-GA2ox groups and challenge previous evolutionary models. In vitro analysis revealed that all GA3ox and GA20ox enzymes prefer substrates of the non-13-hydroxylation pathway. In addition, ectopic expression of GA2ox genes in Arabidopsis thaliana confirmed the activity of their encoded proteins in vivo. The results show that bioactive GA1 accumulates in opening grapevine flowers, whereas at later developmental stages only GA4 is detected in the setting fruit. By studying the expression pattern of the grapevine GA oxidase genes in different organs, and at different stages of flowering and fruit-set, it is proposed that the pool of bioactive GAs is controlled by a fine regulation of the abundance and localization of GA oxidase transcripts.

Evolutionary analysis of three gibberellin oxidase genesin rice, Arabidopsis, and soybean

GAs are plant hormones that play fundamental roles in plant growth and development. GA2ox, GA3ox, and GA20ox are three key enzymes in GA biosynthesis. These enzymes belong to the 2OG-Fe (II) oxygenase superfamily and are independently encoded by different gene families. To date, genome-wide comparative analyses of GA oxidases in plant species have not been thoroughly carried out. In the present work, 61 GA oxidase family genes from rice ( Oryza sativa), Arabidopsis, and soybean ( Glycine max) were identified and a full study of these genes including phylogenetic tree construction, gene structure, gene family expansion and analysis of functional motifs was performed. Based on phylogeny, most of the GA oxidases were divided into four subgroups that reflected functional classifications. Intron/intron average length of GA oxidase genes in rice analysis revealed that GA oxidase genes in rice experienced substantial evolutionary divergence. Segmental duplication events were mainly found in soybean genome. However, in rice and Arabidopsis, no single expansion pattern exhibited dominance, indicating that GA oxidase genes from these species might have been subjected to a more complex evolutionary mechanism. In addition, special functional motifs were discovered in GA20ox, GA3ox, and GA2ox, which suggested that different functional motifs are associated with differences in protein function. Taken together our results suggest that GA oxidase family genes have undergone divergent evolutionary routes, especially at the monocot–dicot split, with dynamic evolution occurring in Arabidopsis thaliana and soybean.

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari, Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA 3 in the culture fluid (2.76–28.4 μg/mL), while two others (B-3828 and B-1725) were inactive. GA 3 levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA 1, at a low level (less than 0.1 μg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.

Function and Expression Analysis of Gibberellin Oxidases in Apple

Three cDNAs, encoding gibberellin (GA) 20-oxidase (MdGA20ox1, identical to AB037114), 3-oxidase (MdGA3ox1), and 2-oxidase (MdGA2ox1), were isolated from apple cv. Fuji (Malus x domestica). Southern blot analysis indicated that each of these genes belongs to a gene family. Standard enzyme assays show that the MdGA20ox1-MBP fusion protein can sequentially oxidize three times at C-20 position of GA12 and GA53 and generate GA9 and GA20; the MdGA3ox1-MBP fusion protein converts GA20 and GA9 to GA4 and GA1, and the MdGA2ox1-MBP fusion protein converts GA4 and GA1 to GA34 and GA8, respectively. In addition, we confirmed that MdGA20ox1 is strongly expressed in immature seeds and scarcely detected in other tissues, whereas MdGA3ox1 and MdGA2ox1 are mainly expressed in flowers. Therefore, all the three cDNAs are localized in reproductive tissues. Functional and expression analysis of the three GA oxidases would provide fundamental molecular information to analyze GA metabolic regulation in apple.

PLANT SIZE CONTROL IN APPLE THROUGH GENETIC ENGINEERING

Tree size control is essential for modern apple production. Chemical controls and different managements have been practically used in commercial production of apple rootstocks and cultivars in order to generate dwarf trees. However, these practices are either inefficient or unfriendly to human health and environment. In contrast, genetic engineering offers a better possibility to improve plant properties. By introducing well-characterised genes that control the growth vigour, it is possible to dwarf an existing genotype without disturbing its main genetic background. Up to now, some genes have been introduced into different plant species and proved to be effective in reducing plant size. Among the genes tested, the rolC, gai and GA oxidase have shown to be able to reduce plant size in most cases. In our previous studies, we have introduced the rolC and gai genes into apple rootstocks and cultivars. Both in vitro and grown analysis in greenhouse have shown that transgenic plants with one of these genes did display reduced plant height in most cases. Since plant height is mainly controlled by gibberellins, regulation of GA biosynthesis would be a better way to control plant size in the future.

Gibberellin Biosynthesis in Developing Pumpkin Seedlings

A gibberellin (GA) biosynthetic pathway was discovered operating in root tips of 7-d-old pumpkin (Cucurbita maxima) seedlings. Stepwise analysis of GA metabolism in cell-free systems revealed the conversion of GA(12)-aldehyde to bioactive GA(4) and inactive GA(34). Highest levels of endogenous GA(4) and GA(34) were found in hypocotyls and root tips of 3-d-old seedlings. cDNA molecules encoding two GA oxidases, CmGA20ox3 and CmGA3ox3, were isolated from root tips of 7-d-old LAB150978-treated seedlings. Recombinant CmGA20ox3 fusion protein converted GA(12) to GA(9), GA(24) to GA(9), GA(14) to GA(4), and, less efficiently, GA(53) to GA(20), and recombinant CmGA3ox3 protein oxidized GA(9) to GA(4). Transcript profiles were determined for four GA oxidase genes from pumpkin revealing relatively high transcript levels for CmGA7ox in shoot tips and cotyledons, for CmGA20ox3 in shoot tips and hypocotyls, and for CmGA3ox3 in hypocotyls and roots of 3-d-old seedlings. Transcripts of CmGA2ox1 were mainly found in roots of 7-d-old seedlings. In roots of 7-d-old seedlings, transcripts of CmGA7ox, CmGA20ox3, and CmGA3ox3 were localized in the cap and the rhizodermis by in situ hybridization. We conclude that hypocotyls and root tips are important sites of GA biosynthesis in the developing pumpkin seedling.

Conversion of gibberellin A20 to gibberellins A1 and A5 in a cell-free system from Phaseolus vulgaris

The soluble fraction of a cell-free system from immature seeds of Phaseolus vulgaris L. converts gibberellin A20 (GA20) to GA1 and GA5. It does however not metabolize GA1 and GA29 to GA5, showing that in this system GA20 is converted directly to GA5. The steps from GA20 to GA1 (3-hydroxylation) and from GA20 to GA5 (Δ(2) double-bond formation) require oxygen, Fe(2+) and α-ketoglutarate, and are stimulated by ascorbate. The enzymes catalyzing these conversions bate. The enzymes catalyzing these conversions have properties similar to those of GA oxidases found in Cucurbita maxima and Pisum sativum.