Isabel López-Díaz

ISOLATION AND TRANSCRIPT ANALYSIS OF GIBBERELLIN 20-OXIDASE GENES IN PEA AND BEAN IN RELATION TO FRUIT DEVELOPMENT

PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgaris). One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv15-11, Pv73-1 and Pv85-26) from bean. Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting [14C]GA12 to [14C]GA9. The intermediates in this three-step reaction, GA15 and GA12, were also identified as products. The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA53 to GA20, as effectively as they did GA12. On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6). Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries. Treatment of unpollinated ovaries with GA3 to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2,4-D, although inducing fruit-set, did not reduce the levels of these transcripts. Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod. The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv15-11 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons, while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant. Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves. Southern blot analysis indicated that the bean GA 20-oxidases are each encoded by single-copy genes, whereas one more gene, homologous to Ps27-12, could also exist in pea.

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.

Molecular, Biochemical and Physiological Characterization of Gibberellin Biosynthesis and Catabolism Genes from Nerium oleander

Genomic and cDNA clones encoding two gibberellin (GA) 20-oxidases (named NoGA20ox1 and -2) and three GA 2-oxidases (NoGA2ox1, -2, and -3) have been isolated and characterized from Nerium oleander L. (oleander), a plant of horticultural importance in the Mediterranean region. NoGA2ox2 and -3 transcripts were abundant in expanding leaves and flower buds, whereas NoGA20ox1, -2 and NoGA2ox1 transcripts were barely detected by Northern blot analysis in the vegetative and reproductive tissues analyzed. The expression of NoGA20ox2, but not that of NoGA20ox1, was subject to negative feedback regulation by active GAs. In contrast, the expression of NoGA2ox2, but not that of NoGA2ox1 and -3, was subject to positive feedforward regulation by active GAs. Multiple transcripts with different 3′ untranslated regions, due to alternative polyadenylation sites, have been identified for each of the isolated genes except NoGA2ox1. Enzyme assays confirmed that NoGA20ox1 and -2 were involved in GA biosynthesis, catalyzing the conversion of GA12 to GA9 and GA53 to GA20, and that NoGA2ox1, -2, and -3 were involved in GA catabolism, converting GA1 to GA8, GA20 to GA29, and GA9 to GA51. NoGA2ox3 catalyzed a further oxidation leading to GA29- and GA51-catabolite. Ectopic expression of NoGA2ox3 in tobacco reduced the level of active GA1 in transgenic plants, and independent transgenic lines displayed a range of dwarf phenotypes correlated with the level of transgene expression. These results define the physiological context of the control of GA metabolism in oleander, and they provide molecular tools for the biotechnological modification of GA metabolism in this species.

Silencing C19-GA 2-oxidases induces parthenocarpic development and inhibits lateral branching in tomato plants

Highlight GA 2-oxidases regulate gibberellin levels in ovaries and axillary buds of tomato plants and their silencing is responsible for parthenocarpic fruit growth and branching inhibition.

Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene beta-cyclase (Dclcyb1) expression.

Highlight The expression of Dclcyb1 in tobacco induces a positive feedback on the isoprenoid, carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness, biomass, photosynthetic efficiency and carotenoid/chlorophyll composition.

microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA-based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and -2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159-overexpressing tomato cv. Micro-Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159-overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA-like genes and the miR167/SlARF8a module. Similarly to SlMIR159-overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159-targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.

Gibberellins negatively modulate ovule number in plants

ABSTRACT Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed. Summary: The first demonstration that gibberellins are involved in the determination of ovule number in plants, via the activity of DELLA proteins – negative regulators of gibberellin signaling.