Genetic analyses of interactions among gibberellin, abscisic acid, and brassinosteroids in the control of flowering time in Arabidopsis thaliana

Abstract

Background: Genetic interactions between phytohormones in the control of flowering time in Arabidopsis thaliana have not been extensively studied. Three phytohormones have been individually connected to the floral-timing program. The inductive function of gibberellins (GAs) is the most documented. Abscisic acid (ABA) has been demonstrated to delay flowering. Finally, the promotive role of brassinosteroids (BRs) has been established. It has been reported that for many physiological processes, hormone pathways interact to ensure an appropriate biological response.

Methodology: We tested possible genetic interactions between GA-, ABA-, and BR-dependent pathways in the control of the transition to flowering. For this, single and double mutants deficient in the biosynthesis of GAs, ABA, and BRs were used to assess the effect of hormone deficiency on the timing of floral transition. Also, plants that over-express genes encoding rate-limiting enzymes in each biosynthetic pathway were generated and the flowering time of these lines was investigated.

Conclusions: Loss-of-function studies revealed a complex relationship between GAs and ABA, and between ABA and BRs, and suggested a cross-regulatory relation between GAs to BRs. Gain-of-function studies revealed that GAs were clearly limiting in their sufficiency of action, whereas increases in BRs and ABA led to a more modest phenotypic effect on floral timing. We conclude from our genetic tests that the effects of GA, ABA, and BR on timing of floral induction are only in partially coordinated action.

Figure 1. Simplified hormone biosynthetic pathways.

The hormone biosynthetic pathways of Arabidopsis for gibberellins A., ABA B., and, brassinolide C.. The biosynthesis mutants used in this study and sites of their lesions are shown. Also, the biosynthetic genes over-expressed to increase the levels of respective hormones are indicated. A. The ga1 mutant is impaired in the first stage of GA-biosynthesis: the cyclization of geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP). B. The aba2 mutant is blocked at the cis-xanthoxin to ABA-aldehyde conversion. C. The conversion of 6-Deoxocathasterone/Cathasterone to 6- Deoxoteasterone/teasterone does not occur in the cpd mutant. A. The GA5 gene encodes a GA 20-oxidase that catalyzes the formation of the GA20 and GA9, the final precursors of the bioactive GAs. B. The NCED3 encodes 9-cis-epoxycarotenoid dioxygenase that catalyzes the oxidative cleavage of a 9-cis isomer of epoxycarotenoid (9-cis-violaxanthin or 9’-cis-neoxanthin) to form xanthoxin. C. The DWF4 gene encodes a 22-a hydroxylase (CYP90B1) that catalyzes the conversion of 6- Oxocampestanol/Campestanol to 6-Deoxocathasterone/Cathasterone. IPP, Isopentenyl pyrophosphate. ABA, abscisic acid. Adapted from .

Figure 2. Floral-timing phenotypes of phytohormone mutants.

A. Floral-timing phenotypes of the wild-type WS, the single aba2, ga1, and cpd mutants and the ga1 aba2, ga1 cpd, and cpd aba2 double mutants. Plants were grown under long days (16 h light/8 h darkness) in controlled greenhouse conditions. Pictures were taken when wild-type plants were flowering. B. Flowering-time analyses of the wild-type WS, the single aba2, ga1, and cpd mutants and the ga1 aba2, ga1 cpd, and the cpd aba2 double mutants. Plants were grown under long days (16 h light/8 h darkness) in the greenhouse. C. As in B., except plants were grown under short days (8 h light/16 h darkness) in the greenhouse. Flowering time was measured as rosette leaf number at bolting for B. or days to flowering for C. Around 12 plants were scored per genotype. The hatched bars denote genotypes that did not flower over duration of measurement. Error bars represent SE. Two experiments were performed, and a representative result is shown.

Figure 3. Overexpression lines for rate-limiting enzymes in various phytohormone pathways.

Transgenic lines harboring 35S::DWF4, 35S::GA5 and 35S::NCED3 constructs. A. Over-expression was confirmed by RT-PCR with primers specific for DWF4, GA5 and NCED3. Primers specific for the elongation factor 1-alpha gene were used as a control. Representative lines are shown. All lines tested showed over-expression of the gene of interest >3 fold. B. Images of 3-weeks-old plants grown under long days (16 h light/8 h darkness) in the greenhouse. The white bar indicates 1 cm.

Figure 4. Floral-timing phenotypes of phytohormone overexpression lines.

Flowering time of the transgenic lines that over-express GA-, BR- and ABA-biosynthetic genes: GA5, DWF4 and NCED3, respectively. A. Long-day conditions (16 h light/8 h darkness). B. Short-day conditions (8 h light/16 h darkness). Flowering time was measured as rosette leaf number at bolting. Around 12 plants were scored per genotype. Error bars represent SE. Student's t-test was applied to test for the differences in flowering time, relative to the wild type, P<0.0001***, P<0.05*.