SHORT VEGETATIVE PHASE reduces gibberellin biosynthesis at the Arabidopsis shoot apex to regulate the floral transition

Abstract

In Arabidopsis thaliana environmental and endogenous cues promote flowering by activating expression of a small number of integrator genes. The MADS box transcription factor SHORT VEGETATIVE PHASE (SVP) is a critical inhibitor of flowering that directly represses transcription of these genes. However, we show by genetic analysis that the effect of SVP cannot be fully explained by repressing known floral integrator genes. To identify additional SVP functions, we analyzed genome-wide transcriptome data and show that GIBBERELLIN 20 OXIDASE 2, which encodes an enzyme required for biosynthesis of the growth regulator gibberellin (GA), is upregulated in svp mutants. GA is known to promote flowering, and we find that svp mutants contain elevated levels of GA that correlate with GA-related phenotypes such as early flowering and organ elongation. The ga20ox2 mutation suppresses the elevated GA levels and partially suppresses the growth and early flowering phenotypes of svp mutants. In wild-type plants, SVP expression in the shoot apical meristem falls when plants are exposed to photoperiods that induce flowering, and this correlates with increased expression of GA20ox2. Mutations that impair the photoperiodic flowering pathway prevent this downregulation of SVP and the strong increase in expression of GA20ox2. We conclude that SVP delays flowering by repressing GA biosynthesis as well as integrator gene expression and that, in response to inductive photoperiods, repression of SVP contributes to the rise in GA at the shoot apex, promoting rapid induction of flowering.

Fig. 1.

The svp-41 mutation accelerates flowering in the absence of functional FT TSF SOC1 FUL genes. (A) Leaf number at flowering of plants grown under LD condition. Data are mean ± SD of at least 10 individual plants. (B) Phenotypes of the quadruple ft-10 tsf-1 soc1-2 ful-2 and of the quintuple svp-41 ft-10 tsf-1 soc1-2 mutant plants around 60 d after germination growing under LDs. See also Fig. S1.

Fig. 2.

SVP reduces GA content through the transcriptional repression of GA20ox2. (A) GA-related genes differentially expressed in svp-41 mutant compared with wild-type plants according to the microarray experiments described (23). (B) Phenotype of seedlings of wild-type and svp-41 mutant (Upper) and ga20ox2-1 mutant and svp-41 ga20ox2-1 double mutants (Lower). Bar = 10 mm. (C) Flowering time and (D) chlorophyll content measurement of wild-type, svp-41, and 35S::SVP plants after treatments with GA₄ (light bars) or mock (dark bars). All plants in A–D were grown under SDs. n = 10–12. (E) Schematic representation of the non–13-hydroxylated GA-biosynthetic pathway in Arabidopsis (adapted from Yamaguchi, ref. 32) ⁽¹⁾GA2ox7 and -8; ⁽²⁾GA2ox1, -2, -3, -4, and -6. (F) Concentration of GAs in aerial part of seedlings grown for 2 wk under SDs. The values are the mean ± SEM of three biological replicates (ng/g fresh weight). Letters shared in common between the genotypes indicate no significant difference in GA concentration (pairwise multiple comparison procedures, Student–Newman–Keuls method, P < 0.05). *Two biological replicates. See also Fig. S2 and Table S1.

Fig. 3.

SVP regulates flowering time through transcriptional regulation of GA20ox2. (A) Flowering time of wild-type plants compared with ga20ox2-1 (Left) and svp-41 compared with svp-41 ga20ox2-1 plants (Right) grown under SDs. The numbers in parentheses indicate the differences in flowering time expressed as a percentage. The ANOVA analysis showed that this difference is statistically significant (Holm–Sidak test, P = 0.003). (B) GA20ox2 mRNA levels in 2-wk-old seedlings of ft-10 tsf-1 and soc1-2 ful-2 in the presence or absence of SVP. Wild-type and svp-41 plants were used as controls. Samples were collected 8 h after dawn under SDs. (C) Effect of GA₄ treatment on flowering phenotype of svp-41, ft-10 tsf-1 soc1-2 ful-2, and svp-41 ft-10 tsf-1 soc1-2 ful-2 mutants growing under LDs. Treatment was carried out with at least 10 individual plants, and wild type was used as control. The asterisk indicates that there is a statistically significant difference between the treated and untreated ft-10 tsf-1 soc1-2 ful-2 plants (P = 0.007).

Fig. 4.

SVP controls floral transition and GA20ox2 transcription in the SAM. (A) Levels of GA20ox2 mRNA in apices and leaves of wild-type and svp-41 plants. (B) Effect of the misexpression of SVP in the SAM on flowering time under LDs (Upper) and SDs (Lower). CL: cauline leaves; RL: rosette leaves. (C) Levels of GA20ox2 mRNA in apices of transgenic plants misexpressing SVP compared with WT and svp-41 mutant grown for 2 wk under SDs.

Fig. 5.

Photoperiodic regulation of GA biosynthesis and transcriptional activation of SPLs. (A) Spatial pattern of SVP mRNA detected by in situ hybridization during a time course of ft-10 tsf-1 soc1-2 ful-2 and svp-41 ft-10 tsf-1 soc1-2 ful-2 mutant plants grown for 3 wk in SDs (0 LD) and then transferred to LDs (3, 5, and 7 LDs). A specific probe was used to detect mRNA of SVP at the shoot apex. (B) Temporal expression pattern of GA20ox2 mRNA in apices of wild-type, ft-10 tsf-1, and soc1-2 ful-2 mutant plants grown for 3 wk in SDs (0 LD) and then shifted to LDs (3, 5, and 7 LDs). All samples were harvested 8 h after dawn. (Scale bar: 50 µm.) (C) Histochemical localization of GUS activity at SAM of pGA20ox2::GA20ox2:GUS seedlings harvested at the beginning (8 LDs), during (11 LDs), and after (14 LDs) the transition to flowering. (Scale bar: 100 µm.) (D) Pattern of expression of SPL4 in ft-10 tsf-1 soc1-2 ful-2 and svp-41 ft-10 tsf-1 soc1-2 ful-2 mutant plants grown for 15 (Upper) and 30 LDs (Lower). (Scale bars: 50 µm.) (E) Quantification of the mRNA levels of SPL5 and SPL3 in wild-type, svp-41, ga20ox2-1, and svp-41 ga20ox2-1 seedlings grown for 2 wk under SDs.

Fig. 6.

Proposed model for the activation of GA biosynthesis in the shoot apical meristem during photoperiodic flowering. In plants exposed to LDs, the transcription of FT and TSF is induced in the leaves. The FT protein moves to the SAM (black dashed line) where FD is expressed. The FT FD module is proposed to activate the transcription of downstream floral promoter genes, such as AP1, SOC1, and FUL. SOC1 (and probably also FUL) directly binds to SVP and contributes to its repression. Downregulation of SVP transcription contributes to increased expression of GA20ox2 and higher GA content at the shoot apex. Higher GA levels increase transcription of the SPL genes and release SPL proteins from DELLA repression during photoperiodic flowering.