AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

Abstract

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.

Figure 1.

GA-dependent trichome formation on mutant seedlings and P_GL1∷GUS expression in mutant embryos. In A, immature seeds of single mutants at 11 to 13 DAP were isolated, sown in petri dishes on Murashige and Skoog medium, and grown for 10 d apart from wild type that was germinated from dry seeds. Immature embryos 11 to 13 DAP of double mutants and ga1-3 were dissected out of immature seeds, allowed to grow like single mutants, and the trichomes of 20 plants were counted for each genotype. Section B shows the GUS activity of the P_GL1∷GUS construct in wild-type, lec2-1, and fus3-8 mutant embryos. Seeds were removed from immature siliques at 11 to 13 DAP, incubated with X-gluc, and embryos were dissected out of seeds.

Figure 2.

The major GA biosynthesis pathway in Arabidopsis and expression of four well-known biosynthetic genes in wild-type, lec2-1, and fus3-8 mutant siliques. Section A describes the main steps of GA biosynthesis. The first committed step in the GA pathway is the biosynthesis of ent-kaurene by AtCPS and AtKS (not represented). AtGA20oxidases catalyze the biosynthesis of inactive substrates GA₉ and GA₂₀ (C₁₉-GAs) from GA₁₂ molecule (C₂₀-GA). AtGA3ox1 and AtGA3ox2 catalyze the biosynthesis of the two bioactive forms in Arabidopsis, GA₁, and GA₄. Multiple arrows indicate multiple biosynthesis steps while single arrows indicate direct synthesis. Section B shows the transcript levels of AtCPS, AtGA20ox1, AtGA3ox1, and AtGA3ox2 in wild-type (white bars), lec2-1 (black bars), and fus3-8 (dashed bars) mutants. Real-time RT-PCR was performed from total RNAs isolated from 10 to 11 DAP siliques. Because AtGA20ox1 is weakly expressed in all backgrounds, the threshold cycle was close to the ultimate cycle (Ct close to 40) in all genetic backgrounds. We considered the putative activation of AtGA20ox1 not reliable in these experimental conditions. Error bars represent sd. Three independent replicates were done from three set of seeds collected independently.

Figure 3.

Expression patterns of three biosynthetic genes in wild-type, lec2-1, and fus3-8 mutant embryos. Section A shows the GUS reporter gene expression patterns in wild-type, lec2-1, and fus3-8 mutants of (first row) AtGA3ox2 in heart-stage or torpedo-stage embryos, (second row) AtGA3ox2 in mature embryos, (third row) AtCPS in mature embryos, and (fourth row) AtGA20ox1 in mature embryos. Section B shows the GUS reporter gene and in situ hybridization of AtGA3ox2 expression in sections of fus3-8 mutant embryos. Two upper images, longitudinal section (top image), and transverse section of the embryo axis (bottom image) of P_AtGA3ox2∷GUS fus3-8 embryos. Arrows on the transverse section indicate staining in epidermal cells and vascular tissues. Two lower images, transverse sections of fus3-8 embryo axis hybridized with the antisense (top image) and sense (bottom image) AtGA3ox2 probe. Arrows on the top image indicate hybridization signal in epidermal cells and vascular tissues.

Figure 4.

Direct interaction of the FUS3 protein with the two RY elements present in the AtGA3ox2 promoter. Section A is a schematic representation of the two RY sites in the AtGA3ox2 promoter. The sequence of the two wild-type sites (RY₁ and RY₂) is indicated together with the two mutated versions (mRY₁ and mRY₂). Section B shows an electrophoretic mobility shift assay experiment with RY₁ site. A radiolabeled probe carrying the RY₁ site (TGCATGCATG) was incubated with the FUS3 protein present in bacterial extracts. Lane 1, radiolabeled probe only; lane 2, rabiolabeled probe incubated with FUS3 extracts; lanes 3 and 4, radiolabeled probe incubated with FUS3 extracts after incubation with an excess of 100- and 500-fold, respectively, of unlabeled probe; lanes 5 and 6, same as in lanes 3 and 4 except that the unlabeled probe carried a mutated RY₁ site (mRY₁). Section C is similar to section B except that the radiolabeled probe (lane 1) carried the RY₂ site (CATGCATG). The probe is retarded by FUS3 extracts (lane 2) and competition experiments were performed with an unlabeled probe carrying the native (lanes 3 and 4) or mutated (lanes 5 and 6) RY₂ site (mRY₂). The arrows indicate FUS3-RY complexes.