The rice StMADS11-like genes OsMADS22 and OsMADS47 cause floral reversions in Arabidopsis without complementing the svp and agl24 mutants

Abstract

During floral induction and flower development plants undergo delicate phase changes which are under tight molecular control. MADS-box transcription factors have been shown to play pivotal roles during these transition phases. SHORT VEGETATIVE PHASE (SVP) and AGAMOUS LIKE 24 (AGL24) are important regulators both during the transition to flowering and during flower development. During vegetative growth they exert opposite roles on floral transition, acting as repressor and promoter of flowering, respectively. Later during flower development they act redundantly as negative regulators of AG expression. In rice, the orthologues of SVP and AGL24 are OsMADS22, OsMADS47, and OsMADS55 and these three genes are involved in the negative regulation of brassinosteroid responses. In order to understand whether these rice genes have maintained the ability to function as regulators of flowering time in Arabidopsis, complementation tests were performed by expressing OsMADS22 and OsMADS47 in the svp and agl24 mutants. The results show that the rice genes are not able to complement the flowering-time phenotype of the Arabidopsis mutants, indicating that they are biologically inactive in Arabidopsis. Nevertheless, they cause floral reversions, which mimic the SVP and AGL24 floral overexpressor phenotypes. Yeast two-hybrid analysis suggests that these floral phenotypes are probably the consequence of protein interactions between OsMADS22 and OsMADS47 and other MADS-box proteins which interfere with the formation of complexes required for normal flower development.

Fig. 1.

Sequence alignment of MADS-box proteins belonging to the StMADS11 subclade. Sequences of SVP and AGL24 of Arabidopsis, OsMADS22, OsMADS47, and OsMADS55 of rice, BM1 and BM10 of barley, and StMADS11 and StMADS16 of potato were used. Black boxes indicate fully conserved residues; shaded boxes indicate similar and partially conserved residues. The MADS-box region spans amino acids 1 to 58.

Fig. 2.

Expression of OsMADS47 and OsMADS22 in rice. (A) Northern blot hybridized using an OsMADS47 specific probe. The RNA samples are derived from wild-type Nipponbare roots (R), mature leaves (L), inflorescences (I), and developing kernels (DK). (B, C) In situ hybridizations using wild-type Nipponbare apical sections probed with an OsMADS22 antisense probe. (B) Expression in a vegetative shoot apical meristem (sam). (C) Expression in an early inflorescence meristem (pm, panicle shoot meristem). The signal is detected throughout the shoot apex and in primary rachis–branch meristems (arrows).

Fig. 3.

Northern blot analysis of plants expressing 35S::OsMADS22 in wt Col-0 and the svp and agl24 mutants. The RNA samples were prepared from young leaves of four independent T₁ transgenic plants for each genotype and tested for the expression level of OsMADS22. Numbers on the top of each lane indicate the transgenic line number. Note the variability in expression level, ranging from plants that do not express OsMADS22 to plants that express it at a very high level. Only plants expressing the transgene at a high level were used in subsequent analyses.

Fig. 4.

Flowering time of Arabidopsis plants overexpressing OsMADS22 and OsMADS47 in wt Col-0, svp-41, and agl24-2. Two independent transgenic lines were tested for each construct. Flowering was determined as the rosette leaf number at bolting of at least 12 individuals per genotype. Error bars indicate the standard deviation from the mean.

Fig. 5.

Morphological analysis of OsMADS22 and OsMADS47 expressing plants. (A, B) Wild-type Columbia inflorescence and flower at anthesis. (C, F) 35S::OsMADS22 (C) and 35S::OsMADS47 (F) inflorescences. (D, G) Flowers of 35S::OsMADS22 (D) and 35S::OsMADS47 (G) at anthesis. Note the abundance of trichomes on sepals and the presence of an ectopic flower inside the 35S::OsMADS47 flower (indicated with an arrow). A sepal was removed in (G) to visualize the inner organs better. (H) A mature 35S::OsMADS47 flower producing extra flowers from the axil of leaf-like sepals. The primary flower is indicated by an arrow. Extra flowers are developing on long pedicels and secondary flowers reiterate the pattern shown by the primary one. (E, I) Mature inflorescences of 35S::OsMADS22 (E) and 35S::OsMADS47 (I), respectively. The sepals converted into leaves are retained on the flower after fertilization. (J–L) SEM analysis on the epidermis of a wild-type sepal (J), a wild-type cauline leaf (K) and a 35S::OsMADS22 sepal (L), respectively. The epidermis of 35S::OsMADS22 sepals resembles that of wild-type leaves (bars = 50 μm). (M) Graph showing the maturation of the siliques of 35S::OsMADS22 and 35S::OsMADS47 as determined by the number of days from anthesis to the beginning of senescence of the valves (mean ±standard deviation).

Fig. 6.

In situ hybridizations on sections of wild-type Columbia (A) and 35S::OsMADS47 (B) inflorescence sections probed with AP1. Numbers refer to the stage of flower development according to Smyth et al. (1990). IM, inflorescence meristem. Bar 50 μm.