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, 135 (4), 2207-19

Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes

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Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes

Fabio Fornara et al. Plant Physiol.

Abstract

MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function.

Figures

Figure 1.
Figure 1.
Sequence alignment of AP1 and FUL of Arabidopsis; ZAP1 of maize; and OsMADS18, OsMADS14, OsMADS15, and OsMADS20 of rice. Black boxes indicate fully conserved residues, shaded boxes indicate similar and partially conserved residues. The MADS box region spans amino acids 1 through 58, the I region spans amino acids 61 through 93, and the K box spans amino acids 96 through 161.
Figure 2.
Figure 2.
OsMADS18 expression in leaves. Northern-blot analysis using wild-type leaves at different developmental stages. 2w, Two weeks after germination; 3w, 3 weeks after germination; 4w, 4 weeks after germination; F, flowering time.
Figure 3.
Figure 3.
OsMADS18 expression analysis in vegetative and reproductive tissues by in situ hybridization. A, Axillary bud meristem 30 d after germination with meristematic leaf primordia. B, Adventitious root primordium protruding from the culm cortex 30 d after germination. C, Developing panicle at the early stage of secondary rachis-branch primordia differentiation. D, Close up of a flower primordium 50 d after germination. The hybridization signal is present in the meristematic domes of the flower and in the procambium forming the vascular bundle. v, Vegetative meristem; l, leaf primordium; s, secondary branch primordium; f, flower primordium; pc, procambium. A and B are bright field pictures, C and D are dark field pictures. Bars represent 100 μm in A and C, bars represent 50 μm in B and D.
Figure 4.
Figure 4.
Expression analysis on OsMADS18 RNAi primary transformants. Total RNA was extracted from leaves of regenerated plants and used for northern-blot analysis. Hybridization was done using a probe specific for OsMADS18. Each lane represents an independent transformant. p1E and p4D are samples taken from two independent plants transformed with the empty vector. RNA quality and equal loading was checked by ethidium bromide staining (lower section).
Figure 5.
Figure 5.
Analysis of 35S:OsMADS18 plants. A, Transgenic plants overexpressing OsMADS18 (a and c) flower earlier compared to wild type (b). The arrows indicate the emerging inflorescences of the transgenic plants. B and C, Stereomicroscope images of a wild-type (B) and 35S:OsMADS18 seedling (C) 5 d after germination. The leaves of the transgenic plant are enclosed in the coleoptile (C), whereas hypocotyl elongation and leaf expansion have already occurred in the wild type (B). Bars represent 1 mm. D, Mean length of adventitious roots (first row), and mean length of the culm (second row) of wild-type (gray columns) and 35S:OsMADS18 lines (black columns) after 7, 10, 15, 20, 25, and 30 d from germination. Bars indicate the ses of the means.
Figure 6.
Figure 6.
Histological analysis of 35S:OsMADS18 transgenic plants (A, D, F, and H) and of wild-type plants (B, C, E, G, and I) at various days from germination. A to C, H, and I are transverse sections, D to G are longitudinal sections. A and B, Axillary bud (arrow) differentiated in 35S:OSMADS18 lines (A) and not in the wild-type plants (B) after 7 d (sections at the same distance from the shoot apex). The differentiation of aerenchyma (a) is more precocious in transgenic than in wild-type plants. C, The axillary bud (arrow) is present in the wild type after 15 d. D and E (day 30), Internodes are shorter in the 35S:OSMADS18 lines (D) compared to the wild type (E). The arrows show the meristematic regions of the nodes. F and G, Close-up pictures of D and E, showing the shoot region with two apical nodes at higher magnification. H and I, Comparison between the adventitious roots of transgenic plants (H) and wild type (I). Root cortex aerenchyma (a) is more developed in transgenic plants. (Bars represent 100 μm in A–E and G; bars represent 50 μm in F and H–I).
Figure 7.
Figure 7.
OsMADS18 overexpression in Arabidopsis. A, Wild-type flower. B, Weakly affected flower showing reduction in the size of petals and sepals. C, Weakly affected flower in which normal petals develop and sepals are converted into leaf-like structures that differentiate stellate trichomes (arrow). D and E, Strongly affected flowers that develop a new flower at the axil of a first whorl organ. F, Severe flower phenotype in which first whorl organs develop carpelloid characteristics. Stigmatic papillae are evident at the tip of the organs and ovules develop along their margins. G, Tertiary and quaternary flowers arise at the axil of the first whorl organs in most affected flowers. H, An ap1-10 mutant flower.
Figure 8.
Figure 8.
RT-PCR on leaves of wild-type and transgenic 35S:OsMADS18 plants. 1, OsMADS18 overexpressing line showing no visible phenotype. 2 to 4, OsMADS18 overexpressing lines showing flower phenotypes described in Figure 7D, F, and G, respectively. +, positive control.
Figure 9.
Figure 9.
In vitro binding of OsMADS18 and OsMADS24. OsMADS18 was produced in E. coli as a TRX fusion protein, and OsMADS24 was translated in vitro in the presence of 35S-Met. OsMADS18 and OsMADS24 were mixed and loaded on a G protein anti-TRX antibody column. The flow-through was collected and the column was washed several times. A control experiment was performed without adding OsMADS18. The protein G-protein complex was separated by SDS-PAGE and 35S-Met labeled OsMADS24 was detected by autoradiography. A, L, 35S-Met labeled OsMADS24 in vitro translation product. B1, Protein G protein complex. FT1, Flow-through fraction. B, Control column. B2, Protein G protein complex. FT2, Flow-through fraction.

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