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Plant Signal Behav. Jul 2008; 3(7): 471–474.
PMCID: PMC2634433

A possible working mechanism for rice SVP-group MADS-box proteins as negative regulators of brassinosteroid responses

Abstract

Most SVP-group MADS-box genes control meristem identity and flowering time. In rice, their roles in regulating the former are well-conserved; however, their involvement in determining the latter is not significant. Characteristically, rice SVP-group MADS-box proteins work as negative regulators in brassinosteroid (BR) responses. To elucidate the molecular mechanism, we studied the localization patterns for these proteins and found that, unexpectedly, they were not specific in the nucleus, whether expressed alone or simultaneously. Interestingly however, OsMADS22 and OsMADS47 were translocated into the nucleus along with OsMADS50 while OsMADS55 inhibited the nuclear localization of OsMADS50. An overlapping cis-element exists between the CArG motif and ARF binding site on the promoter region of OsBLE, which is upregulated by BR treatment and in SVP RNAi plants. These observations suggest that BR-mediated signals may induce target gene expression by removing the SVP-group MADS-box proteins that preoccupy the promoters of BR downstream genes.

Key words: brassinosteroid, BR, MADS, OsMADS22, OsMADS55, rice, SVP, localization

The MADS-box gene family encodes transcription factors with a conserved DNA-binding domain called the MADS-box. Short Vegetative Phase (SVP)-group MADS-box genes work not only as flowering regulators, but also as modulators of meristem identity in various dicot species, including Arabidopsis,16 tomato7 and Antirrhinum.8 Instead, such genes from monocots mainly regulate meristem identity,9,10 although a role in flowering-time control has been suggested in wheat.11,12 Three SVP-group MADS-box genes (OsMADS22, OsMADS47 and OsMADS55) occur in rice. However, flowering time is not changed significantly in single, double, or triple RNAi plants.13

A negative role for OsMADS47 in brassinosteroid (BR) responses has been reported based on certain phenotypes, e.g., reduced root development, enhanced coleoptile growth and increased lamina joint (LJ) inclination in anti-sense plants.14 Analyses of two other SVP-group MADS-box genes have further shown that OsMADS55 works as a major negative regulator and OsMADS22 functions to support OsMADS55.13 For example, whereas single-OsMADS55 RNAi plants display weak BR responses in their lamina joints, OsMADS22-OsMADS55 double and OsMADS22-OsMADS47-OsMADS55 triple RNAi plants manifest dramatic BR responses with regard to LJ inclination, coleoptile elongation and senescence. Stem elongation is also notably reduced in the double and triple RNAi plants, probably because of BR oversensitivity.

Although the roles for three SVP-group MADS-box genes have been elucidated well at the physiological level, detailed molecular mechanisms for repressing BR responses are still unrevealed. They have been deemed not to regulate BR biosynthesis based on the expression levels of Dwarf1 (BRD1) and Ebisu Dwarf (Dwarf2 or D2),15,16 which are not changed significantly in the double/triple RNAi plants.13 Moreover, their expression levels are not altered in transgenic plants that overexpress OsMADS22 and OsMADS55. Therefore, these SVP genes are not likely to be downstream genes of the well-known BR signaling pathway because their expression is not affected by brassinosteroid treatments. Instead, we speculate that SVP proteins may act as endogenous repressors of BR target genes.

OsBLE3, a BR-inducible gene,17 is upregulated in RNAi plants showing BR oversensitivity. Interestingly, the promoter region of OsBLE3 contains three CArG motifs that are putative MADS-box binding sites (Fig. 1, underlined).18 That region also comprises three auxin-responsive elements termed ARF (auxin response factor) binding sites (Fig. 1, boxed).18 ARF binding sites exist on the promoters of genes regulated by both auxin and BR.19,20 Interestingly, one of the CArG motifs overlaps with one of those ARF binding sites (Fig. 1, arrowhead). Therefore, it is possible that AFR and MADS-box proteins compete with each other at the common target site. Hence, to satisfy this assumption, SVP-group MADS-box proteins should be localized in the nucleus.

Figure 1
Promoter region of OsBLE3. CArG motifs are underlined and ARF binding sites are boxed. Arrowhead indicates an overlapping base pair between CArG motif and ARF binding site. Start codon ATG is in bold font.

To test this possibility, we made eGFP- or DsRed-fused constructs at the C-terminal of the SVP MADS-box genes. The molecules were introduced into onion epidermal cells by particle bombardment. Contrary to our expectation, none of the three SVP-group MADS-box proteins showed preferential nuclear localization patterns (Fig. 2A–I). In fact, OsMADS22:eGFP and OsMADS55:eGFP exhibited patterns similar to those of the DsRed controls. GFP and RFP signals were observed in both nucleus and cytosol while being almost absent in the large vacuole. Although the OsMADS22:eGFP signal seemed to be stronger in the nucleus (Fig. 2A), this could, from a statistical viewpoint, be attributed more to the strong expression level and large proportion of nuclear portions in the onion cell compared with the cytosol, rather than because of any preferential localization pattern in the nucleus. By comparison, the OsMADS47:eGFP signal was preferentially localized in the cytosol and on the periphery of the nucleus (Fig. 2D and F). To further confirm our observations, we transformed these fusion constructs into rice protoplasts, and found OsMADS22:eGFP and OsMADS55:eGFP signals in both cytosol and nucleus (Fig. 3A–H).

Figure 2
Localization patterns of SVP-group MADS-box proteins in onion cells. Distributions of eGFP-fused OsMADS22 (A–C), OsMADS47 (D–F) and OsMADS55 (G–I) proteins are represented along with co-expressed DsRed signals. OsMADS50 was co-expressed ...
Figure 3
Localization patterns of SVP-group MADS-box proteins in rice protoplasts. Distributions of eGFP-fused OsMADS22 (A–D) and OsMADS55 (E–H) are represented along with co-expressed RFP proteins containing nuclear localization signal (NLS). ...

Because MADS-box proteins form multimers for nuclear localization,21,22 two SVP-group MADS-box proteins were simultaneously expressed. However, none of our pairings (OsMADS22-OsMADS47, OsMADS22-OsMADS55, OsMADS47-OsMADS55 or OsMADS22-OsMADS47-OsMADS55) affected the localization patterns of any protein (data not shown). Therefore, we examined the MADS-box genes of other groups that showed overlapping expression with those in the SVP group. OsMADS50, a flowering activator,23 was able to recruit OsMADS22:eGFP into the nucleus (Figs. 2J–K and 3I–K). Moreover, the GFP-fusion construct of OsMADS50 demonstrated that this protein alone could be located in the nucleus (data not shown). OsMADS47:eGFP was also translocated into the nucleus with OsMADS50 (data not shown); however, the localization pattern for OsMADS55:DsRed was not changed by OsMADS50:eGFP expression. Rather, OsMADS50:eGFP did not go into the nucleus by being trapped by OsMADS55:DsRed (Fig. 3L–N) 2.

Considering the similar roles and structures of the SVP-group MADS-box genes, the above results were unexpected. However, these phenomena could explain why OsMADS22 and OsMADS55 were not completely redundant. Whereas transgenic plants overexpressing OsMADS22 (22OX) had shorter stems, those of the OsMADS55 overexpressing plants (55OX) were longer.13 We also noted that, although the degree of LJ inclination for flag leaves was reduced in 22OX plants, it was increased in some of the 55OX plants. Therefore, OsMADS55 overexpression might have caused the depletion of OsMADS50 in the nucleus, following the removal of OsMADS22 and OsMADS47. This would then lead to enhanced BR responses, e.g., stem elongation and increased laminar joint angles. However, further confirmation is needed because our OsMADS55 RNAi plants did not show any change in such responses. Thus, OsMADS55 may require another protein(s) for translocation into the nucleus. In fact, the nuclear localization of OsMADS22 and OsMADS47 along with OsMADS50 may support our hypothesis that SVP-group proteins repress brassinosteroid responses by binding to the promoters of BR target genes.

Acknowledgements

We thank Hong Gil Nam and Inhwan Hwang at POSTCH for providing valuable localization vectors and Priscilla Licht for critical reading of the manuscript. This work was supported, in part, by grants from the Crop Functional Genomic Center, the 21st Century Frontier Program (Grant CG1111); from the Biogreen 21 Program, Rural Development Administration (20070401-034-001-007-03-00); and from the Korea Science and Engineering Foundation (KOSEF) through the National Research Laboratory Program funded by the Ministry of Science and Technology (M1060000027006J0000-27010).

Footnotes

Present address: Department of Biochemistry; Purdue University; West Lafayette, Indiana

Present address: Department of Plant and Soil Sciences and Delaware Biotechnology Institute; University of Delaware; Newark, Delaware USA

Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/article/5676

References

1. Hartmann U, Hohmann S, Nettesheim K, Wisman E, Saedler H, Huijser P. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J. 2000;21:351–360. [PubMed]
2. Yu H, Xu Y, Tan EL, Kumar PP. AGAMOUS-LIKE 24, a dosage-dependent mediator of the flowering signals. Proc Natl Acad Sci USA. 2002;99:16336–16341. [PMC free article] [PubMed]
3. Michaels SD, Ditta G, Gustafson Brown C, Pelaz S, Yanofsky M, Amasino RM. AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. Plant J. 2003;33:867–874. [PubMed]
4. Yu H, Ito T, Wellmer F, Meyerowitz EM. Repression of AGAMOUS-LIKE 24 is a crucial step in promoting flower development. Nat Genet. 2004;36:157–161. [PubMed]
5. Gregis V, Sessa A, Colombo L, Kater MM. AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis. Plant Cell. 2006;18:1373–1382. [PMC free article] [PubMed]
6. Lee JH, Yoo SJ, Park SH, Hwang I, Lee JS, Ahn JH. Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Genes Dev. 2007;21:397–402. [PMC free article] [PubMed]
7. Szymkowiak EJ, Irish EE. JOINTLESS suppresses sympodial identity in inflorescence meristems of tomato. Planta. 2006;223:646–658. [PubMed]
8. Masiero S, Li MA, Will I, Hartmann U, Saedler H, Huijser P, Schwarz-Sommer Z, Sommer H. INCOMPOSITA: a MADS-box gene controlling prophyll development and floral meristem identity in Antirrhinum. Development. 2004;131:5981–5990. [PubMed]
9. Sentoku N, Kato H, Kitano H, Imai R. OsMADS22, an STMADS11-like MADS-box gene of rice, is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy. Mol Genet Genom. 2005;273:1–9. [PubMed]
10. Trevaskis B, Tadege M, Hemming MN, Peacock WJ, Dennis ES, Sheldon C. Short vegetative phase-like MADS-box genes inhibit floral meristem identity in barley. Plant Physiol. 2007;143:225–235. [PMC free article] [PubMed]
11. Kane NA, Danyluk J, Tardif G, Ouellet F, Laliberte JF, Limin AE, Fowler DB, Sarhan F. TaVRT-2, a member of the StMADS-11 clade of flowering repressors, is regulated by vernalization and photoperiod in wheat. Plant Physiol. 2005;138:2354–2363. [PMC free article] [PubMed]
12. Kane NA, Agharbaoui Z, Diallo AO, Adam H, Tominaga Y, Ouellet F, Sarhan F. TaVRT2 represses transcription of the wheat vernalization gene TaVRN1. Plant J. 2007;51:670–680. [PubMed]
13. Lee S, Choi SC, An G. Rice SVP-group MADS-box proteins, OsMADS22 and OsMADS55, are negative regulators of brassinosteroid responses. Plant J. 2008 (in press) [PubMed]
14. Duan K, Li L, Hu P, Xu SP, Xu ZH, Xue HW. A brassinolide-suppressed rice MADS-box transcription factor, OsMDP1, has a negative regulatory role in BR signaling. Plant J. 2006;47:519–531. [PubMed]
15. Hong Z, Ueguchi-Tanaka M, Shimizu-Sato S, Inukai Y, Fujioka S, Shimada Y, Takatsuto S, Agetsuma M, Yoshida S, Watanabe Y, Uozu S, Kitano H, Ashikari M, Matsuoka M. Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem. Plant J. 2002;32:495–508. [PubMed]
16. Hong Z, Ueguchi-Tanaka M, Umemura K, Uozu S, Fujioka S, Takatsuto S, Yoshida S, Ashikari M, Kitano H, Matsuoka M. A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450. Plant Cell. 2003;15:2900–2910. [PMC free article] [PubMed]
17. Yang G, Nakamura H, Ichikawa H, Kitano H, Komatsu S. OsBLE3, a brassinolideenhanced gene, is involved in the growth of rice. Phytochemistry. 2006;67:1442–1454. [PubMed]
18. Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 1999;27:297–300. [PMC free article] [PubMed]
19. Goda H, Sawa S, Asami T, Fujioka S, Shimada Y, Yoshida S. Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis. Plant Physiol. 2004;134:1555–1573. [PMC free article] [PubMed]
20. Nemhauser JL, Mockler TC, Chory J. Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol. 2004;2:258. [PMC free article] [PubMed]
21. Immink RG, Gadella TW, Jr, Ferrario S, Busscher M, Angenent GC. Analysis of MADS box protein-protein interactions in living plant cells. Proc Natl Acad Sci USA. 2002;99:2416–2421. [PMC free article] [PubMed]
22. Tonaco IA, Borst JW, de Vries SC, Angenent GC, Immink RG. In vivo imaging of MADS-box transcription factor interactions. J Exp Bot. 2006;57:33–42. [PubMed]
23. Lee S, Kim J, Han JJ, Han MJ, An G. Functional analyses of the flowering time gene OsMADS50, the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog in rice. Plant J. 2004;38:754–764. [PubMed]

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