JXB Advance Access originally published online on August 8, 2003
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Journal of Experimental Botany, Vol. 54, No. 391, pp. 2385-2387,
October 1, 2003
© 2003 Oxford University Press
Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene
Received 3 April 2003; Accepted 1 July 2003
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Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E2, Canada
* Present address: Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada. To whom correspondence should be sent. Fax: +1 306 966 4461. E-mail: bonhamp{at}duke.usask.ca
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Abstract |
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B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.
Key words: Floral development, Brassica napus, BnAP3.
The formation and identity of floral organs in Arabidopsis thaliana is controlled by a well-defined group of transcription factors that function within strictly prescribed domains of the floral meristem (Coen and Meyerowitz, 1991). A subset of this group known as the B-class genes, APETALA3 (AP3) and PISTILLATA (PI), are essential for petal and stamen development (Bowman et al., 1989, 1991, 1993; Hill and Lord, 1989; Jack et al., 1992). Loss of function for AP3 results in sepals in the place of petals and carpels in the place of stamens (Bowman et al., 1989), while the ectopic expression of AP3 results in stamens displacing carpels. Genes with high sequence similarity to AP3, that demonstrate a B-class function, have been identified in a diversity of other plant species (Sommer et al., 1990; Carr and Irish, 1997; Moon et al., 1999; Halfter et al., 1994) suggesting a highly conserved function for AP3-like proteins in plants.
The isolation and characterization of a B. napus cDNA encoding a protein homologous to AP3 of A. thaliana, called BnAP3, are described. It is shown that BnAP3 has a B-class function in B. napus flower development and that BnAP3 complements the ap3-1 mutation in A. thaliana by restoring stamen development.
Screening of a B. napus cDNA library (Bonham-Smith et al., 1992) with a probe derived from A. thaliana AP3 (GeneBank accession number D21125
[GenBank]
) yielded a partial clone, pBAP3-1, which was highly similar to the 3' region of AP3 of A. thaliana (Jack et al., 1992). 5' RACE was used to retrieve the 
250 bp 5' region predicted, by sequence alignment, to be missing from the pBAP3-1 clone. Two different products of 5' RACE were identified: pBAP3 No. 9 and pBAP3 No. 14, providing evidence for at least two transcriptionally active genes of BnAP3. The nucleotides of the overlapping region of pBAP3 No. 9 and pBAP3-1 were identical, and the two clones were combined to yield a full-length clone of BnAP3 (GeneBank accession number AF124814
[GenBank]
). Southern analysis of B. napus genomic DNA probed with BnAP3 DNA lacking the MADS box suggests that up to three copies of the BnAP3 gene occur within the B. napus genome (data not shown). Multiple alleles would be expected since B. napus is an amphidiploid, its genome originating from B. oleracea and B. rapa (U, 1935; Erickson et al., 1983).
The predicted amino acid sequence of BnAP3 shares 98% identity with the B. oleracea AP3, Boi2AP3 (GeneBank accession number U67455 [GenBank] ), 95% with AP3, and 49% with that of OsMADS16 (GeneBank accession number AF077760 [GenBank] ), the AP3 orthologue from the monocot rice (Oryza sativa) (Fig. 1). The MADS box of BnAP3 is identical to that of Boi2AP3, 98% similar to that of AP3, and 77% similar to that of OsMADS16 while the K region of BnAP3 is 97% similar to that of Boi2AP3 and AP3, and 49% similar to that of OsMADS16 (Fig. 1).
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Using RT-PCR, BnAP3 transcripts were detected in floral buds and carpels. These results are consistent with the role of BnAP3 in determining the identity and development of petals and stamens. The presence of a signal in transitional apical meristems collected 28 h following light induction to flowering (TSAMs) suggests that BnAP3 is expressed very early in flower development (Fig. 2). The early onset of BnAP3 expression in flowers is similar to AP3 expression in A. thaliana, which is first expressed during the initiation of sepal primordia (Hill et al., 1998). AP3 transcripts are detected late in ovule development within the integuments of A. thaliana (Jack et al., 1992) and BnAP3 may also be similarly expressed in B. napus, accounting for its presence in the gynoecium. BnAP3 was also detected and confirmed by sequencing, in non-floral tissues, generating a strong signal in roots and a weak signal in bracts (Fig. 2). Neither AP3 (Jack et al., 1992) nor the rice orthologue OsMADS16 (Moon et al., 1999) has been detected in non-floral tissues. These conclusions however, were based on northern analysis, which is not as sensitive as RT-PCR, for detecting transcripts. While it has been previously suggested that the MADS-box genes, AGL21, AG17 and AGL12, may play distinct regulatory roles during A. thaliana root development (Burgeff et al., 2002), whether or not BnAP3 serves a functional role in these tissues remains to be determined.
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Wild-type B. napus, wild-type A. thaliana, and A. thaliana containing the ap3-1 mutation were transformed with the BnAP3 open reading frame under the control of the A. thaliana AGAMOUS (AG) promoter (Fig. 3A). In both A. thaliana (Deyholos and Sieburth, 2000) and B. napus (Pylatuik et al., 2003), the AG promoter restricts expression to the reproductive whorls only. In wild-type A. thaliana (Fig. 3B) and B. napus (Fig. 3C), introduction of the AG::BnAP3 construct resulted in the development of stamen tissue in place of the carpels, while all other parts of the flower were unaffected. In ap3-1 plants, where petals and stamens are replaced with sepals and carpels, respectively, introduction of the AG::BnAP3 construct restored stamen development, but not petal development (Fig. 3B). Furthermore, stamen tissue developed in place of carpels in the fourth whorl. It is clear from these results that BnAP3 has a B-class function in B. napus, and is functionally homologous to AP3 of A. thaliana.
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To summarize, the cloning of a B. napus cDNA encoding a B-class gene which is functionally homologous to the A. thaliana AP3 gene has been reported. Unlike other B-class genes identified, these results suggest that BnAP3 is expressed outside of the floral tissues, namely in the bracts and roots of B. napus plants. Finally, these results show that BnAP3 is involved in stamen development in B. napus, and can restore stamen development in A. thaliana flowers mutant for the AP3 gene.
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Acknowledgements |
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We thank Dr L Sieburth of the University of Utah for providing the AGAMOUS cis-acting control elements and Dennis Dyck for photography. This work was supported by postgraduate scholarships (JDP, DLL) and research grants from the Natural Sciences and Engineering Research Council (PB-S, ARD).
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