JXB Advance Access originally published online on April 11, 2003
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Journal of Experimental Botany, Vol. 54, No. 387, pp. 1637-1639,
June 1, 2003
© 2003 Oxford University Press
Identification of three MADS-box genes expressed in sunflower capitulum
Received 23 December 2002; Accepted 28 February 2003
Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, CC 242 Paraje El Pozo, 3000 Santa Fe, Argentina
1 To whom correspondence should be addressed. Fax: +54 342 4575219. E-mail: rchan{at}fbcb.unl.edu.ar
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Three cDNA clones, HaPI, HaAG and HaAP3, were isolated from sunflower inflorescences at the R2 stage of development. The cDNAs share high sequence similarity with the PISTILLATA, AGAMOUS, and APETALA3 genes from Arabidopsis, respectively, which contain a MADS-box and are involved in floral organ development. Expression of the corresponding genes was analysed by northern blots and in situ hybridization. They are expressed preferentially in the R3 and R4 stages of capitulum development. HaAG accumulates in fertile flowers, mainly in stamens, while HaPI and HaAP3 are preferentially expressed in ray (sterile) flowers and more weakly in petals and stamens of fertile flowers.
Key words: Floral development, MADS-box, sunflower, transcription factor.
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The MADS-box is a consensus DNA sequence that encodes a DNA binding motif found in transcription factors present in several eukaryotic organisms. The first MADS-box containing genes isolated from plants were Antirrhinum DEFICIENS (DEF) and Arabidopsis AGAMOUS (AG). These genes were isolated from homeotic mutants defective in the specification of floral organ identity. Subsequent work revealed the existence of a large family of MADS-box containing genes in plants. Although initially found in floral tissues, it was later established that they also act as regulators of various other aspects of plant development (Rounsley et al., 1995; Kim et al., 2002).
Plant MADS proteins can be divided into several families, according to sequence similarity, expression patterns and function. Based on phylogenetic criteria five main groups were revealed and these were named according to their first-sequenced member as the AGAMOUS (AG), DEFICIENS (DEF), GLOBOSA (GLO), SQUAMOSA (SQUA), and AGL2 groups (Theißen et al., 1996).
Genes from the SQUA family, which include Arabidopsis APETALA1 (AP1) and Antirrhinum SQUA, generally have dual functions: floral meristem identity and floral organ identity specification. Some members are also involved in the floral induction process. The DEF and AG family genes regulate floral organ identity and include APETALA3 (AP3), PISTILLATA (PI) and AGAMOUS (AG) from Arabidopsis. The ABC model of floral development (Weigel and Meyerowitz, 1994) predicts that three classes of homeotic genes, encoding the A, B and C functions, act alone or in combination to give rise to sepals, petals, stamens and carpels. Genes in the AG group include the C function homeotic genes, involved in stamen, and carpel development. Genes in both the DEF and the GLO groups comprise the B function homeotic genes and are involved in petal and stamen development.
The authors are interested in characterizing the expression patterns of genes involved in flower development in sunflower. Sunflower belongs to the Compositae family, with a terminal inflorescence (head or capitulum) composed of hundreds of flowers of two different types: ray (sterile) flowers in the periphery, and rings of disc (fertile) flowers in the centre (actually formed by radiating arcs from the centre of the head) (Seiler, 1997). It is interesting to understand how morphologically and functionally different flowers develop from the same genetic background.
As a first step towards this goal, MADS-box containing cDNAs were cloned from sunflower R2-stage inflorescence RNA by RT-PCR using degenerated oligonucleotides deduced from conserved regions of members of the AG and DEF groups. Only one expressed gene could be identified with oligonucleotides for the AG family genes, while clones representing two different genes were recovered when oligonucleotides for the DEF family genes were used. Full-length cDNAs for two of the identified genes were obtained applying 3'- and 5'-RACE (Frohman, 1994). Sequence analysis revealed that they share significant homology with the AGAMOUS and PISTILLATA genes from Arabidopsis, respectively, and were therefore named HaAG (Helianthus annuus agamous-like) and HaPI (Helianthus annuus pistillata-like). The third gene was named HaAP3 (Helianthus annuus APETALA3-like) since it is related to Arabidopsis APETALA3. The sequences were deposited in the GenBank under the accession numbers AY157724 [GenBank] , AY157725 [GenBank] and AY185363 [GenBank] . HaAG encodes a polypeptide of 248 amino acids and shows 86% amino acid sequence identity with GAGA1 from Gerbera hybrida, another plant that has a composite inflorescence. HaPI encodes a 168-amino acid protein with 91% sequence identity with GGLO1 from Gerbera, HaAP3 is a partial clone and encodes a 67-amino acid peptide with 95% homology with GDEF2 from Gerbera (Yu et al., 1999).
The expression of HaAG and HaPI during flower development was examined by northern blot analysis and in situ hybridization as previously described (Ribichich et al., 2001). Gene-specific DNA probes for northern and riboprobes for in situ hybridizations were used.
RNA from different stages (R1 to R5 according to the classification developed by Schneiter and Miller, 1981) was purified and analysed by RNA blots (Fig. 1). Expression of the three genes was detected at all stages, with a clear increase in transcript levels upon development from the R1 to the R4 stage and a pronounced decrease at R5, which represents an open capitulum (Fig. 1, upper panel). After pollination, HaAG transcripts were detected at very low levels, while the expression of the other two genes was not observed (not shown). A more detailed analysis using RNA prepared from isolated flowers or floral reproductive organs indicated that HaAG was preferentially expressed in stamens of fertile flowers and also in carpels, while HaPI and HaAP3 transcripts were more abundant in ray flowers (Fig. 1, lower panel). To determine the specific cell types that express the HaAG gene, in situ hybridization studies were performed. HaAG expression was observed in reproductive organ primordia at the R2 (not shown) and R3 stages (Fig. 2). Upon development, expression was predominant in anthers and was also detected in developing ovules (Fig. 2E). HaAG expression was also monitored in a sunflower mutant (L207) that produces fertile ray flowers. In this mutant, expression was evident in developing anthers of these flowers (Fig. 2C).
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The expression analysis described here suggests that the sunflower homologues of Arabidopsis AGAMOUS, PISTILLATA and APETALA3 may have functional equivalency with their counterparts, participating in the C and B functions, respectively. It is also evident that the same or very similar genes are expressed in fertile and ray flowers, although at different levels. In addition, HaAG expression is switched on in mutants that develop fertile ray flowers. Future studies will be conducted to evaluate the mechanisms involved in this process.
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We thank Dr Alexander Pershin, Zaporozhye, Ukraine for the gift of L207 mutant seeds. This work was supported by grants from CONICET, ANPCyT, Universidad Nacional del Litoral and Fundación Antorchas (Argentina). RLC and DHG are members of CONICET; MFT and CAD are fellows of CONICET and ANPCyT, respectively.
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Kim S-H, Mizuno K, Fujimura T. 2002. Isolation of MADS-box genes from sweet potato (Ipomea batatas (L.) Lam.) expressed specifically in vegetative tissues. Plant and Cell Physiology 43, 314–322.
Ribichich K, Tioni MF, Chan RL, Gonzalez DH. 2001. Cell-type specific expression of plant cytochrome c mRNA in developing flowers and roots. Plant Physiology 125, 1603–1610.
Rounsley SD, Ditta GS, Yanofsky MF. 1995. Diverse roles of MADS genes in Arabidopsis development. The Plant Cell 7, 1259–1269.[Abstract]
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Seiler GJ. 1997. Anatomy and morphology of sunflower. In: Schneiter AA, ed. Sunflower technology and production. Agronomy Monograph no. 35. Madison, WI: American Society of Agronomy, 67–111.
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