Journal of Experimental Botany, Vol. 53, No. 376, pp. 1991-1993,
September 1, 2002
© 2002 Oxford University Press
The novel rice (Oryza sativa L.) gene OsSbf1 encodes a putative member of the Na+/bile acid symporter family
Received 30 April 2002; Accepted 10 June 2002
Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany
2 To whom correspondence should be addressed. E-mail: msauter{at}botanik.uni-hamburg.de
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PCR-based differential screening was used to identify ethylene-induced genes in deep-water rice (Oryza sativa L.). One of the isolated cDNAs represented a novel protein, OsSBF1, with high homology to mammalian Na+/bile acid transporters and to sodium-dependent transporters from bacteria. One highly homologous protein and three less conserved homologues were identified in Arabidopsis thaliana indicating that Sbf proteins exist in monocot and dicot plant species. Expression of OsSbf1 in deep-water rice was shown to be elevated by growth-inducing treatments. Since bile acids have not been found in plants to date a possible function of SBF proteins may be in the transport of structurally related sulphonated brassinosteroids.
Key words: Key words: Ethylene, gibberellin, growth, rice, sodium/bile acid transporter family (Sbf).
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Deep-water rice (Oryza sativa L.) is well adapted to survive prolonged periods of partial submergence. A major adaptation to hypoxia is submergence-induced rapid elongation of the youngest internode (Sauter, 2000). Internodal growth helps the plant to keep part of its leaves above the water surface and thus ensures access to atmospheric oxygen. The growth response is mediated by ethylene, ABA and gibberellin (Kende et al., 1998). When the plants are submerged, ethylene levels rise. Subsequently, ABA concentration decreases and gibberellin concentration increases. Gibberellin is ultimately responsible for promoting growth of the internode.
To understand hormone regulation of growth and to identify novel proteins involved in this response, a PCR-based differential screening for growth-relevant genes was performed. cDNA libraries were generated from RNA isolated from the growing zone of the rice internode after 0 min, 40 min, and 90 min of treatment with the natural ethylene precursor ACC. Ethylene was chosen as the growth-inducing agent to avoid the isolation of hypoxia-responsive genes induced during submergence while still activating the complete hormonal signalling cascade from ethylene via ABA and gibberellin to growth induction. Subtraction was performed with the 0 min and 40 min cDNA libraries and with the 0 min and 90 min cDNA libraries (Buchanan-Wollaston and Ainsworth, 1997). One clone identified in the 0–40 min subtraction was a partial cDNA that was identical to a rice gene identified on chromosome 1 (accession number BAB40160). Translation of the putative full-length open reading frame revealed a protein of 408 amino acids that was termed OsSBF1 (Fig. 1A).
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Four Sbf homologues were identified from Arabidopsis thaliana. One of the Arabidopsis proteins was highly homologous with more than 70% identical amino acids (AC005168; on chromosome 2). The genomic sequence information available for Arabidopsis was used to clone the corresponding cDNA by RT-PCR using mRNA from whole seedlings and adult plants. The polypeptide sequence that was deduced was longer at two positions than the protein sequence derived from genomic data. A possible explanation could be misannotation of intron/exon transitions in the genomic sequence. The gene was named AtSbf1. The cDNA and the deduced protein sequence were deposited in GenBank under the accession number AF498303. OsSBF1 and AtSBF1 share 73% identical and 84% similar amino acids (Fig. 1A). Three additional Arabidopsis Sbf homologues were identified with sequence identities to OsSBF1 of approximately 40% (accession number AAL24290 located on chromosome 1; accession number NP_566764 located on chromosome 3; and accession number CAA16569 located on chromosome 4). In a domain search, the rice and Arabidopsis proteins were recognized as members of the sodium bile acid cotransporter family (Sbf) of transmembrane proteins present in archaea, bacteria and eukaryotes (Fig. 1C). The rice OsSbf1 gene represents the first Sbf member described from plants. The function of the more distantly related sodium-dependent transporters from bacteria such as Bacillus halodurans (accession number E69902) and Aquifex aeolicus (accession number E70482) is not understood. In bakers yeast, the Sbf-related protein ACR3 (accession number Q06598) is involved in resistance to arsenic compounds (Wysocki et al., 1997). In mammals, Sbf proteins were shown to mediate the co-transport of sodium and bile acids across the plasma membrane in the ileum and in liver cells (Kramer et al., 1999). Sequence similarity between OsSBF1 and human ileal sodium/bile acid transporter (IBAT; accession number I38655) was 56%. The phylogenetic relationship of Sbf proteins from various taxa is shown in Fig. 1C. A more detailed comparison of a few of the sequences is given in Fig. 1A. The highly conserved sodium/bile acid transporter signature domain is indicated by a black bar.
Hydrophobicity plot analysis (Kyte and Doolittle, 1982) also indicated a very similar distribution of predicted alpha helical transmembrane regions in the rice protein compared to the human ileal Na+/bile acid transporter (Fig. 1B), indicating conservation not only at the primary sequence level but also at the structural level. These findings strongly support a role for OsSBF1 as a transmembrane transport protein. In man, the amino acid Pro290 was shown to be an essential residue for bile acid transport (Wong et al., 1995). This residue is conserved in all sequences including the putative plant Sbf proteins (Fig. 1A) further supporting functional conservation.
The regulation of OsSbf1 expression in the growing region, comprising the intercalary meristem and part of the elongation zone of the rice internode, was analysed. First, whole plants were partially submerged and OsSbf1 gene expression was studied up to 18 h after the onset of treatment (Fig. 2A). Elevated transcript levels were observed after 2 h of submergence that is prior to growth induction which occurs after 4 h. Application of ethephon (2-chloroethylphosphonic acid), a chemical precursor of ethylene, induced OsSbf1 expression within 2 h in a similar manner as submergence did (Fig. 2B). The lag phase for ethephon-induced growth is more than 6 h (data not shown). Furthermore, OsSbf1 transcripts also accumulated after gibberellin treatment (Fig. 2C).
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The fact that mRNA accumulation preceded growth induction in submerged and in ethylene-treated tissue favours the view that OsSBF1 may be important for growth to occur. For gibberellin, growth and gene regulation cannot be resolved from the data since both take place in less than 1 h. However, gibberellin accelerates growth rate, but, unlike submergence or ethylene treatment, does not invoke stress responses, further implicating a role for OsSBF1 in stem growth.
How sodium/bile acid symporters may function in plants is unclear. To date, bile acids have not been shown to exist in plants, even though Hortensteiner et al. (1993) reported bile acid transport into plant vacuoles. Uptake of taurocholate and glycocholate was strictly ATP-dependent indicating that this transport was not mediated by a sodium-driven cotransporter of the Sbf-type. Bile acids are sulphonated steroid derivatives. In plants, brassinosteroids possess hormone activity and are involved in a wide range of developmental and growth processes. In rice, brassinosteroids have been implicated in internode elongation (Yamamuro et al., 2000). While active brassinosteroids are not sulphonated, it has been shown previously in Brassica napus that O-sulphonation of brassinosteroids by a steroid sulphotransferase exists and that it results in hormone inactivation (Rouleau et al., 1999). It is conceivable that the transporters predicted to exist for bile acid-like compounds in plants may be involved in the transport of sulphonated brassinosteroids. However, transport of other bile acid-related compounds cannot be excluded and the role of Sbf transporters in plants has yet to be elucidated.
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Acknowledgements |
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The authors would like to acknowledge support through a Research Training Grant from the European Union (contract number HPRN-CT-2000-00090). This work is part of a doctoral thesis by GR at the University of Hamburg.
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