Journal of Experimental Botany, Vol. 53, No. 375, pp. 1829-1830,
August 1, 2002
© 2002 Oxford University Press
Isolation and characterization of the Spindly homologue from tomato
Received 19 December 2001; Accepted 26 April 2002
Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany
1 To whom correspondence should be addressed. Fax: +49 221 5062 413. E-mail: theres{at}mpiz-koeln.mpg.de
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The SPINDLY (SPY) gene is a crucial component of the gibberellin signal transduction pathway in Arabidopsis thaliana (L.) Heynh. In this study, the cloning of the SPINDLY-orthologous gene (LeSpy) from tomato (Lycopersicon esculentum Mill.) and its characterization are reported. SPY and LeSpy show high sequence similarity along their entire lengths, which is reflected in the conservation of exon–intron structure and of all sequence motives previously identified. To analyse the relationship between the Arabidopsis spindly and the tomato procera mutant, which show similar phenotypes, the LeSpy gene was characterized in wild-type and procera tomato plants. These analyses as well as mapping of LeSpy revealed that LeSpy and Procera are different genes.
Key words: Key words: GA signalling, gibberellins, Procera, Spindly.
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Gibberellins, a group of tetracyclic diterpenoids, form one of the major classes of classical plant hormones. They are involved in many processes during plant growth and development, including seed germination, stem elongation, flowering, and fruit development. GA-deficient mutants have played an important role in determining the role of individual GAs and in elucidating the GA biosynthesis pathway (Hedden and Kamiya, 1997). A second group of mutants has been described in which GA signal transduction is disturbed. Besides GA-non-responsive dwarfs this group contains mutants that resemble plants treated with exogenous GA. Among others, the Arabidopsis spindly (spy) mutant (Jacobsen and Olszewski, 1993) and the tomato procera mutant (Jones, 1987) are representatives of this type of mutant. Both mutants respond to exogenous GA and show a partial resistance to GA biosynthesis inhibitors (Jacobsen et al., 1993; van Tuinen et al., 1999). The Arabidopsis SPY gene has been cloned and was shown to code for a protein containing copies of the tetratricopeptide repeat motif, mediating protein–protein interaction, and a segment with homology to N-acetylglucosaminyl transferases (Jacobsen et al., 1996). The objective of this work was the isolation and characterization of the tomato Spindly gene, its mapping by RFLP analysis, and the analysis of its relationship to the procera locus.
Degenerate primers (Spy1, 5'-GCCATTGTAACACATGGAAC NCC-3'; Spy2, 5'-GCNAAGATAACACCTAARGT-3') were deduced from the regions conserved between the Arabidopsis thaliana SPY protein and the Caenorhabditis elegans homologue K04G7.3 (Jacobsen et al., 1996). With these primers a fragment of 276 bp was amplified on cDNA prepared from RNA of vegetative tomato shoot tips. A corresponding cosmid clone was isolated from a tomato genomic cosmid library (Schumacher et al., 1999), subcloned into the pGEM-3Z plasmid vector and sequenced. Restriction enzyme digests of the isolated cosmid clone revealed fragments of the same size as those detected by Southern blot analysis of tomato genomic DNA under low stringency conditions (5xSSPE, 1% SDS, 5xDenhardt’s, 200 µg ml–1 CT-DNA, 61 °C; washing: 2x10 min in 1 x SSPE, 0.1% SDS at 45 °C) using a fragment of the Arabidopsis SPY cDNA as a probe. This result demonstrates that the isolated DNA fragment represents the most similar SPY homologous DNA sequence from tomato (LeSpy).
LeSpy cDNA was assembled from a 5'-partial cDNA clone (729 bp) isolated from a tomato shoot tip cDNA library (Meissner and Theres, 1995) and overlapping cDNA fragments obtained by RT-PCR. Because the 5'-part of the gene was not included in the cosmid clone the genomic sequence was extended by overlapping PCR products amplified on genomic DNA using the cDNA sequence for primer design. Comparison of the cDNA (accession no. AJ312094) and the genomic sequence (accession no. AJ312093) revealed that the LeSpy gene is composed of 18 exons, as already found for the Arabidopsis SPY gene (Jacobsen et al., 1996). Whereas the sizes of exons within the coding region and the positions of introns are perfectly conserved, the sizes of introns in tomato are, on average, about 6-fold larger than those in Arabidopsis. All the sequence motifs identified in the Arabidopsis SPY protein were also found in the LeSpy protein. Besides the Arabidopsis SPY protein (79%) the LeSpy protein shows extensive sequence identity to the SPY proteins from Hordeum vulgare L. (74%; Robertson et al., 1998) and Petunia hybrida Vilm. (91%; Izhaki et al., 2001).
As the phenotypes of the Arabidopsis spindly and the tomato procera mutant suggested that SPY and Pro may be homologous genes, this hypothesis was tested. Sequence analysis of the LeSpy ORFs from wild type and procera did not reveal any sequence deviation. In addition, Northern blot analysis of RNA isolated from wild-type and procera shoot tips demonstrated transcripts of the same size and in very similar amounts. These results suggest that neither sequence alterations in the LeSpy gene nor differences in LeSpy transcript accumulation can be the reason for the procera mutant phenotype.
LeSpy was mapped by RFLP analysis using an F2 population of 33 plants and a framework dataset of 335 RFLP markers distributed over the 12 tomato chromosomes (Tanksley et al., 1992). The mapping revealed cosegregation of the LeSpy gene with RFLP marker CT32 on chromosome 9. As the procera locus has been mapped to tomato chromosome 11 (van Tuinen et al., 1998) mapping of LeSpy to chromosome 9 provides conclusive evidence that Procera and LeSpy are different genes. Because the sequence of LeSpy and its map position are available now, transgenic down-regulation by RNAi or targeted transposon tagging are possible approaches to obtain information about a loss of function phenotype. It is tempting to speculate that the Procera gene of tomato corresponds to a different inhibitor of GA signal signalling, like the GAI (Peng et al., 1997) or RGA (Silverstone et al., 1998) genes characterized in Arabidopsis.
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The authors thank the Tomato Genetic Resource Center for providing seed stocks and Dr Steven Tanksley for providing the segregating F2 population and a framework data set. We are grateful to E Tillmann for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
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