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Journal of Experimental Botany, Vol. 52, No. 355, pp. 375-376, February 2001
© 2001 Oxford University Press

Gene Notes

Isolation and expression analysis of gibberellin 20-oxidase homologous gene in apple

S. Kusaba1, C. Honda and Y. Kano-Murakami

Department of Pomology, National Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan

Received 28 September 2000; Accepted 18 October 2000

Abstract

To characterize the gibberellin (GA) 20-oxidase gene in apple, the genomic and cDNA clone from ‘Fuji’ apple (accession no. AB037114) was isolated. The deduced amino acid sequence of this cDNA showed 71% and 66% identity to those of GA 20-oxidase cloned from French bean and Arabidopsis, respectively. The transcript of this gene was detected mainly in immature seeds between 1–3 months after full bloom. These results suggested that this apple GA 20-oxidase gene might be involved in GA biosynthesis in developing apple seed.

Key words: Malusxdomestica, gibberellin, biosynthesis, GA 20-oxidase, immature seed.

In the biosynthetic pathway of active GA, GA 20-oxidase catalyses the successive oxidation from GA53 or GA12 to GA20 or GA9, respectively (Lange, 1994Go). GA 20-oxidase is thought to be one of the rate-limiting enzymes in GA biosynthesis because the ga5 mutant of Arabidopsis, which is semi-dwarfed has a mutation in the stem GA 20-oxidase gene (Xu et al., 1995Go).

In apple, it is reported that a chemical identification of GAs in immature seed (Hedden et al., 1993Go) and that GA(s) are involved in the spur-type growth habit of ‘McIntosh Wijcik’ (Looney et al., 1988Go). Besides these results, little is known about the genes involved in GA biosynthesis in apple. In this study, the isolation of a GA 20-oxidase gene from apple and the analysis of its expression pattern is reported in order to increase our understanding of the molecular basis of GA biosynthesis in apple.

A 173 bp DNA fragment was obtained by PCR of apple genomic DNA (Fig. 1Go). Sequence analysis revealed that 173 bp DNA fragment showed 80% homology compared to that of French bean GA 20-oxidase (U70530; García-Martínez et al., 1997Go). An apple cDNA library prepared from stem tissue was screened using this PCR product and the tobacco GA 20-oxidase (Kusaba et al., 1998Go) as probes. As a consequence of cDNA screening, a 5'-truncated GA 20-oxidase homologous cDNA clone of 800 bp was obtained. To isolate the genomic clone, a ‘Fuji’ genomic library was screened with this 5'-truncated GA 20-oxidase cDNA as a probe. Four independent clones were isolated. From the results of mapping and sequencing analysis, these four clones were identical. A full-length cDNA clone of apple GA 20-oxidase was obtained by RT-PCR, the sequence of which is shown in Fig. 1Go. Sequencing analysis demonstrated that the genomic clone obtained consisted of three exons and two introns as seen in Arabidopsis (Xu et al., 1995Go). The deduced amino acid sequence of the cDNA showed 70% and 61% identity to those of GA 20-oxidases cloned from French bean (U70530; García-Martínez et al., 1997Go) and Arabidopsis (At2301; Phillips et al., 1995Go), respectively. It is reported that GA 20-oxidases isolated from various plant species exhibit a sequence conservation, with amino acid identities ranging from 50% to 75% (Hedden and Kamiya, 1997Go). Furthermore, the amino acid sequence Leu-Pro-Trp-Lys-Glu-Thr, which is the consensus sequence found in all GA 20-oxidases (Xu et al., 1995Go), is also observed in the deduced amino acid sequence of the putative apple GA 20-oxidase (positions 159–164). From these results, the cDNA appeared to represent a full-length clone of apple GA 20-oxidase.



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  		Fig. 1. DNA sequence and deduced amino acid sequence of apple gibberellin 20-oxidase. The arrowheads indicate the position of the introns. The putative consensus sequence found in all GA 20-oxidase is boxed. The 173 bp DNA fragment was obtained from ‘Fuji’ genomic DNA by PCR at 95 °C for 4 min, then subjected to 35 cycles of 95 °C for 1 min, 35 °C for 1 min and 72 °C for 2 min with degenerate primers (5'-GCAAGCTTAA(TC)TA(TC)TA(TC)CCNCCNTG-3' and 5'-GCGGATCCCC(AGT)AT(AG)TTNACNAC (AG)AA-3'), and is underlined. RT-PCR was performed to isolate cDNA of GA 20-oxidase from immature seed of apple. First strand DNA was synthesized using synthetic oligo nucleotide (5'-GGCCTACCACCTCTGTTCATCTCC-3') as a primer. PCR was performed using synthetic oligonucleotides as primers (5'-GCAAAAATGGCTGTTGAGTGC-3' and 5'-CTCCTCACAGTTTTTGGTTGC-3'). Samples were heated to 95 °C for 4 min, then subjected to 30 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min.

 
Tissue distribution of apple GA 20-oxidase was examined (Fig. 2AGo). The levels of transcript were highest in immature seed, and low in young leaves, mature leaves, and developing stem. No transcript was detected in the flesh of immature fruit (data not shown). The immature seed of apple is rich in GA and the seasonal change of GA in immature seed was investigated (Luckwill et al., 1969Go). This result implies that the apple GA 20-oxidase may also be involved in GA biosynthesis in these organs.



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  		Fig. 2. Expression analysis of apple GA 20-oxidase gene. Ten micrograms of each total RNA preparation was used for Northern blot analysis mentioned elsewhere (Kusaba et al., 1998) using 5'-truncated GA 20-oxidase cDNA as a probe. (A) Tissue distribution of apple GA 20-oxidase mRNA. YL, young leaf; ML, mature leaf; St, developing stem; F; mature flower; Se, immature seed. (B) Temporal changes of apple fruit growth and mRNA accumulation of apple GA 20-oxidase in seed.

 
The seasonal change of mRNA accumulation in immature seed of ‘Fuji’ apple was investigated using mRNA extracted from immature seed in various developing stages of apple fruit (Fig. 2BGo). The mRNA accumulated to high levels in immature seeds 1–3 months after full bloom, and no accumulation was observed in the early developing stages and mature stages of apple fruit. The presence of the transcript coincided with the period of rapid enlargement of the apple fruit (Fig. 2BGo). Luckwill et al. reported that GA first appeared in apple seeds about 5 weeks after full bloom, increased to a maximum concentration at 9 weeks and subsequently decreased again, disappearing completely by the time the seed was mature (Luckwill et al., 1969Go). The temporal change in mRNA accumulation levels of apple GA 20-oxidase in immature seed correlates well with the changes in GA levels in immature seed. Pharis et al. reviewed the control of fruit growth by seed-produced GAs (Pharis et al., 1985). For example, fruit growth rate is often correlated with seed number, but exogenous GA can increase fruit growth and it can substitute for the presence of seeds which have been removed. Apple GA 20-oxidase mRNA accumulation was not observed in the flesh of immature fruit at times when high levels of mRNA were detected in immature seeds. It is likely that the expression of this GA 20-oxidase in immature seed may be involved in the development of apple fruit.

Acknowledgments

We would like to thank Dr M Fukumoto (National Institute of Fruit Tree Science) and Dr S Jackson (Horticulture Research International, UK) for helpful comments, and K Monma and N Ishii (National Institute of Fruit Tree Science) for research assistance.

Notes

1 To whom correspondence should be addressed. (Until May 2001, Horticulture Research International, Plant Genetics and Biotechnology Department, Wellesbourne, Warwick CV35 9EF, UK. Fax: +44 1789 470552); (after May 2001) Shikoku National Agricultural Experiment Station, 2575 Ikano, Zentsuji, Kagawa 765-0053, Japan. Fax: +81 877 62 1130. E-mail: kusa{at}affrc.go.jp Back

References

García-Martínez JL, López-Diaz I, Sánchez-Beltrán MJ, Phillips AL, Ward DA, Gaskin P, Hedden P.1997. Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development. Plant Molecular Biology 33, 1073–1084.[Web of Science][Medline]

Hedden P, Hoad GV, Gaskin P, Lewis MJ, Green JR, Furber M, Mander LN.1993. Kaurenoids and gibberellins, including the newly characterized gibberellin A88, in developing apple seeds. Phytochemistry 32, 231–237.

Hedden P, Kamiya Y.1997. Gibberellin biosynthesis: enzymes, genes and their regulation. Annual Review of Plant Physiology and Plant Molecular Biology 48, 431–460.[Web of Science]

Kusaba S, Fukumoto M, Honda C, Yamaguchi I, Sakamoto T, Kano-Murakami Y.1998. Decreased GA1 content caused by the overexpression of OSH1 is accompanied by suppression of GA 20-oxidase gene expression. Plant Physiology 117, 1179–1184.[Abstract/Free Full Text]

Lange T.1994. Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm. Planta 195, 108–115.[Web of Science][Medline]

Looney NE, Taylor JS, Pharis RP.1988. Relationship of endogenous gibberellin and cytokinin levels in shoot tips to apical form in four strains of ‘McIntosh’ apple. Journal of the American Society for Horticultural Science 113, 395–398.

Luckwill LC, Weaver P, Macmillan J.1969. Gibberellins and other growth hormones in apple seeds. Journal of Horticultural Science 44, 413–424.

Pharis RP, King RW.1985. Gibberellins and reproductive development in seed plants. Annual Review of Plant Physiology 36, 517–558.[Web of Science]

Phillips AL, Ward DA, Uknes S, Appleford NEJ, Lange T, Huttly AK, Gaskin P, Graebe JE, Hedden P.1995. Isolation and expression of three gibberellin 20-oxidase cDNA clone from Arabidopsis. Plant Physiology 108, 1049–1057.[Abstract]

Xu Y-L, Li L, Wu K, Peeters AJM, Gage DA, Zeevaart JAD.1995. The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression. Proceedings of the National Academy of Sciences, USA 92, 6640–6644.[Abstract/Free Full Text]


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