Journal of Experimental Botany, Vol. 52, No. 357, pp. 875-876,
April 15, 2001
© 2001 Oxford University Press
Gene Notes |
Isolation of a VP1 homologue from wheat and analysis of its expression in embryos of dormant and non-dormant cultivars
Tohoku National Agricultural Experimental Station, Morioka, Japan 020-0198
Received 11 October 2000; Accepted 15 November 2000
Abstract
A VP (Viviparous) 1 homologous gene has been cloned from wheat (Triticum aestivumL.). Its expression level was examined in the mature embryos of dormant and non-dormant cultivars. The level of expression was positively correlated with the level of seed dormancy and embryo sensitivity to abscisic acid (ABA).
Key words: Wheat VP1 gene, dormancy, abscisic acid.
Wheat is known as the crop that easily shows pre-harvest sprouting. Pre-harvest sprouting causes devastating damage to the quality of wheat flour due to the degradation of seed starch during germination. Physiological studies have established that pre-harvest sprouting in wheat is caused by the lack of embryo dormancy (Gale and Lenton, 1987
). Therefore, in order to prevent pre-harvest sprouting, it is necessary to know how seed dormancy is regulated. Although the mechanism of dormancy is still largely unknown, genetic studies have revealed several genes that affect the level of seed dormancy (McCarty, 1995). Among them, VP1/ ABI3 (Abscisic Acid Insensitive3)gene plays a pivotal role in seed maturation such as induction of seed dormancy and acquisition of desiccation tolerance (MaCarty, 1995; Giraudat et al., 1992). Recently, Jones et al. revealed that the embryonic expression level of VP1 is correlated with the level of dormancy using inbred lines of Avena fatua (Jones et al., 1997). This result suggested that the VP1 expression level in mature embryos may regulate the level of seed dormancy. Thus, it was intriguing to isolate the wheat VP1 gene and examine its expression level in mature embryos of dormant and non-dormant wheat cultivars.
A wheat mature embryo cDNA library was constructed with RNA from cultivar Minamino. A wheat VP1 homologue was isolated from the cDNA library by PCR with 20 mer-primers at 5' and 3' terminated sequence regions corresponding to the amino acid sequences, MDASAGS and NQMAVSI, respectively. The corresponding 
and 
end sequences of the VP1 genes were confirmed by PCR with internal primers of the VP1 gene and T7 and T3 primers on the vector. The isolated wheat VP1 homologous gene (TaVP1; accession no. AB047554) consists of 2061bp and encodes 687 amino acids (Fig. 1). The sequence has all of the highly conserved domains that have been found in the previously isolated VP1 homologues. Because wheat is an allohexaploid, it is likely that this is a member of a 3 gene family.
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The relationships between seed dormancy and VP1 expression levels in the mature embryos were examined using two wheat cultivars. Minamino is an extremely dormant cultivar, while Tozan 18 is non-dormant. The germination rates of these seeds were determined. After incubation at 22 °C for 5 d, Minamino seeds did not germinate at all, but, all Tozan 18 seeds germinated. It was previously shown that the dormancy level was regulated by ABA sensitivity rather than the amount of ABA in the seeds (Walker-Simmons, 1987
). ABA sensitivity of the embryos of these two cultivars was determined by the assaying germination of half seeds in the presence of exogenous ABA (Kawakami et al., 1997). While the germination of Minamino half seeds was inhibited by 10 µM ABA, all Tozan 18 half seeds germinated under these conditions.
RNA gel blot analysis was performed to examine the expression level of the VP1 genes in the embryos of these two cultivars. While the VP1 homologues did not show any expression in the young leaf, they were expressed in embryos (Fig. 2). This was consistent with the previous description that VP1/ABI3 genes are expressed only in seed and quiescent apexes (Rohde et al., 1999). Moreover, the VP1 gene was much more highly expressed in the embryos of Minamino than that in the embryos of Tozan18 (Fig. 2). These results indicate a positive correlation among the level of seed dormancy, the level of ABA sensitivity and the level of VP1 transcript in mature embryos.
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In the future, it is necessary to determine whether enhancing embryonic VP1 expression is sufficient to increase seed dormancy and ABA sensitivity. In addition, it is also necessary to determine how VP1 expression is regulated in mature embryos.
Acknowledgments
We thank Dr Ruth Finkelstein (University of California, Santa Barbara) for critical reading of the manuscript. Minamino and Tozan 18 seeds were kindly provided by Kyushu experimental station (Japan) and Nagano experimental station (Japan), respectively. This work was supported by a project grant from the Ministry of Agriculture, Forestry and Fisheries, Japan.
Notes
1 To whom correspondence should be addressed. Fax: +81 19 641 7794. E-mail: s_nakamu{at}lifesci.ucsb.edu 
References
Gale MD, Lenton JR.1987. Preharvest sprouting in wheat a complex genetic and physiological problem affecting breadmaking quality in UK wheats. Aspects of Applied Biology 15, 115–124.
Giraudat J, Hauge BM, Valon C, Smalle J, Parcy F, Goodman HM.1992. Isolation of the Arabidopsis ABI3 gene by positional cloning. The Plant Cell 4, 1251–1261.
Jones HD, Peters NCB, Holdsworth MJ.1997. Genotype and environment interact to control dormancy and differential expression of the VIVIPAROUS 1 homologue in embryos of Avena fatua. The Plant Journal 12, 911–920.[Web of Science][Medline]
Kawakami N, Miyake Y, Noda K.1997. ABA insensitivity and low ABA levels during seed development of non-dormant wheat mutants. Journal of Experimental Botany 48, 1415–1421.
McCarty DR.1995. Genetic control and integration of maturation and germination pathways in seed development. Annual Review of Physiology and Plant Molecular Biology 46, 71–93.[Web of Science]
Rohde A, Montagu V, Boerjan W.1999. The ABSCISIC ACID-INSENSITIVE3(ABI3) gene is expressed during vegetative quiescence processes in Arabidopsis. Plant, Cell and Environment 22, 261–270.
Walker-Simmons M.1987. ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars. Plant Physiology 84, 61–66.
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