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Journal of Experimental Botany, Vol. 55, No. 396, pp. 387-395, February 1, 2004
© 2004 Oxford University Press

Cell and Molecular Biology, Biochemistry and Molecular Physiology

Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells

Received 13 June 2003; Accepted 16 October 2003

Ikuko Yonamine, Kazuya Yoshida*, Keiji Kido, Atsushi Nakagawa, Hideki Nakayama and Atsuhiko Shinmyo

Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0101, Japan

* To whom correspondence should be addressed. Fax: +81 743 72 5469. E-mail: kazz{at}bs.aist-nara.ac.jp

The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.

Key words: HAL3 genes, HAL3 proteins, overexpression, proline biosynthesis, salt tolerance, tobacco cells.


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