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J. Biol. Chem., Vol. 281, Issue 46, 35369-35380, November 17, 2006

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Evolution of a Basic Helix-Loop-Helix Protein from a Transcriptional Repressor to a Plastid-resident Regulatory Factor

INVOLVEMENT IN HYPERSENSITIVE CELL DEATH IN TOBACCO PLANTS^*

From the Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0192, Japan

The tobacco gene NtWIN4 (Nicotiana tabacum wound-induced clone 4) is transiently up-regulated in response not only to wounding but also to pathogen attack. NtWIN4 encodes a putative basic helix-loop-helix protein with an apparent molecular mass of 28 kDa that exhibited clear nuclear transcription repression activity in Dual-Luciferase assays. However, immunoblotting indicated the existence of a 17-kDa form of NtWIN4 localized exclusively in tobacco leaf chloroplasts. Subsequent peptide dissection analyses with green fluorescent protein fusions revealed that a polypeptide of 81 amino acids starting at position 13 from the N terminus is maximally necessary for this localization. Further fine dissection analysis strongly suggested that the protein actually begins at the second Met located at position 27, yielding a signal peptide of 67 amino acids. However, the last C-terminal 15 amino acids overlap with the conserved basic region critical for DNA binding, so NtWIN4 presumably does not function as a transcription factor in planta. Transgenic tobacco plants constitutively overexpressing NtWIN4 demonstrated mortality with abnormal features, including albinism, and transient expression upon agroinfiltration resulted in distinct necrosis with a sharp decrease in chlorophyll content, consistent with the phenomenon known as chlorosis. Transgenic RNA interference tobacco plants exhibited reduced hypersensitive cell death, showing delayed tissue necrosis upon pathogen infection. These results suggest that NtWIN4 arose by divergence, becoming a chloroplast-resident factor from a nuclear transcriptional repressor by obtaining a transit peptide sequence, and that, upon translocation, it interacts with chloroplast components to induce hypersensitive cell death through chloroplast disruption, thereby contributing to plant stress responses.

Received for publication, May 1, 2006 , and in revised form, September 8, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB258537 [GenBank] and AB258538 [GenBank] .

^* This work was supported by grants from the Research for the Future Program and for creative scientific research from the Japan Society for the Promotion of Science and by a grant in-aid for scientific research for plant graduate students (to Y. K.) from the Nara Institute of Science and Technology, supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-743-72-5650; Fax: 81-743-72-5659; E-mail: sano{at}gtc.naist.jp.

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