Journal of Experimental Botany, Vol. 54, No. 384, pp. 961-969,
March 1, 2003
© 2003 Oxford University Press
Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco
Received 1 July 2002; Accepted 22 November 2002
1 Laboratory of Molecular and Cellular Biology, Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502 Japan
2 Department of Plant Gene and Totipotency, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto, 606-8502 Japan
3 Present address: Research and Education Center for Genetic Informations, Nara Institute of Science and Technology (NAIST), Takayama, Ikoma, Nara, 630-0101, Japan.
4 Present address: Akita Prefectural University, Akita, 010-0195, Japan.
5 To whom correspondence should be addressed at Laboratory of Molecular and Cellular Biology of Totipotency, Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8502, Japan. Fax: +81 75 753 6398. e-mail: fumihiko{at}kais.kyoto-u.ac
Abbreviations: CAM, crassulacean acid metabolism; CaMV, cauliflower mosaic virus; GUS, ß-glucuronidase; PCR, polymerase chain reaction; PEPC, phosphoenolpyruvate carboxylase; RT, reverse transcriptase; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Phosphoenolpyruvate carboxylase (PEPC), which catalyses the carboxylation of phosphoenolpyruvate using HCO3– to generate oxaloacetic acid, is an important enzyme in the primary metabolism of plants. Although the PEPC genes (ppc) comprise only a small gene family, the function of each gene is not clear, except for roles in C4 photosynthesis and CAM. Three PEPC genes (Nsppc1–3) from the C3 plant Nicotiana sylvestris were used to investigate their roles and regulation in a C3 plant, and their regulation by phosphorus depletion in particular. First, the induction of PEPC by phosphorus depletion was confirmed. Next, Nsppc1 was determined to be mainly responsive to phosphorus deficiency at the transcriptional level. Further studies using transgenic tobacco harbouring a chimeric gene consisting of the 2.0 kb promoter region of Nsppc1 and the ß-glucuronidase (GUS) reporter showed that PEPC is transcriptionally induced. It was also found that sucrose had a synergistic effect on the induction of PEPC by phosphorus deficiency. A series of transgenic tobacco containing 5'-deletion mutants of Nsppc1 promoter::GUS fusion revealed that the –539 to –442 bp Nsppc1 promoter region, relative to the translation start site, was necessary for the response to phosphorus deficiency. Gain-of-function analysis using a construct containing three tandem repeats of the –539 to –442 bp region confirmed that this region was sufficient to induce the phosphorus-deficiency response in tobacco.
Key words: Cis-acting element, Nicotiana tabacum, PEPC, phosphoenolpyruvate carboxylase, phosphorus deficiency, sucrose effect, tobacco, transcriptional regulation.
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