JXB Advance Access originally published online on June 4, 2004
Journal of Experimental Botany 2004 55(403):1761-1763; doi:10.1093/jxb/erh182
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GENE NOTES |
Differential expression of the nitrite reductase gene family in tobacco as revealed by quantitative competitive RT-PCR
1Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
* To whom correspondence should be addressed. Fax: +81 824 24 0749. E-mail: hmorikaw{at}sci.hiroshima-u.ac.jp
Received 19 February 2004; Accepted 21 April 2004
Abstract
Tobacco (Nicotiana tabacum L. cv. Xanthi XHFD8) possesses four nitrite reductase (NiR) genes: nii1, nii2, nii3, and nii4. Their differential expression in leaves and roots was investigated by quantitative competitive RT-PCR using gene-specific primer pairs. These results appear to contradict existing views on the expression of these NiR genes: (i) the mRNA of each of the four NiR genes was distinguishable both in leaves and roots; (ii) nitrate treatment increased nii1 and nii3 mRNA in leaves and roots by at least 4-fold (at least 5-fold in nii2 and nii4 mRNA); and (iii) the steady-state levels of nii1 and nii3 mRNA were almost the same in leaves (6–7x105 and about 3x106 copies µg–1 of total RNA before and after nitrate treatment, respectively) and in roots (3–4x104 and 3–6x105 copies µg–1 of total RNA before and after nitrate treatment, respectively). Very similar relationships were obtained for the steady-state levels of nii2 and nii4 mRNA in roots (2–4x105 and 8x106 copies µg–1 of total RNA before and after nitrate treatment, respectively), and in leaves (5–9x104 and 4x105 copies µg–1 of total RNA before and after nitrate treatment, respectively). These results demonstrate that nii1 and nii3 transcripts are a dominating, but not exclusive, NiR mRNA in leaves, and the same is true for nii2 and nii4 transcripts in roots.
Key words: Differential gene expression, nitrite reductase (NiR), quantitative competitive RT-PCR, tobacco
Nitrogen is one of the major growth-limiting nutrients for plants, and its source in most higher plants is nitrate taken up through roots. Nitrate can be reduced both in roots and shoots. A ferredoxin-nitrite reductase (NiR) catalyses the reduction of nitrite to ammonium in the second step of the nitrate-assimilation pathway.
Tobacco is unique in possessing four NiR genes: nii1, nii2, nii3, and nii4 (Kronenberger et al., 1993
). Homologues of these genes have been found in two ancestral species of tobacco: those of nii1 and nii2 in Nicotiana tomentosiformis, and those of nii3 and nii4 in Nicotiana sylvestris (Kronenberger et al., 1993). It has been suggested that two of the four NiR genes (nii1 and nii3) from tobacco are predominantly expressed in leaves, and the other two (nii2 and nii4) are predominant in roots, and thus they have been termed ‘leaf NiR genes’ and ‘root NiR genes’, respectively (Kronenberger et al., 1993). The analysis of the differential expression of these four genes is vital to an understanding of the physiological significance of multiple isoforms of NiR existing in the plant kingdom. However, the whole picture of the open reading frames (ORFs) of these four genes is not available because not all of them have been cloned. Therefore, in this study NiR genes were cloned, and PCR primers specific to each gene were constructed and used to analyse the differential expression in roots and leaves.
The ORF for nii1, a leaf NiR gene (1764 bp long, encoding 587 amino acids), was reported by Vaucheret et al. (1992). A tobacco (Nicotiana tabacum L. cv. Xanthi XHFD8) genomic library (CLONTEC) was used to obtain the genomic clones of nii3 and nii4 used in this study, and the ORFs for the respective genes were deduced (accession numbers AB093533 and AB093534, respectively). The ORF for nii2 (accession number AB103507) was determined from a genomic sequence obtained by PCR cloning in this study.
The ORFs for nii2 and nii3 appeared to be the same length as that of nii1, whereas that for nii4 was 1755 bp long, and encoded 584 amino acids. The homology of nucleotide sequences for the ORF within leaf genes (nii1 and nii3) was estimated to be 97%, and that within root genes (nii2 and nii4) was 95%. However, the homology of nucleotide sequences for the ORF with four combinations between leaf and root NiR appeared to be 81–84%, indicating that the ORFs for leaf and root NiR genes are distinct.
PCR primer pairs specific to each of the four NiR genes were designed based on the NiR ORF sequences, and they were used for further analysis (Table 1). The mRNA levels determined by quantitative competitive RT-PCR in the leaves and roots of tobacco before and after nitrate treatment (Fig. 1) indicated that the expression pattern of the four NiR genes differed from the conventional view of ‘organ-specific’ expression of the genes (Kronenberger et al., 1993).
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Clearly, the mRNA of each of the four NiR genes was detectable both in leaves and roots (Fig. 1). In particular, the combined steady-state levels of nii2 and nii4 mRNA accounted for approximately 10% of total NiR mRNA in leaves both before and after nitrate treatment (Fig. 1). This indicates that each of the four NiR genes makes a small, but definite, contribution both in shoots and roots. Nitrate treatment increased the nii1 and nii3 mRNA in leaves and roots by at least 4-fold, and nii2 and nii4 mRNA in leaves and roots by at least 5-fold (Fig. 1). This indicates that all of the four NiR genes are inducible by nitrate.
The steady-state levels of nii1 and nii3 mRNA were almost the same in leaves (6–7x105 and 3x106 copies µg–1 of total RNA before and after nitrate treatment, respectively) and in roots (3–4x104 and 3–6x105 copies µg–1 of total RNA before and after nitrate treatment, respectively). Very similar relationships were obtained for the steady-state levels of nii2 and nii4 mRNA: 2–4x105 and about 8x106 copies µg–1 of total RNA in roots before and after nitrate treatment, respectively, and 5–9x104 and about 4x105 copies µg–1 of total RNA in leaves before and after nitrate treatment, respectively (Fig. 1). These steady-state mRNA levels indicate that the dominating (but not exclusive) NiR mRNA in leaves and roots originates, respectively, from nii1 and nii3 and from nii2 and nii4.
Although the results described above were obtained with 9-week-old plants that were cultured for 10 d in nitrogen-starved medium (see legend of Fig. 1), it is unlikely that the observed nii2 and nii4 expression in leaves (or nii1 and nii3 expression in roots) resulted from the prolonged nitrogen starvation because very similar results were obtained when tobacco plants were grown in the same medium supplemented with 10 mM ammonium succinate and 10 mM KCl (data not shown).
To date 14 NiR ORFs (including four tobacco ones) from 11 plant species have been reported: they are all 1749–1791 bp long, and encode 582–596 amino acids. Interestingly, nii2, nii4, and a hot pepper NiR gene (accession number AF065616) appeared to form a tight phylogenetic cluster. It is likely that hot pepper has another NiR gene that is evolutionally closer to nii1 and nii3, and that Solanaceae species possess leaf and root NiR genes like nii1 and nii3 and like nii2 and nii4, respectively, in tobacco.
Acknowledgements
This work was supported in part by the Research for the Future Program, Japanese Society for the Promotion of Science (JSPS-RFTF96L00604) and by a Grant-in-Aid for Creative Scientific Research (no. 13GS0023) from the Japan Society for the Promotion of Science. DNA was analysed with CREST-Akita Plant Molecular Science Satellite Laboratory, Life Research Support Center, Akita Prefectural University.
Footnotes
Abbreviations: NiR, nitrite reductase; ORF, open reading frame.
References
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