Abstract
The rhodophyte seaweed Porphyra yezoensis, known more commonly world-wide as “nori”, is an important commercial crop in Japan. Cultivation of nori in Japan is often affected by outbreaks of “iroochi”, a discoloration of the thalli due to a decrease in inorganic nutrients in the culture area that in turn decreases the amount of photosynthetic pigments in the thalli. Treating thalli with inorganic nitrogen can reverse iroochi. In this paper, we report on the characterization of three P. yezoensis genes, a nitrate transporter (PyNRT2) and two urea transporters (PyUT1 and PyUT2), which may be involved in reversing iroochi. The predicted length of the PyNRT2 protein was 479 amino acids (AA), while PyUT1 and PyUT2 were 740 and 680 AA, respectively. PyNRT2 was more similar to NRT2 from a chromophyte than to NRTs from Chlamydomonas and higher plants. The two P. yezoensis UTs had 56% AA identity to each other, and showed the closest relationship to higher plant and yeast DUR3 proteins which formed a subfamily of the sodium-solute symporter protein family. Hydrophobicity plots of the AA sequences showed that the PyNRT2, PyUT1, and PyUT2 included 12, 15, and 16 transmembrane domains, respectively. Southern blot analysis indicated that the P. yezoensis genome has a single NRT2-encoding gene and at least four UT-encoding genes. Expression analysis of PyNRT2 and PyUT genes showed that the messenger RNA level of the PyNRT2 gene reached a maximum after 48 h in the nitrate starvation condition and was then restored to the constitutive level, while expression of the PyUT genes was induced in proportion to treatment times in the nitrate starvation condition. These results suggest that the PyNRT2 and PyUT are responsible for the high-affinity nitrate/urea transport systems that operate under low external nitrate concentrations.
Similar content being viewed by others
Abbreviations
- AA:
-
amino acid(s)
- DIN:
-
dissolved inorganic nitrogen
- EST:
-
expressed sequence tag
- NNP:
-
nitrate-nitrite porter
- NRT:
-
nitrate transporter
- nt:
-
nucleotide(s)
- ORF:
-
open reading frame
- PCR:
-
polymerase chain reaction
- PES:
-
Provasoli’s enriched seawater
- RACE:
-
rapid amplification of cDNA ends
- ss:
-
single-stranded
- SSS:
-
sodium-solute symporter
- TM:
-
transmembrane domain
- UT:
-
urea transporter
References
Amano H, Noda H. Effect of nitrogenous fertilization on the recovery of discoloured fronds of Porphyra yezoensis. Bot Mar 1987;30:467–73.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10.
Altschul SF, Madden TL, Schäffer AA, Zhang J, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402.
Arai M, Mitsuke H, Ikeda M, Xia JX, Kikuchi T, Satake M, Shimizu T. ConPred II: a consensus prediction method for obtaining transmembrane topology models with high reliability. Nucleic Acids Res 2004;32:W390–3.
Crawford NM, Glass ADM. Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci 1998;3:389–95.
ElBerry HM, Majumdar ML, Cunningham TS, Sumrada RA, Cooper TG. Regulation of the urea active transporter gene (DUR3) in Saccharomyces cerevisiae. J Bacteriol 1993;175:4688–98.
Filleur S, Daniel-Vedele F. Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display. Planta 1999;207:461–9.
Forde BG. Nitrate transporters in plants: structure, function and regulation. Biochim Biophys Acta 2000;1465:219–35.
Galván A, Fernández E. Eukaryotic nitrate and nitrite transporters. Cell Mol Life Sci 2001;58:225–33.
Hildebrand M, Dahlin K. Nitrate transporter genes from the diatom Cylindrotheca fusiformis (Bacillariophyceae): mRNA levels controlled by nitrogen source and by the cell cycle. J Phycol 2000;36:702–13.
Ito K, Sato S, Sato Y, Matsumoto F. Biochemical studies on the edible seaweed, Porphyra tenera-II. On the utilization of various nitrogenous compounds. Bull Japan Soc Sci Fish 1960;26:938–43.
Iwasaki H, Matsudaira C. Studies on cultural grounds of a laver, Porphyra tenera Kjellman in Matsulawa-ura intel-I. Environmental characteristics effecting upon nitrogen and phosphorus contents of laver. Bull Japan Soc Sci Fish 1954;20:112–9.
Jung H. The sodium/substrate symporter family: structural and functional features. FEBS Lett 2002;529:73–7.
Kojima S, Bohner A, von Wirén N. Molecular mechanisms of urea transport in plants. J Membrane Biol 2006;212:83–91.
Lejay L, Tillard P, Lepetit M, Olive FD, Filleur S, Daniel-Vedele F, Gojon A. Molecular and functional regulation of two \({\text{NO}}_3^ - \) uptake systems by N- and C-status of Arabidopsis plants. Plant J 1999;18:509–19.
Lejay L, Gansel X, Cerezo M, Tillard P, Müller C, Krapp A, von Wirén N, Daniel-Vedele F, Gojon A. Regulation of root ion transporters by photosynthesis: functional importance and relation with Hexokinase. Plant Cell 2003;15:2218–32.
Liu LH, Ludewig U, Frommer WB, von Wirén N. AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis. Plant Cell 2003;15:790–800.
Nozawa K. Nutrient uptake and fertilizing of Porphyra. Aquaculture 1959;7:1–12 (in Japanese).
Okamoto M, Vidmar JJ, Glass ADM. Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: response to nitrate provision. Plant Cell Physiol 2003;44:304–17.
Orsel M, Filleur S, Fraisier V, Daniel-Vedel F. Nitrate transport in plants: which gene and which control? J Exp Bot 2002;53:825–33.
Pao SS, Paulsen IT, Saier MH. Major facilitator superfamily. Microbiol Mol Biol Rev 1998;62:1–34.
Provasoli L. Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A, editors. Culture and collection of algae: Proceedings of US-Japan conference in Hakone. Tokyo: Japanese Society of Plant Physiologists; 1968. p. 63–75.
Quesada A, Fernández E. Expression of nitrate assimilation related genes in Chlamydomonas reinhardtii. Plant Mol Biol 1994;24:185–94.
Quesada A, Galván A, Fernández E. Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J 1994;5:407–19.
Quesada A, Hidalgo J, Fernández E. Three Ntr2 genes are differentially regulated in Chlamydomonas reinhardtii. Mol Gen Genet 1998;258:373–7.
Ranamalie Amarasinghe BHR, de Bruxelles GL, Braddon M, Onyeocha I, Forde BG, Udvardi MK. Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max). Planta 1998;206:44–52.
Sakaguchi K, Ochiai N, Park CS, Kakinuma M, Amano H. Evaluation of discoloration in harvested laver Porphyra yezoensis and recovery after treatment with ammonium sulfate enriched seawater. Nippon Suisan Gakkaishi 2003;69:399–404 (in Japanese).
Sano T. Studies on the colour changes of cultured lavers. I. On the change of hydrochrome. Bull Tohoku Reg Fish Res Lab 1955;4:243–61 (in Japanese).
Tamura S, Seki T, Sato M, Aoki K. Experimental fertilizing of Porphyra culturing area. Rep Chiba Pref Fish Exp St Tokyo Bay 1963;5:37–53 (in Japanese).
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80.
Trueman LJ, Onyeocha I, Forde BG. Recent advances in the molecular biology of a family of eukaryotic high affinity nitrate transporters. Plant Physiol Biochem 1996;34:621–7.
Tsay YF, Chiu CC, Tsai CB, Ho CH, Hsu PK. Nitrate transporters and peptide transporters. FEBS Lett 2007;581:2290–300.
Zemke-White WL, Ohno M. World seaweed utilization: an end-of-century summary. J Appl Phycol 1999;11:369–76.
Zhuo DG, Okamoto M, Vidmar JJ, Glass ADM. Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana. Plant J 1999;17:563–8.
Acknowledgments
This study was supported by the National Research Institute of Fisheries Science, Fisheries Research Agency, Japan, and was funded in part by Grant-in-Aids from the Fisheries Agency, Government of Japan and the Japan Society for the Promotion of Science. We thank Dr. M. Iwabuchi and Dr. T. Fukunaga of the Fukuoka Fisheries and Marine Technology Research Center, Japan, for supplying P. yezoensis strain FA-89 and for help with its culture. We also thank Dr. M. Kobayashi and M. Yoshikawa of National Research Institute of Fisheries Science, Japan, for help with the experiments.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kakinuma, M., Coury, D.A., Nakamoto, C. et al. Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta). Cell Biol Toxicol 24, 629–639 (2008). https://doi.org/10.1007/s10565-007-9053-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10565-007-9053-7