Coordinated Diurnal Regulation of Low- and High-Affinity Nitrate Transporters in Tomato

Abstract:

Two new members of the NRT2 gene family encoding high-affinity NO₃ ⁻ transporters were isolated from tomato by heterologous screening. Both cDNAs, LeNRT2;1 and LeNRT2;2, are highly similar in the coding region, however, significant nucleotide differences were found in the 3Œ-untranslated region. High-stringent Southern analysis with gene-specific probes indicated that the two genes are linked in the genome and are most probably derived from a recent gene duplication. Expression of both genes was restricted to roots. Although no difference in expression between both genes could be demonstrated by Northern analysis, LeNRT2;1 and LeNRT2;2 were isolated 56 and 3 times, respectively, from a cDNA library of root hairs, indicating a dominant expression of LeNRT2;1 in root hairs of tomato seedlings. Maximum transcript levels of LeNRT2 peaked 4 h after NO₃ ⁻ induction and coincided with those of the putative low-affinity NO₃ ⁻ transporter gene, LeNRT1;2. In contrast, 4 h after NO₃ ⁻ supply LeNRT1;1 transcription was maximally repressed, reflecting an opposite regulation. As transcript levels of LeNRT2 also increased independently from NO₃ ⁻ induction, diurnal gene expression was monitored. All investigated genes, LeNRT2, LeNRT1;1, LeNRT1;2 and LeNR, showed the highest peak in transcript levels 4 h after the onset of the light period and a second peak at the end of the light period. Although expression of these genes was highly coordinated at these times of the day, LeNRT1;1, LeNRT1;2 and LeNR showed additional peaks. This indicates that diurnal oscillation of these genes is most likely based on multiple factors, such as carbohydrate supply to the roots, feedback regulation from N assimilates and phytochrome-mediated regulation.

Abbreviations:

Le: Lycopersicon esculentum At: Arabidopsis thaliana Np: Nicotiana plumbaginifolia N: nitrogen NR: nitrate reductase NRT: nitrate transporter UTR: untranslated region

Key words:

Transcriptional regulation - diurnal expression - nitrate induction - NRT1 - NRT2 - nitrate reductase - tomato

References

• 01 Amarasinghe,  B. H. R. R.,, de Bruxelles,  G. L.,, Braddon,  M.,, Onyeocha,  I.,, Forde,  B. G.,, and Udvardi,  M. K.. (1998);  Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max). .  Planta. 206 44-52
• 02 Aslam,  M.,, Travis,  R. L.,, and Huffaker,  R. C.. (1992);  Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley (Hordeum vulgare L.) seedlings.  Plant Physiol.. 99 1124-1133
• 03 Becker,  T. W.,, Foyer,  C.,, and Caboche,  M.. (1992);  Light-regulated expression of the nitrate reductase and nitrite reductase genes in tomato and in the phytochrome-deficient aurea mutant of tomato.  Planta. 188 39-47
• 04 Bucher,  M.,, Schroeer,  B.,, Willmitzer,  L.,, and Riesmeier,  J.. (1997);  Two genes encoding extensin-like proteins are predominantly expressed in tomato root hair cells.  Plant Mol. Biol.. 35 497-508
• 05 Cardenas-Navarro,  R.,, Adamowicz,  S.,, and Robin,  P.. (1998);  Diurnal nitrate uptake in young tomato (Lycopersicon esculentum Mill.) plant: test of a feedback-based model.  J. Exp. Bot.. 49 721-730
• 06 Cheng,  C.-L.,, Acedo,  G. N.,, Dewdney,  J.,, Goodman,  H. M.,, and Conkling,  M. A.. (1991);  Differential expression of the two Arabidopsis nitrate reductase genes.  Plant Physiol.. 96 275-279
• 07 Crawford,  N. M.. (1995);  Nitrate: Nutrient and signal for plant growth.  Plant Cell. 7 859-868
• 08 Delhon,  P.,, Gojon,  A.,, Tillard,  P.,, and Passama,  L.. (1995);  Diurnal regulation of NO₃ ^- uptake in soybean plants: I. Changes in NO₃ ^- influx, efflux, and N utilization in the plant during the day/night cycle.  J. Exp. Bot.. 46 1585-1594
• 09 Deng,  M.-D.,, Moureaux,  T.,, Leydecker,  M.-T.,, and Caboche,  M.. (1990);  Nitrate reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves.  Planta. 180 257-261
• 10 Doddema,  H., and Telkamp,  G. P.. (1979);  Uptake of nitrate by mutants of Arabidopsis thaliana disturbed in uptake or reduction of nitrate. II. Kinetics.  Physiol. Plant.. 45 332-338
• 11 Filleur,  S., and Daniel-Vedèle,  F.. (1999);  Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display.  Planta. 207 461-469
• 12 Galangau,  F.,, Daniel-Vedèle,  F.,, Moureaux,  T.,, Dorbe,  M.-F.,, Leydecker,  M.-T.,, and Caboche,  M.. (1988);  Expression of leaf nitrate reductase genes from tomato and tobacco in relation to light-dark regimes and nitrate supply.  Plant Physiol.. 88 383-388
• 13 Geiger,  M.,, Walch-Liu,  P.,, Engels,  C.,, Harnecker,  J.,, Schulze,  E. D.,, Ludewig,  F.,, Sonnewald,  U.,, Scheible,  W.-R.,, and Stitt,  M.. (1998);  Enhanced carbon dioxide leads to a modified diurnal rhythm of nitrate reductase activity in older plants, and a large stimulation of nitrate reductase activity and higher levels of amino acids in young tobacco plants.  Plant Cell Environ.. 21 253-268
• 14 Godon,  C.,, Krapp,  A.,, Leydecker,  M.-T.,, Daniel-Vedèle,  F.,, and Caboche,  M.. (1996);  Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport.  Mol. Gen. Genet.. 250 357-366
• 15 Huang,  N.-C.,, Chiang,  C.-S.,, Crawford,  N. M.,, and Tsay,  Y.-F.. (1996);  CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots.  Plant Cell. 8 2183-2191
• 16 Johnstone,  I. L.,, McCabe,  P. C.,, Greaves,  P.,, Gurr,  S. J.,, Cole,  G. E.,, Brow,  M. A. D.,, Unkles,  S. E.,, Clutterbuck,  A. J.,, Kinghorn,  J. R.,, and Innis,  M. A.. (1990);  Isolation and characterization of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. .  Gene. 90 181-192
• 17 Keltjens,  W. G., and Nijenstein,  J H.. (1987);  Diurnal variations in uptake, transport and assimilation of nitrate and efflux of OH^- in maize plants.  J. Plant Nutr.. 10 887-900
• 18 King,  B. J.,, Siddiqi,  M. Y.,, Ruth,  T. J.,, Warner,  R. L.,, and Glass,  A. D. M.. (1993);  Feedback regulation of nitrate influx in barley roots by nitrate, nitrite and ammonium.  Plant Physiol.. 102 1279-1286
• 19 Krapp,  A.,, Fraisier,  V.,, Scheible,  W.-R.,, Quesada,  A.,, Gojon,  A.,, Stitt,  M.,, Caboche,  M.,, and Daniel-Vedèle,  F.. (1998);  Expression studies of Nrt2;1Np, a putative high-affinity nitrate transporter: evidence for its role in nitrate uptake.  Plant J.. 14 723-731
• 20 Lauter,  F. R.,, Ninnemann,  O.,, Bucher,  M.,, Riesmeier,  J.,, and Frommer,  W. B.. (1996);  Preferential expression of an ammonium transporter and two putative nitrate transporters in root hairs of tomato.  Proc. Natl. Acad. Sci. USA. 93 8139-8144
• 21 LeBot,  J., and Kirkby,  A. E.. (1992);  Diurnal uptake of nitrate and potassium during the vegetative growth of tomato plants.  J. Plant Nutr.. 15 247-264
• 22 Lejay,  L.,, Tillard,  P.,, Lepetit,  M.,, Olive,  F. D.,, Filleur,  S.,, Daniel-Vedèle,  F.,, and Gojon,  A.. (1999);  Molecular and functional regulation of two uptake systems for NO₃ ^- by N- and C-status of Arabidopsis plants.  Plant J.. 18 509-519
• 23 Liu,  K.-H.,, Huang,  C.-Y.,, and Tsay,  Y.-F.. (1999);  CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.  Plant Cell. 11 865-874
• 24 Perez,  M. D.,, Gonzalez,  C.,, Avila,  J.,, Brito,  N.,, and Siverio,  J. M.. (1997);  The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite and nitrate reductase and its disruption causes inability to grow in nitrate.  Biochem. J.. 321 397-403
• 25 Peuke,  A. D.,, and Jeschke,  W. D.. (1998);  The effects of light on induction, time courses, and kinetic patterns of net nitrate uptake in barley.  Plant Cell Environ.. 21 765-774
• 26 Prescott,  A., and Martin,  C.. (1987);  A rapid method for the quantitative assessment of levels of specific mRNAs in plants.  Plant Mol. Biol. Rep.. 4 219-224
• 27 Quesada,  A.,, Galvan,  A.,, and Fernandez,  E.. (1994);  Identification of nitrate transporter genes in Chlamydomonas reinhardtii. .  Plant J.. 5 407-419
• 28 Quesada,  A.,, Krapp,  A.,, Trueman,  L. J.,, Daniel-Vedèle,  F.,, Fernandez,  E.,, Forde,  B. G.,, and Caboche,  M.. (1997);  PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.  Plant Mol. Biol.. 34 265-274
• 29 Siddiqi,  M. Y.,, Glass,  A. D. M.,, Ruth,  T. J.,, and Rufty,  T. W. Jr.. (1990);  Studies of the uptake of nitrate in barley. I. Kinetics of ¹³NO₃ ^- influx.  Plant Physiol.. 93 1426-1432
• 30 Smart,  D. R., and Bloom,  A. J.. (1993);  Relationships between the kinetics of NH₄ ⁺ and NO₃ ^- absorption and growth in the cultivated tomato (Lycopersicon esculentum Mill. Cv T-5).  Plant Cell Environ.. 16 259-267
• 31 Swofford,  D. L.. (1997) PAUP: Phylogenetic analysis using parsimony, version 4 d 65. 
• 32 Trueman,  L. J.,, Richardson,  A.,, and Forde,  B. G.. (1996);  Molecular cloning of higher plant homologues of the high affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. .  Gene. 175 223-231
• 33 Tsay,  Y.,, Schroeder,  J. I.,, Feldmann,  K. A.,, and Crawford,  N. M.. (1993);  The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter.  Cell. 72 705-713
• 34 Unkles,  S. E.,, Hawker,  K. L.,, Grieve,  C.,, Campbell,  E. I.,, Montague,  P.,, and Kinghorn,  J. R.. (1991);  crnA encodes a nitrate transporter in Aspergillus nidulans. .  Proc. Natl. Acad. Sci. USA. 88 204-208
• 35 von Wirén,  N.,, Gazzarrini,  S.,, and Frommer,  W. B.. (1997);  Regulation of mineral nitrogen uptake in plants.  Plant Soil. 196 191-199
• 36 von Wirén,  N.,, Lauter,  F.-R.,, Ninnemann,  O.,, Gillisen,  B.,, Walch-Liu  P.,, Engels,  C.,, Jost,  W.,, and Frommer,  W. B.. (2000);  Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato.  Plant J..
• 37 Wang,  R., and Crawford,  N. M.. (1996);  Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.  Proc. Natl. Acad. Sci. USA. 93 (17) 9297-9301
• 38 Zhang,  D., and Lefebvre,  P. A.. (1997);  FAR1, a negative regulatory locus required for the repression of the nitrate reductase gene in Chlamydomonas reinhardtii. .  Genetics. 146 121-133
• 39 Zhou,  D.,, Okamoto,  M.,, Vidmar,  J. J.,, and Glass,  A. D. M.. (1999);  Regulation of a putative high-affinity nitrate transporter (NRT2;1At) in roots of Arabidopsis thaliana. .  Plant J.. 17 563-568