Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis

Science

Volumn 285, Issue 5431, 1999, Pages 1256-1258

  Apse, Maris P ^a   Aharon, Gilad S ^a   Snedden, Wayne A ^a   Blumwald, Eduardo ^a  

^a UNIVERSITY OF TORONTO   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

SODIUM CHLORIDE; SODIUM ION;

SALINITY TOLERANCE; SALT;

AGRICULTURE; ARABIDOPSIS; ARTICLE; BIOTECHNOLOGY; CELLULAR DISTRIBUTION; CYTOSOL; GOLGI COMPLEX; NONHUMAN; PLANT GROWTH; PRIORITY JOURNAL; PROTON SODIUM EXCHANGE; SALINITY;

ARABIDOPSIS; ARABIDOPSIS PROTEINS; CATION TRANSPORT PROTEINS; CHLORIDE CHANNELS; CHLORIDES; GENE EXPRESSION; HYDROGEN; PLANT LEAVES; PLANT PROTEINS; PLANTS, GENETICALLY MODIFIED; SODIUM; SODIUM CHLORIDE; SODIUM-HYDROGEN ANTIPORTER; VACUOLES;

EID: 0033588033     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5431.1256     Document Type: Article

References (22)

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12. C. P. Darley, D. van Wuyt Swinkel, K. Van der Woude, P. Mager, B. de Boer, ibid., p. 8.
13. R. A. Gaxiola et al., Proc. Natl. Acad. Sci. U.S.A. 96, 1480 (1999). GeneBank accession number AF106324.
14. Membrane proteins were purified from 5-week-old Arabidopsis thaliana plants using the method described (5) with the following modifications. Tonoplast-, Golgi/ER-, and plasma membrane-enriched fractions were obtained from the 0%/20%, 20%/ 30%, and 30%/40% interfaces, respectively. Chymostatin, leupeptin, aprotinin, and pepstatin (each to a 10 μM final concentration) were added to the resuspension buffer.
15. The open reading frame sequence obtained by our lab is identical to that recently described by Gaxiola et al. (13). The AtNHX1 putative open reading frame was amplified by polymerase chain reaction (PCR) from the cDNA template and cloned into the Sal I/Sma I sites of pBISN1 [S. B. Narasimhulu, X. Deng, R. Sarria, S. B. Gelvin, Plant Cell 8, 873 (1996)] in a sense orientation using the following primers; Se-ATX1-5′-CGCGTCGACATGTTGGATTCTCTAGTGTCG-3′ and AtXCT2-5′-CGGAATTCTCAAAGCTTTTCTTCCACG-3′. The resulting vector contained AtNHX1 under the control of the supermas promoter. Agrobacterium-mediated transformation was performed using flowering plants with primary bolts reaching 15 cm. Plants were dunked into a bacterial solution, for 5 min, containing 0.5× Murashige and Skoog (MS) salts; 0.5 g/liter MES; 5% sucrose and 0.03% Silwet. The same procedure was repeated 12 days later. Transgenic plants were selected by plating the seeds on 0.5× MS agar plates containing 25 mg/ liter kanamycin. Plants were carried for two more generations in order to identify plants homozygous for the transgene.
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20. M. P. Apse, G. S. Aharon, E. Blumwald, data not shown.
21. Antibodies were raised in rabbits against a glutathione S-transferase (GST)-fusion protein of the COOH-terminal region of AtNHX1. These fusion proteins were produced in Escherichia coli (strain BL21pLysS) that had been transformed with the pGEX2TK, into which we had subcloned the region encoding the final 95 amino acids of AtNHX1. The Eco RI-Bam HI fragment of the PCR product amplified from the cDNA template using the PCR primers, forward-5′-CGGGATCCCCGCACCAGAACGCCACC3′ and reverse-AtXCT2 (15), was ligated into the same sites on the pGEX2TK vector. Induction and purification of the recombinant GST-fusion was as per manufacturers instructions (Pharmacia). Antibodies were purified by first passing the antiserum over an immobilized GST column (Pierce), and then by affinity blot purification [E. Harlow and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988), p. 498].
22. iase and M. Manolson for antibodies raised against the V-ATPase. Supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to E.B.