Vacuolar Na⁺/H⁺ Antiporter from Barley: Identification and Response to Salt Stress

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A. V. Vasekina¹, P. V. Yershov¹, O. S. Reshetova¹, T. V. Tikhonova¹, V. G. Lunin¹, M. S. Trofimova², and A. V. Babakov^1*

¹All-Russia Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, ul. Timiryazevskaya 42, 127550 Moscow, Russia; fax: (7-095) 977-0947; E-mail: avb@iab.ac.ru

²Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, 127276 Moscow, Russia; fax: (7-095) 977-8018; E-mail: pmembrane@ippras.ru

^* To whom correspondence should be addressed.

Received May 14, 2004; Revision received August 4, 2004

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One of the protective mechanisms used by plants to survive under conditions of salt stress caused by high NaCl concentration is the removal of Na⁺ from the cytoplasm. This mechanism involves a number of Na⁺/H⁺-antiporter proteins that are localized in plant plasma and vacuolar membranes. Due to the driving force of the electrochemical H⁺ gradient created by membrane H⁺-pumps (H⁺-ATPases and vacuolar H⁺-pyrophosphatases), Na⁺/H⁺-antiporters extrude sodium ions from the cytoplasm in exchange for protons. In this study, we have identified the gene for the barley vacuolar Na⁺/H⁺-antiporter HvNHX2 using the RACE (rapid amplification of cDNA ends)-PCR (polymerase chain reaction) technique. It is shown that the identified gene is expressed in roots, stems, and leaves of barley seedlings and that it presumably encodes a 59.6 kD protein composed of 546 amino acid residues. Antibodies against the C-terminal fragment of HvNHX2 were generated. It is shown that the quantity of HvNHX2 in tonoplast vesicles isolated from roots of barley seedlings remains the same, whereas the rate of Na⁺/H⁺ exchange across these membranes increases in response to salt stress. The 14-3-3-binding motif Lys-Lys-Glu-Ser-His-Pro (371-376) was detected in the HvNHX2 amino acid sequence, which is suggestive of possible involvement of the 14-3-3 proteins in the regulation of HvNHX2 function.

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KEY WORDS: barley, tonoplast, Na⁺/H⁺-antiporter, cDNA, salt stress

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