Molecular characterization of a novel Na+/H+antiporter cDNA from
Fabiola Baltierraa, Mabel Castillob, María Cecilia Gamboab, Matías Rothhammera, Erwin Krauskopfa,b,⇑
aFundación Ciencia & Vida, Zañartu 1482, Ñuñoa, Santiago, Chile
bUniversidad Andres Bello, Centro de Biotecnología Vegetal, Facultad de Ciencias Biológicas, República 239, Santiago, Chile
a r t i c l ei n f o
Received 15 November 2012
Available online 8 December 2012
a b s t r a c t
Environmental stress factors such as salt, drought and heat are known to affect plant productivity. How-
ever, high salinity is spreading throughout the world, currently affecting more than 45 million ha. One of
the mechanisms that allow plants to withstand salt stress consists on vacuolar sequestration of Na+,
through a Na+/H+antiporter. We isolated a new vacuolar Na+/H+antiporter from Eucalyptus globulus from
a cDNA library. The cDNA had a 1626 bp open reading frame encoding a predicted protein of 542 amino
acids with a deduced molecular weight of 59.1 KDa. Phylogenetic and bioinformatic analyses indicated
that EgNHX1 localized in the vacuole. To assess its role in Na+exchange, we performed complementation
studies using the Na+sensitive yeast mutant strain Dnhx1. The results showed that EgNHX1 partially
restored the salt sensitive phenotype of the yeast Dnhx1 strain. However, its overexpression in transgenic
Arabidopsis confers tolerance in the presence of increasing NaCl concentrations while the wild type plants
exhibited growth retardation. Expression profiles of Eucalyptus seedlings subjected to salt, drought, heat
and ABA treatment were established. The results revealed that Egnhx1 was induced significantly only by
drought. Together, these results suggest that the product of Egnhx1 from E. globulus is a functional vac-
? 2012 Elsevier Inc. All rights reserved.
Plants as sessile organisms have developed several mechanisms
that allow them to tolerate unfavorable environmental conditions
such as high temperature, drought and high salinity. Even though
salt accumulation occurs as a natural process in the environment,
the presence of high concentrations severely affectagricultural pro-
ductivity and biodiversity. Therefore, the maintenance of ion
homeostasis bythe plantsis essentialto survive inthese conditions.
In order to avoid Na+toxicity, the plant cell may either transport
the ions outside the cell or store them inside the vacuole, processes
mediated by specialized proteins. Some of them belong to the fam-
ily of Na+/H+exchangers, which can be located in the plasma mem-
brane or vacuole . Na+/H+antiporters have been identified in
several mammals, bacteria and plants . These transporters play
roles in pumping out Na+from the cytoplasm by exchanging it for
H+at the expense of the proton gradient generated by specialized
pumps in the cell and vacuolar membrane . This mechanism al-
lows plant cells to accumulate Na+in the vacuole and therefore
maintain the appropriate ion concentration in the cytoplasm. In
plants, these Na+/H+antiporters consist on a family of proteins
known as NHX, with eight members (NHX1-8) being NHX7/SOS1
located in the plasma membrane and the other seven in intracellu-
lar membranes [4,5]. Furthermore, transporters found in intracel-
lular membranes are classified as Class I or Class II proteins,
depending on their location. Class I transporters are located in
the vacuolar membrane (NHX1-4) while class II members are lo-
cated in the endosome (NHX5, 6 and 8) .
One of the most studied vacuolar transporters is NHX1, which
carries Na+to the inside of the vacuole and exchanges it for H+, pre-
venting sodium toxicity in the cytoplasm [6,7]. Overexpression of
Atnhx1 in Arabidopsis thaliana and other species such as Lycopersi-
cum esculentum confers high salinity tolerance [8,9]. Moreover,
mutant Atnhx1 Arabidopsis plants have shown an increased sensi-
tivity to salt stress compared to wild type Arabidopsis . Addition-
ally, an ortholog of Atnhx1 in Oryza sativa (Osnhx1) was found to
confer salt stress tolerance when it was overexpressed in the same
Eucalyptus globulus is considered the most important temperate
hardwood plantation species in the world due to its wood proper-
ties that make it suitable for the pulp and paper industry .
Moreover, this tree species has fast growth rates and ability to
adapt to a broad range of geographic locations. Most importantly,
Eucalyptus has been listed as one of the candidate biomass energy
crops by the U.S. Department of Energy . However, little infor-
0006-291X/$ - see front matter ? 2012 Elsevier Inc. All rights reserved.
⇑Corresponding author at: Universidad Andres Bello, Centro de Biotecnología
Vegetal, Facultad de Ciencias Biológicas, República 239, Santiago, Chile. Fax: +56 2
E-mail address: firstname.lastname@example.org (E. Krauskopf).
Biochemical and Biophysical Research Communications 430 (2013) 535–540
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