Download The Vacuolar Proton-Cation Exchanger EcNHX1

The Vacuolar Proton-Cation Exchanger EcNHX1
Generates pH Signals for the Expression of Secondary
Metabolism in Eschscholzia californica1
Sophie Weigl, Wolfgang Brandt, Renate Langhammer, and Werner Roos*
Institute of Pharmacy, Department of Pharmaceutical Biology, Laboratory of Molecular Cell Biology
(S.W., W.R.), and Institute of Genetics, Department of Molecular Genetics (R.L.), Martin Luther University
Halle-Wittenberg, 06120 Halle (Saale), Germany; and Leibniz Institute of Plant Biochemistry,
Department of Bioorganic Chemistry, 06120 Halle (Saale), Germany (W.B.)
ORCID IDs: 0000-0002-0125-1974 (S.W.); 0000-0002-0825-1491 (W.B.); 0000-0003-2687-5844 (R.L.).
Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine
alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at
the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons,
resulting in a peak of cytosolic H+. This study demonstrates that one of the Na+/H+ antiporters acting at the tonoplast of
E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA
(EcNHX1–EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell
lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered
overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na+-dependent H+ efflux
in a Dnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene
displayed Na+-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of
vacuolar H+. This finding was supported by three-dimensional protein homology models that predict a plausible recognition site
for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated
expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.
Plants react to microbial pathogens with the overproduction of antimicrobial secondary metabolites, so-called
phytoalexins (Bennett and Wallsgrove, 1994; Wink, 2003;
Zhao et al., 2005; Watts et al., 2011). This response is initiated by contact with pathogen-derived, low-molecular
elicitors and proceeds to the overexpression of enzymes
relevant to secondary biosynthesis (Dittrich and Kutchan,
1991; Viehweger et al., 2006; Ren et al., 2008; Boller and
Felix, 2009; Angelova et al., 2010). The signal paths
extending from elicitor recognition to gene expression
are not known in sufficient detail. This gap constrains
our understanding of how secondary metabolism complies with the fitness of the producing plant (Wink, 2003;
1
This work was supported by the German Research Council
(grant no. 889/12 to W.R.).
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Werner Roos ([email protected]).
S.W., supervised by W.R. and R.L., conducted most of the exper+
imental work, such as cloning of the EcNHX antiporters, Na+/H
antiport assays in permeabilized cells, RNAi-based gene silencing,
and complementation of yeast mutants; W.B. did the molecular modeling of EcNHX and ScNHX; W.R. coordinated the work and wrote
the article.
www.plantphysiol.org/cgi/doi/10.1104/pp.15.01570
Heinze et al., 2015) and biotechnological attempts aimed
to accumulate valuable plant metabolites (Leonard et al.,
2009; Aharoni and Galili, 2011).
The elucidation of signal paths that activate genes of
secondary metabolism in whole plants is often complicated by their integration into a hierarchically organized series of pathogen-triggered reactions, known as
the hypersensitive response (Lamb and Dixon, 1997).
Orchestrated by an oxidative burst, genes of secondary
metabolism are coregulated with those relevant to the
overproduction and polymerization of phenolics, cell
wall-localized proteins, and other defense constituents
(Tsunezuka et al., 2005; Truman et al., 2007). This
complexity is based on widely ramified signal cascades
that use ubiquitous intermediates such as jasmonates
(Blechert et al., 1995; Memelink et al., 2001), calcium
ions, and salicylates and are concatenated by various
modes of cross talk (Sudha and Ravishankar, 2002;
Aharoni and Galili, 2011).
In plant cell cultures, used as model systems of lower
complexity, two signal components related to the upregulation of secondary metabolism were investigated
in greater detail: (1) hormones or hormone-like signal
molecules and (2) regulatory proteins that control the
activation of genes relevant to a distinct secondary biosynthesis. The first group is dominated by oxylipins of
the jasmonate family (Gundlach et al., 1992; Memelink
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Weigl et al.
et al., 2001; Pauwels et al., 2009) and lysophosphatidylcholine (LPC; Viehweger et al., 2002, 2006). The second
group contains an increasing number of transcription factors, concentrated in the MYB, WRKY, bHLH, and AP2/
ERF families (van der Fits and Memelink, 2001, Pauw et al.,
2004a; Shoji et al., 2010; Yang et al., 2012; Schluttenhofer
and Yuan, 2015).
Signal pathways that selectively activate genes of a
secondary biosynthesis have long been supposed to
exist, either as distinguishable branches within complex
defense responses or as solitary mechanisms toward
the overproduction of distinct, specialized metabolites.
Tentative evidence arose from the induction of secondary biosynthetic enzymes independent of an oxidative burst and/or elevated levels of jasmonates (van
der Fits et al., 2000; Färber et al., 2003; Pauw et al.,
2004b). Transient shifts of the intracellular pH that
preceded the induction of secondary biosynthetic genes
were earlier considered as potential components of
signal pathways toward the expression of secondary
biosynthesis (Kuchitsu et al., 1997; Roos et al., 1998;
Roos, 2000; Shibuya and Minami, 2001). This evoked
activities to define the spatial and temporal properties of
pH transients necessary and sufficient for gene activation and the subcellular mechanism of their generation.
These topics were intensively investigated with cell
suspension cultures of Eschscholzia californica. The rootderived cells retain the plant’s ability to overexpress the
biosynthesis of benzophenanthridines, antimicrobial
alkaloids of the benzylisoquinoine class, in response to
pathogens or elicitors. Contact with a yeast glycoprotein elicits the induction of biosynthetic enzymes by a
signal path that involves a transient acidification of the
cytoplasm as an essential intermediary step. This was
proven by bracketing the vacuolar and cytoplasmic pH,
which stops the elicitation process, and by artificially
induced pH shifts, which mimic the elicitor effects
(Roos et al., 1998, 2006; Viehweger et al., 2006; Angelova
et al., 2010).
Prior to the pH shifts, the activation of a plasma
membrane-localized phospholipase A2 (PLA2) is
detectable in elicited cells, which gives rise to a transient peak of LPC in the cytoplasm (Roos et al., 1999;
Viehweger et al., 2002, 2006; Schwartze and Roos,
2008). In order to localize the interference of this molecule
with the intracellular signal transfer, we established an
osmotic procedure that permeabilized the plasma membrane for micromolecules less than 10 kD and left the
tonoplast functionally intact, as confirmed by the
vacuolar accumulation of pH probes, the ATP- or
Figure 1. pH shifts monitored in vacuoles of E. californica by confocal pH
topography (used with permission from
Viehweger et al., 2002). In situ vacuoles preloaded with the pH probe
DM-NERF were perfused with isotonic
medium containing the indicated ion
concentrations. A, pH maps obtained
at 100 mM KCl plus 10 mM NaCl show
ATP-triggered acidification (images A
and B) and LPC-triggered, transient alkalinization (images B–D), as quantified in the inset. B, In vacuoles perfused
with 70 mM KCl and 80 mM sorbitol,
1 mM LPC causes no pH peak unless
10 mM NaCl is present. C, pH shifts
caused by different extravacuolar Na+
concentrations, normalized to the pH
gradient between a particular vacuole
and the external medium (7.4). In the
presence of 1 mM LPC, the shifts of vacuolar pH (pHVac) occur at lower extravacuolar Na+ concentrations. Graph D, In
an experiment as in B, 1 mM LPC plus
10 mM NaCl were added together with
40 mM ethyl isopropyl amiloride (EIPA).
No pH peak was detectable.
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EcNHX Antiporter: Signals for Secondary Metabolism
pyrophosphate-powered acidification Fig. 1A, and the
loss of protons caused by inhibitors of the vacuolar H+
pumps (Viehweger et al., 2002). Upon perfusion with
isotonic media, these in situ vacuoles show an efflux of
preaccumulated protons that increases with the extravacuolar Na+ in the range from 10 to 30 mM. In the presence of LPC, this efflux requires less Na+ to occur (from 2
to 10 mM) but saturates at the same maximum velocity
(Fig. 1C). Thus, LPC would allow an efflux of vacuolar
protons at Na+ concentrations that are likely present in
the cytoplasm (this concentration was estimated to
5 mM; Viehweger, 2003). If Na+ is absent from the external medium (in situ vacuoles perfused with 70 mM
KCl), LPC causes no proton efflux unless 10 mM Na+ is
added (Fig. 1B).
The Na+-dependent efflux of vacuolar protons, initiated by either LPC or high Na+ concentrations, is
completely inhibited by amiloride (Fig. 1D), a longknown inhibitor of plant NHX-encoded antiporters
(Darley et al., 2000). Together with the aforementioned
data, this was taken as an indication that one or more
vacuolar Na+/H+ antiporters are required to generate
the pH signal in the cytosol. The causal sequence: elicitor contact/activation of PLA2/peak of LPC/efflux of
vacuolar protons/activation of genes encoding biosynthetic enzymes, was termed the LPC/pH signal
path (Roos et al., 2006; Viehweger et al., 2006).
The essential role of PLA2 as the initial signal generator of this pathway was further evidenced by a series
of knockdown experiments. First, silencing of PLA2
completely prevents elicitor-triggered alkaloid production (Heinze et al., 2015). Second, the elicitor activation
of this enzyme is controlled by surrounding Ga proteins
(Heinze et al., 2007), and the antisense-mediated knockdown of Ga inhibits the following elicitor-initiated events:
(1) activation of PLA2, (2) generation of vacuolar pH
shifts, and (3) overexpression of alkaloid biosynthetic
enzymes (Viehweger et al., 2006). Finally, the produced
benzophenanthridine alkaloids exert a long-range feedback inhibition at PLA2, thereby preventing continuous,
unlimited overexpression (Heinze et al., 2015).
In contrast to the LPC/pH signal path, the jasmonatetriggered expression of alkaloid biosynthesis, which can
be evoked in the same cell culture by higher elicitor
concentrations, involves elements of the hypersensitive
response (see above) but neither includes nor requires
vacuolar/cytosolic pH shifts (Roos et al., 1998; Färber
et al., 2003; Viehweger et al., 2006; Angelova et al., 2010).
This study addresses the question of how the efflux of
vacuolar protons, an essential element of the LPC/pH
signal path, is generated in elicitor-treated cells. Based
on the aforementioned inhibition by amiloride, we identified Na+/H+ antiporters of E. californica cells and investigated their susceptibility to elicitor-derived signal transfer
intermediates.
As a prerequisite for the assay of proton fluxes
across the tonoplast, we adapted our previously
established, microscopically validated methods of permeabilizing the plasma membrane for micromolecules
and fluorescence measurement of vacuolar pH (see
above; Viehweger et al., 2002, 2006) for use in microplatehosted cell suspensions (Fig. 2; see “Materials and
Methods”).
Na+/H+ antiporters of the plant tonoplast are
encoded by NHX genes that constitute a well-defined
group within the CPA1 (cation proton antiporter 1)
family of ion transporters. This family evolved from
bacterial NhaP antiporters that mediate an electroneutral exchange of Na+ for H+ and are found in
all kingdoms of life (Chanroj et al., 2012). The common origin of the catalytic domain explains why
Figure 2. Fluorescence detection of cells with permeabilized plasma
membranes. Cells were loaded with the pH indicator 59-carboxyfluorescein diacetate acetoxymethyl ester and subjected to the permeabilization procedure described in “Materials and Methods.” After
adding the membrane-impermeable stain propidium iodine, fluorescence images were obtained at excitation 480 6 10 nm/emission 525 6
50 nm (to detect the green fluorescence of 59-carboxyfluorescein diacetate acetoxymethyl ester) and at excitation 560 6 40 nm/emission
greater than 600 nm (to detect the red fluorescence of propidium
iodine), and the resulting images were overlaid in silico. Microscopy
was done with a Nikon Optiphot equipped with a Sony 3 CCD camera.
Images were obtained and processed with Optimas 6.2 software. A and
B, E. californica 6-d-old wild-type culture before (A) and after (B) permeabilization. Fluorescence overlay images are combined with the
transmission image. The objective lens was a Nikon Fluor 40, with
numerical aperture of 0.85. C and D, Fluorescence images of
S. cerevisiae cells (strain B4741) obtained under the same conditions
before (C) and after (D) permeabilization. The objective lens was a
Nikon Fluor 100 oil, with numerical aperture of 1.3. Arrows mark some
nuclear/cytoplasmic areas (n/c) and vacuoles (v). Accumulation of
59-carboxyfluorescein diacetate acetoxymethyl ester (green) indicates an ion-impermeable vacuolar membrane, and staining of DNA and
cytoskeleton areas by propidium iodine (red) indicates the lack of the ion
barrier function of the plasma membrane.
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Weigl et al.
Figure 3. A, Protein sequence alignment of the Na+/H+ antiporters EcNHX1 to EcNHX4 with selected NHX antiporters from
higher plants. The NHX genes detected in cDNA of E. californica were sequenced and translated in silico (rows 1–4). The EcNHX1
protein sequence was used to search for the nearest homologs among the completely annotated, NHX-encoded Na+/H+ antiporters in the National Center for Biotechnology Information (NCBI) protein database (rows 5–15). Identical amino acids appear in black.
(See text for percentage identity values.) The box marks amiloride-binding sites. Underlined sequences are encoded by the DNA
used for the RNA interference (RNAi)-based silencing of NHX genes (target sequence 1 in blue and control target sequence in
black). Arrows point to the LPC-binding amino acids Lys-397 and Gln-266 (see text). B and C, Phylograms of selected plant NHX
protein sequences aligned with the antiporters EcNHX1 to EcNHX4 from E. californica. Trees were generated by the neighborjoining algorithm with CLC viewer 7.6 software. Numbers are bootstrap values from 100 iterations. The length of each branch
corresponds to the rate of evolution of the named protein. B, The nearest homologs of EcNHX1 to EcNHX4 (for alignment, see A)
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EcNHX Antiporter: Signals for Secondary Metabolism
Figure 4. RNAi-based silencing of genes encoding Na+/H+ antiporters
in E. californica cells, shown by reverse transcription (RT)-PCR. RNA
isolated from the cell strains transformed with RNAi target sequence
1 (G11–G13) or target sequence 2 (G0) and the wild type (wt) was
subjected to the reverse transcriptase reaction, and the resulting cDNA
was probed with NHX gene-specific primers (Supplemental Table S1).
Previous experiments had confirmed that these primers allowed the
selective amplification of the expected parts of the named NHX genes.
The PCR products shown here were separated on agarose gels, ethidium
stained, and quantified densitometrically. Bands from one typical experiment are shown. Numbers give the cDNA content of each band,
averaged from four different culture batches, in percentage of the wildtype value (first column). SE ranges between 5% and 10% of the average
value. The actin cDNA band served as a loading control.
NHX-encoded plant antiporters share a molecular
heritage with animal Na+ channels, as exemplified by
the highly conserved binding site of the potent inhibitor
amiloride (Darley et al., 2000), which is in medical use
for the treatment of renal hypertension.
RESULTS
The Na+/H+ Antiporters of E. californica Are Homologous
to, But Well Distinguishable from, Other Members of the
Plant NHX Family
The sequence similarity between the published NHX
genes was instrumental to our PCR-based identification
of NHX homologs in E. californica. Starting from consensus sequence primers that included the amiloridebinding site, a series of PCR and RACE experiments led
us to the amplification of four different open reading
frames (ORFs) from complementary DNA (cDNA) of
E. californica. The sequences of these newly discovered
genes were translated in silico and showed high homology to known plant NHX sequences in the 12
transmembrane domains but much variability in the
N- and C-terminal regions (Fig. 3, A and B). The nearest
NHX homolog is found in Theobroma cacao and displays
an overall amino acid identity with the E. californica antiporters between 77% (EcNHX1) and 83% (EcNHX4).
As displayed in Figure 3B, the EcNHX antiporters form
two phylogenetically distinct pairs: EcNHX1 with
EcNHX3 (82% sequence identity) and EcNHX2 with
EcNHX4 (87%). Between these pairs, substantially lower
sequence identities exist (72%–74%). It also may be argued from this phylogram that EcNHX1 was subjected to
a faster evolution than the other homologs.
Sequence comparisons with plant NHX isoforms that
are known to be localized at either tonoplast or endosomal membranes confirm that the E. californica NHX
antiporters are part of the vacuolar NHX clade. As seen
in the phylogram in Figure 3C, the antiporters EcNHX1
to EcNHX4 are much more closely related to the vacuolar
isoforms of Arabidopsis (Arabidopsis thaliana; AtNHX1–
AtNHX4) or tomato (Lycopersicon esculentum; LeNHX1,
LeNHX3, and LeNHX4) than to the endosomally located
isoforms AtNHX5, AtNHX6 (Bassil et al., 2011, 2012), and
LeNHX2 (Venema et al., 2003). This coincides with our
earlier pH topographies, which suggested that the LPCstimulated Na+/H+ antiport occurs at the central vacuole
of the E. californica cells (see introduction).
RNA Interference-Mediated Silencing Reveals an Essential
Role of EcNHX Antiporters in the LPC/pH Signal Path
Based on the identification of four NHX genes that
encode Na+/H+ antiporters in E. californica, we attempted the simultaneous knockdown of most of their
transcripts in order to confirm whether any of these
antiporters are required for the elicitor-initiated signal
transfer. To this aim, some fairly conserved gene sequences were amplified, inserted into an expression
vector, and used as targets for RNAi-based gene silencing (see “Materials and Methods). The most consistent result was obtained with the so-named target
sequence 1 (Fig. 3A), which encoded part of the
amiloride-binding site and the following approximately 100 amino acids. About 50 mutant colonies
were isolated via antibiotic resistance and displayed
reduced alkaloid contents, as indicated by their white
color compared with the light-red wild type. In nine of
them, the presence of RNAi transcripts was confirmed
by PCR with vector-specific sequences. Cell suspension
Figure 3. (Continued.)
C, The NHX antiporters of E. californica compared with the homologs from Arabidopsis and tomato (alignment not shown).
EcNHX1 to EcNHX4 cluster with the tonoplast-located isoforms AtNHX1 to AtNHX4 and LeNHX1, LeNHX3, and LeNHX4. They
are separated from the endoplasmic reticulum-located isoforms AtNHX5, AtNHX6, and LeNHX2, which form their own subclade. AtNHX7 (SOS1), which is located at the plasma membrane, forms an outgroup.
Plant Physiol. Vol. 170, 2016
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Weigl et al.
cultures were established from these strains, and three
of them were selected for a more detailed characterization. As shown in Figure 4, the cell lines established
by RNAi against NHX mRNAs display different degrees of silencing among the isoforms, with the lowest
mRNA level of EcNHX1 in strain G13, of EcNHX3
in strain G11, and of EcNHX4 in strain G12. The
total Na+/H+ antiporter activity, assayed as the Na+dependent efflux of vacuolar protons, was diminished
to 30% to 50% of the wild type (Fig. 5A). The same
cultures lack most of the stimulation of the Na+/H+
antiport by LPC or after yeast elicitor treatment (Fig. 5,
B and C). The elicitor-triggered alkaloid production is
drastically reduced to similarly low levels in all RNAi
strains (Fig. 5D).
A similar procedure with a target sequence of lower
similarity between the EcNHX isogenes (target sequence 2 in Fig 3A) yielded cell cultures that displayed
less than 25% or even no loss in EcNHX mRNA levels,
alkaloid content, and vacuolar Na+/H+ antiport activity
as well as unimpaired alkaloid production in response
to elicitor. They were used as an internal process control
(strain G0 in Figs. 4 and 5) and indicated that the nongene-specific effects of the RNAi-silencing process were
negligible.
At this point, it appears that the knockdown of
Na+/H+ antiporters impairs the elicitor-triggered signal
transfer, but not proportional to the loss of antiport
activity at the tonoplast. For instance, cell lines G11 and
G13 display significantly different total Na+/H+ antiport activities (Fig. 5A) but a similarly low alkaloid
response to elicitor (Fig. 5D). This suggests that either a
threshold capacity of Na+/H+ antiport or the activity of
only one or two distinct antiporter(s) is required for
LPC-triggered signaling. Therefore, we conducted a
search for LPC-sensitive individual Na+/H+ antiporters
by yeast complementation studies.
EcNHX Antiporters Can Be Expressed in Yeast and
Complement the Dnhx Null Mutant
The yeast Saccharomyces cerevisiae contains only one
Na+/H+ antiporter of the NHX family, ScNHX1. A
yeast strain carrying the null mutation is available
(nhx1::kanMX4) and was used for complementation
experiments. Successful gene transfer into this background was documented by RT-PCR showing the
presence of transcripts of each of the plant’s NHX genes
in the yeast cells (Fig. 6, inset). The plasma membrane of
wild-type and transgenic yeast cells could be permeabilized by a slightly modified version of the protocol used for E. californica cells (see “Materials and
Methods”), and the resulting in situ vacuoles (Fig. 2D)
allowed the assay of Na+-dependent changes in the
vacuolar pH. As expected, the nhx1::kanMX4 strain
(Dnhx null mutant) displayed a negligible Na+/H+ antiporter activity, which could be substantially increased
by transformation with any of the four NHX genes of
E. californica (Fig. 6). The resulting transgenic strains all
show an Na+-dependent efflux of protons from the
vacuolar interior, which saturates at about 20 mM Na+,
similar to the wild type. Therefore, the successful
complementation of a phenotype that lacks Na+/H+
exchange processes indicates that the plant antiporters
are correctly expressed, targeted to, and inserted into
the yeast vacuolar membrane.
Figure 5. Effects of the silencing of NHX genes at
Na+/H+ antiport and elicitor-triggered events in
E. californica cells. The cell strains G11 to G13,
generated by RNAi with target sequence 1, are compared with the cell strain G0, which represents the
RNAi strains obtained with target sequence 2 and
serves as a negative control treatment. A, Na+dependent efflux of H+ from vacuoles of permeabilized cells. B, LPC-triggered efflux of H+ from
vacuoles of permeabilized cells at 5 mM external Na+.
Data from LPC-free controls are subtracted. LPC was
added to a final concentration of 2 mM. C, Elicitortriggered efflux of H+ from vacuoles in intact cells.
Yeast elicitor was present at 1 mg mL21. Data from
elicitor-free controls are subtracted. D, Elicitortriggered alkaloid production of intact cells, in
percentage of similarly treated wild-type (wt) cultures (100% represents an amount between 10
and 20 ng mg21 fresh weight in different culture
batches.) Yeast elicitor was present at 1 mg mL21. All
data are means 6 SE; n = 3 experiments from a single
culture batch of each strain. Repetition with two
different culture batches yielded similar relationships between the two mutant classes. Asterisks indicate significant (P , 0.05) differences with the
wild type.
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EcNHX Antiporter: Signals for Secondary Metabolism
Homology Modeling Supports a Selective Effect of LPC
Figure 6. Functional complementation of the yeast Dnhx mutant by the
NHX genes of E. californica. The yeast strains obtained by transformation of the Dnhx1 mutant 4290 with the genes EcNHX1 to EcNHX4
(termed Dnhx1+EcNHX1 to Dnhx1+EcNHX4) were assayed for the Na+dependent efflux of vacuolar protons and are compared with the
nontransformed mutant nhx1::kanMX4 (termed Dnhx1) and the corresponding wild-type BY4741. Data are means 6 SE; n = 5 cell suspension
cultures per mutant strain. The experiment was repeated with another
culture batch per strain and yielded similar relations between the antiport activities. The inset shows mRNA of Na+/H+ antiporters from E.
californica expressed in S. cerevisiae by RT-PCR. The ORFs of EcNHX1
to EcNHX4 were transferred to the S. cerevisiae Dnhx1 mutant 4290
(BY4741nhx1::kanMX4) as described in “Materials and Methods.” From
the resulting transgenic yeast clones nhx1::kanMX4 + EcNHX1 to
EcNHX4 (termed +EcNHX1 to +EcNHX4) and the nontransformed
mutant (termed Dnhx1), RNA was extracted and subjected to the reverse
transcriptase reaction, and the generated cDNA was probed with genespecific primers (Supplemental Table S1). The four PCR products shown
here are of the expected size (i.e. 556, 523, 525, and 410 bp). A typical
experiment is displayed, which was repeated with another culture batch
and yielded bands of similar size.
The selective stimulation of only one out of four
EcNHX antiporters by LPC raised the question of their
structural peculiarities. As x-ray crystallographic structures of sufficient resolution are not available for plant
NHX antiporters, a bacterial transporter was chosen as a
template, which bears the basal properties of the Na+/H+
exchange domain (see “Materials and Methods”). The
obtained three-dimensional (3D) model of EcNHX1 was
used for docking studies, which predicted a recognition
site for LPC close to the active center (Fig. 8). It consists of
three perfectly positioned side chains that detect the
phosphate group, the quaternary nitrogen, and the -OH
of this molecule (Fig. 9, left).
The non-LPC-stimulated antiporter EcNHX3 yields a
model with a dysfunctional LPC docking site (Fig. 9,
right): Gln is replaced by Glu, which likely establishes a
strong salt bridge to the phosphate-binding Lys of
NHX1 and thus strongly aggravates phosphate recognition. The other non-LPC-stimulated antiporters, EcNHX2
and EcNHX4, lack the critical Lys and, instead, bear Asn
in a homologous position (compare with the alignment in
EcNHX1 Selectively Reacts to the Signal Molecule LPC
The yeast clones obtained by complementation with
the E. californica NHX genes were tested for the effect
of the signal molecule LPC on the activity of the recombinant Na + /H + antiporters. The experiments
were performed at an extravacuolar Na+ concentration
of 5 mM, which is about its cytosolic concentration in
E. californica cells (Viehweger et al., 2002). Only the
strain transformed with EcNHX1 shows a clear stimulation of ion-exchange activity by LPC (Fig. 7). The
maximum effect is caused by about 4 mM LPC, as tested
in preliminary experiments. Under such conditions, the
transgenic EcNHX1 strain reaches a similar activity of
Na+/H+ antiport to the LPC-stimulated yeast wild type
(Fig. 7). As no plant genes other than the specified
EcNHX were transferred to the yeast cells, this finding
reveals a unique property of EcNHX1 among the Na+/H+
antiporters of E. californica.
Figure 7. Effects of LPC on the Na+/H+ antiport activity of transgenic
yeast strains. The transgenic yeast strains expressing EcNHX1 to
EcNHX4 (termed +EcNHX1 to +EcNHX4) were assayed for their impact
on the Na+/H+ antiport activity of 2 mM LPC (white columns) and 4 mM
LPC (hatched columns). Basal activities measured in the absence of LPC
are subtracted. The extravacuolar Na+ concentration was 5 mM in all
experiments. The yeast host strain nhx1::kanMX4 (termed Dnhx1) and
the corresponding wild-type BY4741 (termed wt) are included to show
nonspecific effects of LPC in cells lacking NHX-encoded antiporters
and in cells with the active yeast antiporter ScNHX1, respectively. Data
are means 6 SE; n = 3 cell suspension cultures per mutant strain. The
experiment was repeated twice with other culture batches and yielded
similar relations between the transgenic yeast strains.
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Weigl et al.
Figure 8. 3D model of the Na+/H+ antiporter EcNHX1 with docked LPC. The
model was calculated using the x-ray
structural data of the bacterial antiporter
NapA as a template. In accordance with
this molecule, the antiporter acts as a
dimer (one monomer appears in gray) in
which the amino acid Asp-154 is exposed as an essential cation-binding site
(Lee et al., 2013). LPC (shown as a spacefilling model; green carbon atoms) docks
close to this area.
Fig. 3A), which also would hamper a recognition of LPC’s
phosphate moiety.
Interestingly, the yeast Na+/H+ antiporter ScNHX1 is
also stimulated by LPC (Fig. 7). Homology modeling of
this antiporter to a yeast template (see “Materials and
Methods”) likewise predicts a 3D structure with a wellfitting LPC recognition site (Fig. 10). Although the
amino acid sequence identity with EcNHX1 is low
(31%), phosphate and quaternary nitrogen of LPC appear to be recognized by Lys and Glu as well (Fig. 11).
Figure 9. The LPC recognition site of EcNHX1 and the homologous structure in EcNHX3. In EcNHX1 (left), the model of Figure 8
predicts a precise recognition of LPC (in color) through its phosphate moiety by Lys-397 and Gln-266 and its trimethyl-ammonium
group by Glu-263. The fatty acid fits to the hydrophobic side chains of Phe-79, Tyr-14 (of chain B), and Tyr-396. The hydroxyl
group of LPC is recognized by Thr-400 as well as by Ser-11 (of chain B). In EcNHX3 (right), Lys-400, which is at the homologous
position to Lys-397 in EcNHX1, is blocked by a strong salt bridge to Glu-269 (which attains the position homologous to Gln-266 in
NHX1). This likely prevents recognition of the PO4 moiety of LPC.
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EcNHX Antiporter: Signals for Secondary Metabolism
Thus, the selective activation by LPC of both the recombinant EcNHX1 and the genuine ScNHX1 antiporter, as measured at the level of Na+/H+ antiport, is
in line with the structural peculiarities predicted by homology models.
DISCUSSION AND CONCLUSION
Our data concordantly indicate that one of the four
Na+/H+ antiporters of E. californica cells is selectively
stimulated by LPC, a product of PLA2, and thus constitutes an essential element in the signal transfer toward the expression of alkaloid biosynthesis. As the
stimulation could be transferred with the EcNHX1 gene
to a yeast strain, proteins that might be associated with
the EcNHX1 protein in E. californica cells would not
obscure this result. The involvement in the stimulation
process of one or more protein(s) conserved between
plant and yeast is not excluded. However, activation
via protein phosphorylation, as supposed for the longknown stimulation by LPC of the plasma membrane
H+-transporting ATPase (Xing et al., 1996), is less likely,
as the protein kinase inhibitor staurosporine and changes
in Ca2+ did not influence the LPC-triggered proton fluxes
(Viehweger et al., 2002).
The higher concentration of LPC required for a full
stimulation of recombinant EcNHX1 in yeast (about
4 mM) compared with genuine EcNHX1 in E. californica
(about 2 mM; Viehweger et al., 2002) might reflect differences between the plant and yeast systems (e.g.
compartmentation and metabolism of LPC). Based
on earlier results about the metabolism of LPC in
E. californica cell cultures, it is likely that the LPC molecule
itself, rather than a rapidly made metabolite, stimulates
the Na+/H+ antiporter: the only metabolite detectable after a 20-min feeding of radiolabeled LPC was
phosphatidylcholine (made by reacylation; Schwartze
and Roos, 2008). This compound does not stimulate the
Na+/H+ antiport activity, and the same holds true for
potential degradation products such as lysophosphatidic acid, phosphorylcholine, and fatty acids (tested by
Viehweger et al. [2002]).
The stimulation by LPC of the recombinant antiporter EcNHX1 is compatible with the data from
E. californica cells: in the RNAi mutants that show decreased elicitor-triggered proton fluxes and lack of
elicited alkaloid production (G11–G13), EcNHX1 is the
Figure 10. 3D model of the Na+/H+ antiporter ScNHX1 with docked LPC. The 3D model, obtained with a template protein of
archaebacterial origin (see “Materials and Methods”), predicts the antiporter to act as a dimer (one monomer appears in gray) in
which the amino acid Asp-201 is exposed as a conserved cation-binding site (Lee et al., 2013). Recognition of LPC (shown as a
space-filling model; green carbon atoms) might occur close to this area.
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Weigl et al.
Figure 11. Recognition of LPC in the yeast Na+/H+
antiporter ScNHX1. LPC (in color) is supposed to interact through its phosphate moiety with Lys-242, His244, and Asn-310 and the quaternary nitrogen with
Glu-238. The fatty acid fits well to the hydrophobic side
chains of Phe-120 and Leu-450. The hydroxy group of
LPC is detected by Gln-453 as well as by Lys-242.
only NHX gene that is significantly silenced in each cell
line, although to different degrees. We thus conclude that
EcNHX1 is the searched-for component of the signal
chain (Roos et al., 1998) that recruits the proton gradient
across the tonoplast for the cytoplasmic acidification.
It appears that even a moderate loss of EcNHX1
strongly impairs the signal transfer: while the remaining mRNA content of EcNHX1 in the RNAi cell lines
ranges from 17% to 75% (Fig. 4, first lane), all strains
show the same phenotype (i.e. the elicitor-triggered
alkaloid production drops to near zero; Fig. 5D). Although the expressed antiporter proteins and individual activities in the RNAi strains are not quantified
(antibodies of sufficient isoform selectivity are not available), it is tempting to assume that the full expression of
EcNHX1 is required to generate a pH shift sufficient to
induce alkaloid biosynthetic genes. Earlier data show that
the extent and duration of elicitor-triggered or artificial H+
peaks in the cytoplasm need to surpass a threshold to
overexpress alkaloid biosynthesis. According to a large
number of observations, an H+ peak to be recognized as a
signal for gene expression requires cytoplasmic pH to stay
below 7.1 for at least 10 min (Viehweger et al., 2006).
Vacuoles with only three-fourths of EcNHX1 capacity
(e.g. in strain G12) might be unable to generate this critical
efflux.
As found with individual vacuoles, LPC increases the
sensitivity of the vacuolar Na+/H+ exchange process
toward extravacuolar Na+ but does not affect the
maximum exchange capacity (Fig. 1C; Viehweger et al.,
2002). This is well compatible with our data here from
the recombinant EcNHX1 and explains why the LPCstimulated antiporter (Fig. 7) reaches a similar activity
at 5 mM Na+ to the Na+-saturated antiporter in the absence of LPC (Fig. 6).
In several plants, antiporters of the vacuolar NHX
clade are known to catalyze both Na+/H+ antiport and
K+/H+ antiport, the latter being an important component of pH control in growth and developmental processes (Bassil et al., 2011; Barragán et al., 2012). At the in
situ vacuoles of E. californica used in this study, LPC
likely stimulates the Na+/H+ antiport: at the extravacuolar K+ concentration of 100 mM, LPC triggers no
measurable pH shift unless Na+ is added. NaCl at 5 mM
causes a measurable proton efflux that is stimulatable
by LPC (Fig. 5A), and comparable data exist from individual cells (Fig. 1B). Therefore, it appears justified to
conclude that LPC acts at EcNHX1 by increasing its
affinity toward Na+ at the cytoplasmic side (but not or
less toward K+) and, thus, allows an exchange with
vacuolar H+ even at the low cytoplasmic Na+ concentration of about 5 mM. This might not exclude the idea
that, in intact cells, K+ gradients exist across the tonoplast that allow EcNHX1 to act in the K+/H+ antiport
mode and be stimulatable by LPC.
The in silico models of EcNHX and ScNHX antiporters still await validation by site-directed mutations
and/or direct binding assays. Therefore, the suggestive
docking data do not finally prove the binding of LPC. Here
is clearly scope for forthcoming studies. Nevertheless,
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EcNHX Antiporter: Signals for Secondary Metabolism
the existence at EcNHX1 of a precise recognition site for
LPC indicates a structural peculiarity not seen in the
homologs: this molecule appears to be selected by a 3fold interaction (i.e. phosphate to Lys, quaternary
nitrogen to Glu, and -OH to Thr and Ser). Each of the
three non-LPC-stimulated antiporters of E. californica
lacks at least one of these potential sites of interaction.
Thus, the risk of a misfit in the in silico procedures of
modeling and docking (see “Materials and Methods”)
appears low, although not negligible. Based on the 3D
models, it appears that only a few sequence deviations
from the other EcNHX proteins are required to create an
LPC recognition site in EcNHX1, notably the replacement of Glu-269 by Gln (Fig. 9).
Despite the evolutionary distance between E. californica
and yeast, both organisms harbor Na+/H+ antiporters
that are activated by LPC (Fig. 7) and likely contain
specific recognition sites for this signal molecule in the
neighborhood of the catalytic site (compare Figs. 9 and
11). ScNHX1 marks the phylogenetic origin of the NHX
family (Chanroj et al., 2012), and its stimulation by LPC
might thus indicate an original property of the NHX
progenitor protein. It is tempting to speculate that the
susceptibility to LPC was maintained or reinvented in
one NHX antiporter of E. californica because of its advantageous function in the LPC/DpH signal path: the
ability to selectively express the biosynthesis of phytoalexins (i.e. independent of the hypersensitive cell
death) might cause a positive selection pressure. This
idea would be compatible with a faster evolution rate
of EcNHX1, as suggested by the phylogenetic tree in
Figure 3B.
The vacuolar NHX antiporters of plants are long
known as crucial players in cellular pH regulation and
ion homeostasis (Apse et al., 2003; Pardo et al., 2006).
The complementation of physiological experiments by
genetic knockout techniques (Casey et al., 2010; Bassil
et al., 2011; Barragán et al., 2012) and the discovery of
endosomal isoforms of NHX antiporters allowed a
broader understanding of how these elementary functions are instrumental to a variety of growth and developmental processes. Acting coordinately with vacuolar
and endosomal proton pumps, NHX antiporters control
pH-dependent steps in organellar development, endosomal trafficking, cell expansion, and responses to abiotic
stress (for review, see Bassil et al., 2012). In contrast with
this progress, not much is known about the control of
NHX-encoded Na+/H+ antiporters at the activity level.
Their regulation by phosphorylation is deduced from
phosphoprotein screens but not finally proven (Endler
et al., 2009). Clear evidence exists for the regulation of
Na+/K+ selectivity at the C terminus of AtNHX1, which
extrudes into the vacuolar lumen. Here, the calmodulinlike protein AtCaM15 binds in a pH- and Ca2+-dependent
manner, thereby inhibiting the Na+/H+, but not the K+/H+,
antiporter activity (Yamaguchi et al., 2005)
To our knowledge, genuine, low-molecular effectors
that control Na+/H+ exchange activities at plant intracellular membranes are not yet reported. The activation
by LPC, as demonstrated in this study, points to a novel
function of NHX-encoded antiporters in pathogen defense and phytoalexin biosynthesis.
It appears worthwhile to test a variety of plant
Na+/H+ antiporters for this particular property in order
to reveal potential roles in the transmission of elicitorlike signals and determinants of their molecular evolution. A recent finding that might motivate such research is
the activation of monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus cells by both LPC and yeast
elicitor (Heinze et al., 2015).
MATERIALS AND METHODS
Plant and Yeast Cultures
Submerged growing cell cultures of Eschscholzia californica, established
previously from upper root tissue of greenhouse plants (according to Angelova
et al. [2010]), were maintained in Linsmayer and Skoog medium in a 9-d growth
cycle in rotary shakers (for details, see Viehweger et al., 2002). Cells were
transferred to new medium by filtration with mild suction through a nylon
mesh of 50 mm2, and the cell density was kept at 50 mg fresh weight mL21, if not
indicated otherwise. For biolistic transformation, callus cultures were used after
pregrowing them on culture medium with 2% agar.
Yeast elicitor is a glycoprotein fraction prepared from baker’s yeast
(Saccharomyces cerevisiae) by ethanol precipitation (according to Schumacher
et al. [1987]) and purified by ultrafiltration, FPLC, and SDS-PAGE. It contains
about 40% Man (Heinze et al., 2007). Dosage refers to the dry weight of the
crude elicitor preparation.
The yeast strain BY4741 and the related Dnhx mutant 4290 (nhx1::kanMX4)
were obtained from the Euroscarf strain collection (http://euroscarf.de). Yeast
were grown in 1% yeast extract, 2% peptone, and 2% Glc or synthetic minimal
medium (0.67% yeast nitrogen base without amino acids) mixed with amino
acid/base supplement according to Sherman et al. (1986). The carbon sources
Glc and Gal were added to a final concentration of 2%. For selection of transformants, the corresponding amino acid or base was omitted.
Fluorescence Assay of Benzophenanthridine Alkaloids
Elicited cell cultures of E. californica accumulate mainly dihydrobenzophenanthridines (about 80%) and benzophenanthridines (about 20%). They
were quantified by their fluorescence at excitation 360 nm/emission 460 nm
(dihydroalkaloids) and excitation 490 nm/emission 570 nm (benzophenanthridines) using authentic dihydrosanguinarine and sanguinarine as reference
compounds. The assay is described in detail by Angelova et al. (2010). In brief,
500-mL samples were withdrawn from cell suspensions, thoroughly mixed with
500 mL of methanol containing 0.3 M KOH, extracted by mild shaking (20 min at
40°C), and centrifuged (10 min at 13,000 rpm). A total of 150 mL of supernatant
was mixed with 15 mL of 5 N H2SO4 and transferred to black quartz microtiter
plates, and the fluorescence was read in the Microplate Fluorescence Reader
FLX 800 (BioTek).
Activity of Vacuolar Na+/H+ Antiporters
Permeabilization of the Plasma Membrane for Micromolecules
in Plant and Yeast Cells
This procedure takes advantage of the higher flexibility against osmotic
pressure of the tonoplast compared with the plasma membrane and is based on
microscopic studies of Viehweger et al. (2002). Cells loaded with the pH indicator 5-carboxyfluorescein (see below) were incubated for 15 min in hypertonic
medium 1 (300 mM KCl, 20 mM K-HEPES, and 5 mM reduced glutathione [GSH],
pH adjusted to 7.4 with KOH), followed by a further 15 min in the slightly
hypotonic medium 2 (100 mM KCl, 20 mM K-HEPES, and 5 mM GSH, pH adjusted to 7.4 with KOH), and finally resuspended in the near-isotonic maintenance medium (100 mM KCl, 50 mM MOPS/1,3-bis(tris[hydroxymethyl]
methylamino) propane, 5 m M GSH, 5 m M NaCl, 0.5 m M sodium citrate,
0.5 mM Na2HPO4, 2 mM ascorbic acid, 10 mM KCN, and 0.1% [w/v] bovine
serum albumin, pH adjusted to 7.4 with KOH). The enhanced permeability of
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Weigl et al.
the plasma membrane to micromolecules (the above procedure generates
pores that allow the permeation of fluorescent dextrans up to 10 kD; Viehweger
et al., 2002) was tested by adding 75 nM propidium iodine. This fluorescent
cation stains double-stranded DNA and cytoskeleton of permeable cells, while
the intactness of the tonoplast is indicated by the vacuolar accumulation of
5-carboxyfluorescein (Fig. 2). Yeast cells were permeabilized by the same procedure except that medium 1 contained 400 mM instead of 300 mM KCl.
Assay of Na+/H+ Antiport
This assay relies upon the vacuolar trapping of the pH indicator
59-carboxyfluorescein, which is liberated from 59-carboxyfluorescein diacetate
acetoxymethyl ester during the incubation of intact cells. It was shown earlier
that acetoxymethyl esters are split in the cytoplasm, whereas the vacuole lacks
this esterase but contains other deacylating esterases that cleave the absorbed
59-carboxyfluorescein diacetate, giving rise to the vacuolar trapping of the trianion
(Roos, 2000).
After 7 to 9 d of culture, cells were suspended in phosphate-free, 75% (v/v)
culture liquid and incubated in rotary shakers with 100 nM carboxyfluorescein
diacetate acetoxymethyl ester plus 100 mM eserine (to inhibit extracellular
cleavage of the fluorescein esters). Incubation was terminated if at least 90% of
the intracellular fluorescence was accumulated in the vacuoles (30–60 min), as
confirmed by microscopy of test samples. The carboxyfluorescein diacetateloaded cells were immediately subjected to the selective permeabilization of
the plasma membrane (see above). Of the resulting cell suspension, 100-mL
aliquots were pipetted onto a quartz microtiter plate with light-shielded compartments, supplied with the indicated effectors, and mounted in the Microplate Fluorescence Reader FLX 800 (BioTek). pH-dependent fluorescence was
assayed by excitation ratioing, based on simultaneous measurements at excitation 435 6 20 nm (channel 1), excitation 485 6 20 nm (channel 2), and emission
520 6 20 nm (both channels). The emission intensity was ratioed (channel 2 to
channel 1) and converted into pH using a calibration graph obtained with
similarly treated cells that were suspended in nutrient solutions containing
40 mM sodium-MES buffers to fix the external pH at eight different values. To
facilitate the equilibration of external and vacuolar pH in the calibration experiments, each medium contained 5 mM pivalic acid (in the pH range 4–5.5) or
80 mM methylamine (in the pH range 5–7.5).
Proton efflux from the vacuole was quantified as the change in H+ concentration between two measurements (at time intervals of 0.75 to 2 min) and
ratioed to the direct driving force (i.e. the difference between the pH of the
vacuole at the first time point and the external medium; the latter was kept at
pH 7.4 throughout the experiment).
Routine tests with cell suspensions treated by the above permeabilization
procedure and subsequent pH assay confirmed essential properties of the
vacuolar H+ pool, such as ATP-fueled acidification and bafilomycin-triggered
alkalinization, similar to the in situ vacuoles investigated earlier in single cells
(Viehweger et al., 2002).
Cloning and Sequencing of NHX Genes
Standard procedures were followed if not indicated in detail, and kit systems
were used as advised by the manufacturers.
To search for active NHX genes, total RNA was isolated from E. californica cell
cultures (RNeasy Plant Mini Kit; Qiagen) and used to generate double-stranded
cDNA (RevertAid M-MulV Reverse Transcriptase Kit; Thermo Scientific). The
cDNA served as a template for a series of PCRs with primers derived from
conserved regions of plant NHX genes (searched in the NCBI nonredundant
library). The resulting amplimers were sequenced and used to design primers
for new PCRs, including sequences that extended from overlapping areas.
PCRs were run for about 30 cycles under conditions recommended by the
manufacturer of each polymerase used (mostly Taq polymerase [Fermentas] or
Phusion High-Fidelity DNA-Polymerase II [Finnzyme]). In parallel, incomplete
NHX cDNAs were subjected to 39 and 59 RACE-PCR with the Marathon cDNA
Amplification Kit (Clontech). These procedures led to a stepwise disclosure of
four ORFs with high homology to plant NHX antiporters.
For the detection of NHX mRNAs by RT-PCR, cDNA was obtained from
RNA of transformed and wild-type cells as described above and used as a
template in PCRs with gene-specific primers (Supplemental Table S1) that were
designed from the divergent sequences in the C termini of the EcNHX antiporters (Fig. 3A). Each primer combination was tested at optimized melting
temperature and different dilutions for a potential cross-amplification of undesired NHX cDNAs. No such PCR products were found with different templates, as proved by sequencing. The amplified cDNA bands were separated by
DNA agarose electrophoresis, ethidium stained, and quantified by fluorescence
densitometry. The actin mRNA served as an internal load control.
RNAi-Based Silencing of NHX Genes
In principle, DNA sequences intended to generate hairpin RNA for the
targeted degradation of EcNHX mRNAs were selected from the known cDNAs
(Fig. 3A), amplified by PCR with gene-specific primers (Supplemental Table
S1), and cloned in an expression vector that was finally delivered to cultured
cells by biolistic gene transfer. Using Gateway cloning (Invitrogen TOPO
cloning procedure) and following the manufacturer’s advice, the target sequence was first introduced into the entry vector pCR8/GW/TOPO and
transferred to the RNAi destination vector pK7GWIWG2(II) by LR Clonase II.
Positive bacterial clones, resulting from the replacement of the vector’s lethal
ccdB gene by the target sequence, were selected, and the desired new vector was
isolated using the QIAprep Spin Miniprep Kit (Qiagen). As confirmed by DNA
sequencing, the isolated vector contained the target sequence twice, in opposite
directions, separated by an intron and each flanked by an attR2 site. The map of
pK7GWIWG2(II) is available at http://www.uoguelph.ca/~jcolasan/pdfs/
gateway_protocols_and_plasmids.pdf.
The RNAi vector DNA was fixed at gold particles and used for the biolistic
bombardment of E. californica wild-type cells by the Bio-Rad PDS-1000/He
Particle Delivery System, following a recently published protocol (Heinze
and Roos, 2013). The stably transformed cell lines were grown over at least three
9-d passages on selection medium containing 100 mg mL21 paromomycin and
later maintained by growth cycles in medium with 50 mg mL21 paromomycin.
The presence in the transgenic plant cells of DNA encoding the intended
double-stranded RNA sequences was confirmed by PCR with genomic DNA
(extracted by the cetyl-trimethyl-ammonium bromide (CTAB) method) and
specific primers that amplified sequences extending from a flanking region of
the destination vector to the adjacent target sequence.
Transformation of Yeast Dnhx Mutants by EcNHX Genes
The ORF of EcNHX1 was amplified by PCR, and the product was cloned
into the pBluescript II vector (In-Fusion HD Cloning Kit; Clontech), thereby
attaching the restriction sites XbaI (59) and EcoRI (39). The ORFs of EcNHX2,
EcNHX3, and EcNHX4 received the restriction sites XbaI (59) and XhoI (39) via
PCR and were cloned into the pJET1.2/blunt vector (CloneJet PCR Cloning Kit;
Thermo Scientific). The primers used are listed in Supplemental Table S1. In a
second step, the genes were cloned into the yeast expression vector p416GAL1
and isolated for further use in the transformation/complementation of yeast cells.
The yeast Dnhx null mutant 4290 (nhx1::kanMX4) was transformed with the
expression vector p416GAL1, carrying the ORFs of EcNHX1 to EcNHX4 (prepared as described above), according to Akada et al. (2000). Yeast cultures
transformed with the empty p416GAL1 vector served as controls. Transformed
yeast colonies were selected via the uracil prototrophy conferred by the URA3
gene in the expression vector. The recombinant yeast strains obtained were
further characterized by RT-PCR with primers that allowed the specific amplification of EcNHX isoforms (Fig. 6, inset).
Molecular Modeling of NHX Antiporters
All 3D models were established with the YASARA software, version 14.7.17
(Krieger et al., 2009). The underlying algorithm selects potential templates not
only by their sequence identity to the target (which amounts here to about 20%)
but includes similarities of secondary structures as predicted by PSI-BLAST and
PSI-PRED (Jones,1999). By applying these criteria to the homology modeling of
EcNHX1 and EcNHX3, the bacterial Na+/H+ antiporter NapA (Lee et al., 2013;
Protein Data Bank code 4BWZ) was found to be the best template protein
available, with sufficient resolution (3 Å). The comparison of this template with
the targets EcNHX1 and EcNHX3 yields expect values of 0.015 and 0.042, respectively (i.e. fairly below the minimum level of 0.5, which is required by
YASARA’s homology-modeling procedure). The resulting models were positively evaluated, as suggested by Z scores of 21.488 (EcNHX1) and 21.454
(EcNHX3).
For the modeling of ScNHX1, four promising templates (each with an expect
value below 1e-19) were identified among proteins of archaebacterial origin (e.g.
MjNhaP1 [Paulino et al., 2014; Wöhlert et al., 2014]; Protein Data Bank codes
4CZB, 4CZ8, 4CZA, and 4CZ9) and used to create a best-ranging hybrid model
that was evaluated positively with a Z score of 21.595.
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EcNHX Antiporter: Signals for Secondary Metabolism
The stereochemical quality of all models was finally checked with the
PROCHECK software (Laskowski et al., 1993). In the cases of EcNHX1 and
EcNHX3, 93.3% of all residues are located in the most favored area of the
Ramachandran plot, with two outliers. The model of ScNHX1 shows 90% of all
residues in most favored areas, with six outliers. All outliers are located in loop
regions outside the binding site of LPC. The putative binding site of LPC was
identified with the site-finder module in the MOE software, version 2013.08001
(Chemical Computing Group). Subsequently, LPC was docked to the active site
with MOE, and the protein ligand complex was optimized with the AMBER10:
EHT force field implemented in MOE.
The cDNA and protein sequences of the new antiporters EcNHX1 to
EcNHX4 have been deposited in GenBank with the following accession numbers: KU156822 (EcNHX1), KU156823 (EcNHX2), KU156824 (EcNHX3), and
KU156825 (EcNHX4).
Supplemental Data
The following supplemental materials are available.
Supplemental Table S1. (Primers used for the detection of EcNHX genes).
ACKNOWLEDGMENTS
We thank Dr. Michael Heinze for help with the assay of benzophenanthridine alkaloids and RNAi-based procedures in E. californica cell cultures as well
as Gabriele Danders and Ursula Klokow for expert technical assistance.
Received October 6, 2015; accepted November 13, 2015; published November
17, 2015.
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