The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH … · The Vacuolar Proton-Cation...

The Vacuolar Proton-Cation Exchanger EcNHX1Generates pH Signals for the Expression of SecondaryMetabolism 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 UniversityHalle-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 benzophenanthridinealkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 atthe 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 ofE. 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 celllines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggeredoverproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na+-dependent H+ effluxin a Dnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 genedisplayed Na+-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux ofvacuolar H+. This finding was supported by three-dimensional protein homology models that predict a plausible recognition sitefor lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiatedexpression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.

Plants react to microbial pathogens with the overpro-duction of antimicrobial secondary metabolites, so-calledphytoalexins (Bennett and Wallsgrove, 1994; Wink, 2003;Zhao et al., 2005; Watts et al., 2011). This response is ini-tiated by contact with pathogen-derived, low-molecularelicitors and proceeds to the overexpression of enzymesrelevant to secondary biosynthesis (Dittrich andKutchan,1991; Viehweger et al., 2006; Ren et al., 2008; Boller andFelix, 2009; Angelova et al., 2010). The signal pathsextending from elicitor recognition to gene expressionare not known in sufficient detail. This gap constrainsour understanding of how secondary metabolism com-plies with the fitness of the producing plant (Wink, 2003;

Heinze et al., 2015) and biotechnological attempts aimedto accumulate valuable plantmetabolites (Leonard et al.,2009; Aharoni and Galili, 2011).

The elucidation of signal paths that activate genes ofsecondary metabolism in whole plants is often com-plicated by their integration into a hierarchically orga-nized series of pathogen-triggered reactions, known asthe hypersensitive response (Lamb and Dixon, 1997).Orchestrated by an oxidative burst, genes of secondarymetabolism are coregulated with those relevant to theoverproduction and polymerization of phenolics, cellwall-localized proteins, and other defense constituents(Tsunezuka et al., 2005; Truman et al., 2007). Thiscomplexity is based on widely ramified signal cascadesthat use ubiquitous intermediates such as jasmonates(Blechert et al., 1995; Memelink et al., 2001), calciumions, and salicylates and are concatenated by variousmodes of cross talk (Sudha and Ravishankar, 2002;Aharoni and Galili, 2011).

In plant cell cultures, used as model systems of lowercomplexity, two signal components related to the up-regulation of secondary metabolism were investigatedin greater detail: (1) hormones or hormone-like signalmolecules and (2) regulatory proteins that control theactivation of genes relevant to a distinct secondary bio-synthesis. The first group is dominated by oxylipins ofthe jasmonate family (Gundlach et al., 1992; Memelink

1 This work was supported by the German Research Council(grant no. 889/12 to W.R.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed 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 mod-eling of EcNHX and ScNHX; W.R. coordinated the work and wrotethe article.

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et al., 2001; Pauwels et al., 2009) and lysophosphati-dylcholine (LPC; Viehweger et al., 2002, 2006). The secondgroup contains an increasing number of transcription fac-tors, concentrated in the MYB, WRKY, bHLH, and AP2/ERF families (vander Fits andMemelink, 2001, Pauwet al.,2004a; Shoji et al., 2010; Yang et al., 2012; Schluttenhoferand Yuan, 2015).

Signal pathways that selectively activate genes of asecondary biosynthesis have long been supposed toexist, either as distinguishable branches within complexdefense responses or as solitary mechanisms towardthe overproduction of distinct, specialized metabolites.Tentative evidence arose from the induction of sec-ondary biosynthetic enzymes independent of an oxi-dative burst and/or elevated levels of jasmonates (vander Fits et al., 2000; Färber et al., 2003; Pauw et al.,2004b). Transient shifts of the intracellular pH thatpreceded the induction of secondary biosynthetic geneswere earlier considered as potential components ofsignal pathways toward the expression of secondarybiosynthesis (Kuchitsu et al., 1997; Roos et al., 1998;Roos, 2000; Shibuya and Minami, 2001). This evokedactivities to define the spatial and temporal properties ofpH transients necessary and sufficient for gene activa-tion and the subcellular mechanism of their generation.

These topics were intensively investigated with cellsuspension cultures of Eschscholzia californica. The root-derived cells retain the plant’s ability to overexpress thebiosynthesis of benzophenanthridines, antimicrobialalkaloids of the benzylisoquinoine class, in response topathogens or elicitors. Contact with a yeast glycopro-tein elicits the induction of biosynthetic enzymes by asignal path that involves a transient acidification of thecytoplasm as an essential intermediary step. This wasproven by bracketing the vacuolar and cytoplasmic pH,which stops the elicitation process, and by artificiallyinduced pH shifts, which mimic the elicitor effects(Roos et al., 1998, 2006; Viehweger et al., 2006; Angelovaet al., 2010).

Prior to the pH shifts, the activation of a plasmamembrane-localized phospholipase A2 (PLA2) isdetectable in elicited cells, which gives rise to a tran-sient 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 thismoleculewith the intracellular signal transfer, we established anosmotic procedure that permeabilized the plasma mem-brane for micromolecules less than 10 kD and left thetonoplast functionally intact, as confirmed by thevacuolar accumulation of pH probes, the ATP- or

Figure 1. pH shifts monitored in vacu-oles of E. californica by confocal pHtopography (used with permission fromViehweger et al., 2002). In situ vacu-oles preloaded with the pH probeDM-NERF were perfused with isotonicmedium containing the indicated ionconcentrations. A, pH maps obtainedat 100 mM KCl plus 10 mM NaCl showATP-triggered acidification (images Aand B) and LPC-triggered, transient al-kalinization (images B–D), as quanti-fied in the inset. B, In vacuoles perfusedwith 70 mM KCl and 80 mM sorbitol,1 mM LPC causes no pH peak unless10 mM NaCl is present. C, pH shiftscaused by different extravacuolar Na+

concentrations, normalized to the pHgradient between a particular vacuoleand the external medium (7.4). In thepresence of 1 mM LPC, the shifts of vac-uolar pH (pHVac) occur at lower extrav-acuolar Na+ concentrations. Graph D, Inan experiment as in B, 1 mM LPC plus10 mM NaCl were added together with40 mM ethyl isopropyl amiloride (EIPA).No pH peak was detectable.

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pyrophosphate-powered acidification Fig. 1A, and theloss of protons caused by inhibitors of the vacuolar H+

pumps (Viehweger et al., 2002). Upon perfusion withisotonic media, these in situ vacuoles show an efflux ofpreaccumulated protons that increases with the extrav-acuolar Na+ in the range from 10 to 30 mM. In the pres-ence of LPC, this efflux requires less Na+ to occur (from 2to 10 mM) but saturates at the same maximum velocity(Fig. 1C). Thus, LPC would allow an efflux of vacuolarprotons at Na+ concentrations that are likely present inthe cytoplasm (this concentration was estimated to5 mM; Viehweger, 2003). If Na+ is absent from the ex-ternal medium (in situ vacuoles perfused with 70 mM

KCl), LPC causes no proton efflux unless 10 mM Na+ isadded (Fig. 1B).The Na+-dependent efflux of vacuolar protons, initi-

ated by either LPC or high Na+ concentrations, iscompletely inhibited by amiloride (Fig. 1D), a long-known inhibitor of plant NHX-encoded antiporters(Darley et al., 2000). Together with the aforementioneddata, this was taken as an indication that one or morevacuolar Na+/H+ antiporters are required to generatethe pH signal in the cytosol. The causal sequence: elic-itor contact/activation of PLA2/peak of LPC/efflux ofvacuolar protons/activation of genes encoding bio-synthetic enzymes, was termed the LPC/pH signalpath (Roos et al., 2006; Viehweger et al., 2006).The essential role of PLA2 as the initial signal gen-

erator of this pathwaywas further evidenced by a seriesof knockdown experiments. First, silencing of PLA2completely prevents elicitor-triggered alkaloid produc-tion (Heinze et al., 2015). Second, the elicitor activationof this enzyme is controlled by surrounding Ga proteins(Heinze et al., 2007), and the antisense-mediated knock-downofGa inhibits the following elicitor-initiated events:(1) activation of PLA2, (2) generation of vacuolar pHshifts, and (3) overexpression of alkaloid biosyntheticenzymes (Viehweger et al., 2006). Finally, the producedbenzophenanthridine alkaloids exert a long-range feed-back inhibition at PLA2, thereby preventing continuous,unlimited overexpression (Heinze et al., 2015).In contrast to the LPC/pH signal path, the jasmonate-

triggered expression of alkaloid biosynthesis, which canbe evoked in the same cell culture by higher elicitorconcentrations, involves elements of the hypersensitiveresponse (see above) but neither includes nor requiresvacuolar/cytosolic pH shifts (Roos et al., 1998; Färberet 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/pHsignal path, is generated in elicitor-treated cells. Basedon the aforementioned inhibition by amiloride, we identi-fied Na+/H+ antiporters of E. californica cells and investi-gated their susceptibility to elicitor-derived signal transferintermediates.As a prerequisite for the assay of proton fluxes

across the tonoplast, we adapted our previouslyestablished, microscopically validated methods of per-meabilizing the plasma membrane for micromoleculesand fluorescence measurement of vacuolar pH (see

above; Viehweger et al., 2002, 2006) for use inmicroplate-hosted cell suspensions (Fig. 2; see “Materials andMethods”).

Na+/H+ antiporters of the plant tonoplast areencoded by NHX genes that constitute a well-definedgroup within the CPA1 (cation proton antiporter 1)family of ion transporters. This family evolved frombacterial NhaP antiporters that mediate an electro-neutral exchange of Na+ for H+ and are found inall kingdoms of life (Chanroj et al., 2012). The com-mon origin of the catalytic domain explains why

Figure 2. Fluorescence detection of cells with permeabilized plasmamembranes. Cells were loaded with the pH indicator 59-carboxy-fluorescein diacetate acetoxymethyl ester and subjected to the per-meabilization procedure described in “Materials and Methods.” Afteradding the membrane-impermeable stain propidium iodine, fluores-cence imageswere obtained at excitation 4806 10 nm/emission 525650 nm (to detect the green fluorescence of 59-carboxyfluorescein di-acetate acetoxymethyl ester) and at excitation 560 6 40 nm/emissiongreater than 600 nm (to detect the red fluorescence of propidiumiodine), and the resulting images were overlaid in silico. Microscopywas done with a Nikon Optiphot equipped with a Sony 3 CCD camera.Images were obtained and processed with Optimas 6.2 software. A andB, E. californica 6-d-old wild-type culture before (A) and after (B) per-meabilization. Fluorescence overlay images are combined with thetransmission image. The objective lens was a Nikon Fluor 40, withnumerical aperture of 0.85. C and D, Fluorescence images ofS. cerevisiae cells (strain B4741) obtained under the same conditionsbefore (C) and after (D) permeabilization. The objective lens was aNikon Fluor 100 oil, with numerical aperture of 1.3. Arrows mark somenuclear/cytoplasmic areas (n/c) and vacuoles (v). Accumulation of59-carboxyfluorescein diacetate acetoxymethyl ester (green) indi-cates an ion-impermeable vacuolar membrane, and staining of DNA andcytoskeleton areas by propidium iodine (red) indicates the lack of the ionbarrier function of the plasma membrane.

Plant Physiol. Vol. 170, 2016 1137

EcNHX Antiporter: Signals for Secondary Metabolism



Figure 3. A, Protein sequence alignment of the Na+/H+ antiporters EcNHX1 to EcNHX4 with selected NHX antiporters fromhigher plants. TheNHX genes detected in cDNA of E. californicawere sequenced and translated in silico (rows 1–4). The EcNHX1protein sequence was used to search for the nearest homologs among the completely annotated, NHX-encoded Na+/H+ anti-porters in theNational 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 DNAused for the RNA interference (RNAi)-based silencing of NHX genes (target sequence 1 in blue and control target sequence inblack). Arrows point to the LPC-binding amino acids Lys-397 and Gln-266 (see text). B and C, Phylograms of selected plant NHXprotein sequences aligned with the antiporters EcNHX1 to EcNHX4 from E. californica. Trees were generated by the neighbor-joining algorithm with CLC viewer 7.6 software. Numbers are bootstrap values from 100 iterations. The length of each branchcorresponds 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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NHX-encoded plant antiporters share a molecularheritage with animal Na+ channels, as exemplified bythe highly conserved binding site of the potent inhibitoramiloride (Darley et al., 2000), which is in medical usefor the treatment of renal hypertension.

RESULTS

The Na+/H+ Antiporters of E. californica Are Homologousto, But Well Distinguishable from, Other Members of thePlant NHX Family

The sequence similarity between the published NHXgeneswas instrumental to our PCR-based identificationof NHX homologs in E. californica. Starting from con-sensus sequence primers that included the amiloride-binding site, a series of PCR and RACE experiments ledus to the amplification of four different open reading

frames (ORFs) from complementary DNA (cDNA) ofE. californica. The sequences of these newly discoveredgenes were translated in silico and showed high ho-mology to known plant NHX sequences in the 12transmembrane domains but much variability in theN- and C-terminal regions (Fig. 3, A and B). The nearestNHX homolog is found in Theobroma cacao and displaysan overall amino acid identity with the E. californica an-tiporters between 77% (EcNHX1) and 83% (EcNHX4).As displayed in Figure 3B, the EcNHX antiporters formtwo phylogenetically distinct pairs: EcNHX1 withEcNHX3 (82% sequence identity) and EcNHX2 withEcNHX4 (87%). Between these pairs, substantially lowersequence identities exist (72%–74%). It also may be ar-gued from this phylogram that EcNHX1was subjected toa faster evolution than the other homologs.

Sequence comparisons with plant NHX isoforms thatare known to be localized at either tonoplast or endo-somal membranes confirm that the E. californica NHXantiporters are part of the vacuolar NHX clade. As seenin the phylogram in Figure 3C, the antiporters EcNHX1to EcNHX4 are muchmore closely related to the vacuolarisoforms of Arabidopsis (Arabidopsis thaliana; AtNHX1–AtNHX4) or tomato (Lycopersicon esculentum; LeNHX1,LeNHX3, and LeNHX4) than to the endosomally locatedisoformsAtNHX5, AtNHX6 (Bassil et al., 2011, 2012), andLeNHX2 (Venema et al., 2003). This coincides with ourearlier pH topographies, which suggested that the LPC-stimulated Na+/H+ antiport occurs at the central vacuoleof the E. californica cells (see introduction).

RNA Interference-Mediated Silencing Reveals an EssentialRole of EcNHX Antiporters in the LPC/pH Signal Path

Based on the identification of four NHX genes thatencode Na+/H+ antiporters in E. californica, we attemp-ted the simultaneous knockdown of most of theirtranscripts in order to confirm whether any of theseantiporters are required for the elicitor-initiated signaltransfer. To this aim, some fairly conserved gene se-quences were amplified, inserted into an expressionvector, and used as targets for RNAi-based gene si-lencing (see “Materials and Methods). The most con-sistent result was obtained with the so-named targetsequence 1 (Fig. 3A), which encoded part of theamiloride-binding site and the following approxi-mately 100 amino acids. About 50 mutant colonieswere isolated via antibiotic resistance and displayedreduced alkaloid contents, as indicated by their whitecolor compared with the light-red wild type. In nine ofthem, the presence of RNAi transcripts was confirmedby 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. Theyare separated from the endoplasmic reticulum-located isoforms AtNHX5, AtNHX6, and LeNHX2, which form their own sub-clade. AtNHX7 (SOS1), which is located at the plasma membrane, forms an outgroup.

Figure 4. RNAi-based silencing of genes encoding Na+/H+ antiportersin E. californica cells, shown by reverse transcription (RT)-PCR. RNAisolated from the cell strains transformed with RNAi target sequence1 (G11–G13) or target sequence 2 (G0) and the wild type (wt) wassubjected to the reverse transcriptase reaction, and the resulting cDNAwas probed with NHX gene-specific primers (Supplemental Table S1).Previous experiments had confirmed that these primers allowed theselective amplification of the expected parts of the named NHX genes.The PCR products shown herewere separated on agarose gels, ethidiumstained, and quantified densitometrically. Bands from one typical ex-periment are shown. Numbers give the cDNA content of each band,averaged from four different culture batches, in percentage of the wild-type value (first column). SE ranges between 5% and 10% of the averagevalue. The actin cDNA band served as a loading control.




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cultures were established from these strains, and threeof them were selected for a more detailed characteri-zation. As shown in Figure 4, the cell lines establishedby RNAi against NHX mRNAs display different de-grees of silencing among the isoforms, with the lowestmRNA level of EcNHX1 in strain G13, of EcNHX3in strain G11, and of EcNHX4 in strain G12. Thetotal Na+/H+ antiporter activity, assayed as the Na+-dependent efflux of vacuolar protons, was diminishedto 30% to 50% of the wild type (Fig. 5A). The samecultures 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 isdrastically reduced to similarly low levels in all RNAistrains (Fig. 5D).

A similar procedure with a target sequence of lowersimilarity between the EcNHX isogenes (target se-quence 2 in Fig 3A) yielded cell cultures that displayedless than 25% or even no loss in EcNHX mRNA levels,alkaloid content, and vacuolar Na+/H+ antiport activityas well as unimpaired alkaloid production in responseto elicitor. Theywere used as an internal process control(strain G0 in Figs. 4 and 5) and indicated that the non-gene-specific effects of the RNAi-silencing process werenegligible.

At this point, it appears that the knockdown ofNa+/H+ antiporters impairs the elicitor-triggered signaltransfer, but not proportional to the loss of antiportactivity at the tonoplast. For instance, cell lines G11 andG13 display significantly different total Na+/H+ anti-port activities (Fig. 5A) but a similarly low alkaloidresponse to elicitor (Fig. 5D). This suggests that either athreshold capacity of Na+/H+ antiport or the activity of

only one or two distinct antiporter(s) is required forLPC-triggered signaling. Therefore, we conducted asearch for LPC-sensitive individual Na+/H+ antiportersby yeast complementation studies.

EcNHX Antiporters Can Be Expressed in Yeast andComplement the Dnhx Null Mutant

The yeast Saccharomyces cerevisiae contains only oneNa+/H+ antiporter of the NHX family, ScNHX1. Ayeast strain carrying the null mutation is available(nhx1::kanMX4) and was used for complementationexperiments. Successful gene transfer into this back-ground was documented by RT-PCR showing thepresence of transcripts of each of the plant’s NHX genesin the yeast cells (Fig. 6, inset). The plasmamembrane ofwild-type and transgenic yeast cells could be per-meabilized by a slightly modified version of the pro-tocol used for E. californica cells (see “Materials andMethods”), and the resulting in situ vacuoles (Fig. 2D)allowed the assay of Na+-dependent changes in thevacuolar pH. As expected, the nhx1::kanMX4 strain(Dnhx null mutant) displayed a negligible Na+/H+ an-tiporter activity, which could be substantially increasedby transformation with any of the four NHX genes ofE. californica (Fig. 6). The resulting transgenic strains allshow an Na+-dependent efflux of protons from thevacuolar interior, which saturates at about 20 mM Na+,similar to the wild type. Therefore, the successfulcomplementation of a phenotype that lacks Na+/H+

exchange processes indicates that the plant antiportersare correctly expressed, targeted to, and inserted intothe yeast vacuolar membrane.

Figure 5. Effects of the silencing of NHX genes atNa+/H+ antiport and elicitor-triggered events inE. californica cells. The cell strains G11 to G13,generated by RNAi with target sequence 1, are com-pared with the cell strain G0, which represents theRNAi strains obtained with target sequence 2 andserves as a negative control treatment. A, Na+-dependent efflux of H+ from vacuoles of per-meabilized cells. B, LPC-triggered efflux of H+ fromvacuoles of permeabilized cells at 5 mM external Na+.Data from LPC-free controls are subtracted. LPC wasadded to a final concentration of 2 mM. C, Elicitor-triggered efflux of H+ from vacuoles in intact cells.Yeast elicitor was present at 1 mg mL21. Data fromelicitor-free controls are subtracted. D, Elicitor-triggered alkaloid production of intact cells, inpercentage of similarly treated wild-type (wt) cul-tures (100% represents an amount between 10and 20 ng mg21 fresh weight in different culturebatches.) Yeast elicitor was present at 1 mg mL21. Alldata are means6 SE; n = 3 experiments from a singleculture batch of each strain. Repetition with twodifferent culture batches yielded similar relation-ships between the two mutant classes. Asterisks in-dicate significant (P , 0.05) differences with thewild type.


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EcNHX1 Selectively Reacts to the Signal Molecule LPC

The yeast clones obtained by complementation withthe E. californica NHX genes were tested for the effectof the signal molecule LPC on the activity of the re-combinant Na+/H+ antiporters. The experimentswere performed at an extravacuolar Na+ concentrationof 5 mM, which is about its cytosolic concentration inE. californica cells (Viehweger et al., 2002). Only thestrain transformed with EcNHX1 shows a clear stimu-lation of ion-exchange activity by LPC (Fig. 7). Themaximum effect is caused by about 4 mM LPC, as testedin preliminary experiments. Under such conditions, thetransgenic EcNHX1 strain reaches a similar activity ofNa+/H+ antiport to the LPC-stimulated yeast wild type(Fig. 7). As no plant genes other than the specifiedEcNHX were transferred to the yeast cells, this findingreveals a unique property of EcNHX1 among theNa+/H+

antiporters of E. californica.

Homology Modeling Supports a Selective Effect of LPC

The selective stimulation of only one out of fourEcNHX antiporters by LPC raised the question of theirstructural peculiarities. As x-ray crystallographic struc-tures of sufficient resolution are not available for plantNHX antiporters, a bacterial transporter was chosen as atemplate,which bears the basal properties of theNa+/H+

exchange domain (see “Materials and Methods”). Theobtained three-dimensional (3D) model of EcNHX1 wasused for docking studies, which predicted a recognitionsite for LPC close to the active center (Fig. 8). It consists ofthree perfectly positioned side chains that detect thephosphate group, the quaternary nitrogen, and the -OHof this molecule (Fig. 9, left).

The non-LPC-stimulated antiporter EcNHX3 yields amodel with a dysfunctional LPC docking site (Fig. 9,right): Gln is replaced by Glu, which likely establishes astrong salt bridge to the phosphate-binding Lys ofNHX1 and thus strongly aggravates phosphate recogni-tion. The other non-LPC-stimulated antiporters, EcNHX2and EcNHX4, lack the critical Lys and, instead, bear Asnin a homologous position (compare with the alignment in

Figure 6. Functional complementation of the yeast Dnhxmutant by theNHX genes of E. californica. The yeast strains obtained by transforma-tion of the Dnhx1 mutant 4290 with the genes EcNHX1 to EcNHX4(termedDnhx1+EcNHX1 toDnhx1+EcNHX4)were assayed for theNa+-dependent efflux of vacuolar protons and are compared with thenontransformed mutant nhx1::kanMX4 (termed Dnhx1) and the corre-spondingwild-type BY4741. Data are means6 SE; n = 5 cell suspensioncultures per mutant strain. The experiment was repeated with anotherculture batch per strain and yielded similar relations between the an-tiport activities. The inset shows mRNA of Na+/H+ antiporters from E.californica expressed in S. cerevisiae by RT-PCR. The ORFs of EcNHX1to EcNHX4 were transferred to the S. cerevisiae Dnhx1 mutant 4290(BY4741nhx1::kanMX4) as described in “Materials andMethods.” Fromthe resulting transgenic yeast clones nhx1::kanMX4 + EcNHX1 toEcNHX4 (termed +EcNHX1 to +EcNHX4) and the nontransformedmutant (termedDnhx1), RNAwas extracted and subjected to the reversetranscriptase reaction, and the generated cDNAwas probed with gene-specific primers (Supplemental Table S1). The four PCR products shownhere are of the expected size (i.e. 556, 523, 525, and 410 bp). A typicalexperiment is displayed, whichwas repeatedwith another culture batchand yielded bands of similar size.

Figure 7. Effects of LPC on the Na+/H+ antiport activity of transgenicyeast strains. The transgenic yeast strains expressing EcNHX1 toEcNHX4 (termed +EcNHX1 to +EcNHX4) were assayed for their impacton the Na+/H+ antiport activity of 2 mM LPC (white columns) and 4 mM

LPC (hatched columns). Basal activities measured in the absence of LPCare subtracted. The extravacuolar Na+ concentration was 5 mM in allexperiments. The yeast host strain nhx1::kanMX4 (termed Dnhx1) andthe corresponding wild-type BY4741 (termed wt) are included to shownonspecific effects of LPC in cells lacking NHX-encoded antiportersand in cells with the active yeast antiporter ScNHX1, respectively. Dataare means 6 SE; n = 3 cell suspension cultures per mutant strain. Theexperiment was repeated twice with other culture batches and yieldedsimilar relations between the transgenic yeast strains.






Fig. 3A), which alsowould hamper a recognition of LPC’sphosphate moiety.

Interestingly, the yeast Na+/H+ antiporter ScNHX1 isalso stimulated by LPC (Fig. 7). Homology modeling ofthis antiporter to a yeast template (see “Materials and

Methods”) likewise predicts a 3D structure with a well-fitting LPC recognition site (Fig. 10). Although theamino acid sequence identity with EcNHX1 is low(31%), phosphate and quaternary nitrogen of LPC ap-pear to be recognized by Lys and Glu as well (Fig. 11).

Figure 8. 3D model of the Na+/H+ an-tiporter EcNHX1 with docked LPC. Themodel was calculated using the x-raystructural data of the bacterial antiporterNapA as a template. In accordance withthis molecule, the antiporter acts as adimer (one monomer appears in gray) inwhich the amino acid Asp-154 is ex-posed as an essential cation-binding site(Lee et al., 2013). LPC (shown as a space-filling model; green carbon atoms) docksclose to this area.

Figure 9. The LPC recognition site of EcNHX1 and the homologous structure in EcNHX3. In EcNHX1 (left), the model of Figure 8predicts a precise recognition of LPC (in color) through its phosphatemoiety by Lys-397 andGln-266 and its trimethyl-ammoniumgroup by Glu-263. The fatty acid fits to the hydrophobic side chains of Phe-79, Tyr-14 (of chain B), and Tyr-396. The hydroxylgroup 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 homologousposition to Lys-397 in EcNHX1, is blocked bya strong salt bridge toGlu-269 (which attains the position homologous toGln-266 inNHX1). This likely prevents recognition of the PO4 moiety of LPC.


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Thus, the selective activation by LPC of both the re-combinant EcNHX1 and the genuine ScNHX1 anti-porter, as measured at the level of Na+/H+ antiport, isin line with the structural peculiarities predicted by ho-mology models.

DISCUSSION AND CONCLUSION

Our data concordantly indicate that one of the fourNa+/H+ antiporters of E. californica cells is selectivelystimulated by LPC, a product of PLA2, and thus con-stitutes an essential element in the signal transfer to-ward the expression of alkaloid biosynthesis. As thestimulation could be transferred with the EcNHX1 geneto a yeast strain, proteins that might be associated withthe EcNHX1 protein in E. californica cells would notobscure this result. The involvement in the stimulationprocess of one or more protein(s) conserved betweenplant and yeast is not excluded. However, activationvia protein phosphorylation, as supposed for the long-known stimulation by LPC of the plasma membraneH+-transporting ATPase (Xing et al., 1996), is less likely,as the protein kinase inhibitor staurosporine and changes

inCa2+ did not influence the LPC-triggered proton fluxes(Viehweger et al., 2002).

The higher concentration of LPC required for a fullstimulation of recombinant EcNHX1 in yeast (about4 mM) compared with genuine EcNHX1 in E. californica(about 2 mM; Viehweger et al., 2002) might reflect dif-ferences between the plant and yeast systems (e.g.compartmentation and metabolism of LPC). Basedon earlier results about the metabolism of LPC inE. californica cell cultures, it is likely that the LPCmoleculeitself, rather than a rapidly made metabolite, stimulatesthe Na+/H+ antiporter: the only metabolite detect-able after a 20-min feeding of radiolabeled LPC wasphosphatidylcholine (made by reacylation; Schwartzeand Roos, 2008). This compound does not stimulate theNa+/H+ antiport activity, and the same holds true forpotential degradation products such as lysophospha-tidic acid, phosphorylcholine, and fatty acids (tested byViehweger et al. [2002]).

The stimulation by LPC of the recombinant anti-porter EcNHX1 is compatible with the data fromE. californica cells: in the RNAi mutants that show de-creased elicitor-triggered proton fluxes and lack ofelicited 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 ofarchaebacterial origin (see “Materials and Methods”), predicts the antiporter to act as a dimer (one monomer appears in gray) inwhich the amino acid Asp-201 is exposed as a conserved cation-binding site (Lee et al., 2013). Recognition of LPC (shown as aspace-filling model; green carbon atoms) might occur close to this area.





only NHX gene that is significantly silenced in each cellline, although to different degrees.We thus conclude thatEcNHX1 is the searched-for component of the signalchain (Roos et al., 1998) that recruits the proton gradientacross the tonoplast for the cytoplasmic acidification.

It appears that even a moderate loss of EcNHX1strongly impairs the signal transfer: while the remain-ing mRNA content of EcNHX1 in the RNAi cell linesranges from 17% to 75% (Fig. 4, first lane), all strainsshow the same phenotype (i.e. the elicitor-triggeredalkaloid production drops to near zero; Fig. 5D). Al-though the expressed antiporter proteins and individ-ual activities in the RNAi strains are not quantified(antibodies of sufficient isoform selectivity are not avail-able), it is tempting to assume that the full expression ofEcNHX1 is required to generate a pH shift sufficient toinduce alkaloid biosynthetic genes. Earlier data show thatthe extent andduration of elicitor-triggered or artificialH+

peaks in the cytoplasm need to surpass a threshold tooverexpress alkaloid biosynthesis. According to a largenumber of observations, an H+ peak to be recognized as asignal for gene expression requires cytoplasmic pH to staybelow 7.1 for at least 10 min (Viehweger et al., 2006).Vacuoles with only three-fourths of EcNHX1 capacity(e.g. in strainG12)might be unable to generate this criticalefflux.

As foundwith individual vacuoles, LPC increases thesensitivity of the vacuolar Na+/H+ exchange processtoward extravacuolar Na+ but does not affect themaximum exchange capacity (Fig. 1C; Viehweger et al.,

2002). This is well compatible with our data here fromthe recombinant EcNHX1 and explains why the LPC-stimulated antiporter (Fig. 7) reaches a similar activityat 5 mM Na+ to the Na+-saturated antiporter in the ab-sence of LPC (Fig. 6).

In several plants, antiporters of the vacuolar NHXclade are known to catalyze both Na+/H+ antiport andK+/H+ antiport, the latter being an important compo-nent of pH control in growth and developmental pro-cesses (Bassil et al., 2011; Barragán et al., 2012). At the insitu vacuoles of E. californica used in this study, LPClikely stimulates the Na+/H+ antiport: at the extrava-cuolar K+ concentration of 100 mM, LPC triggers nomeasurable pH shift unless Na+ is added. NaCl at 5 mM

causes a measurable proton efflux that is stimulatableby LPC (Fig. 5A), and comparable data exist from in-dividual cells (Fig. 1B). Therefore, it appears justified toconclude that LPC acts at EcNHX1 by increasing itsaffinity toward Na+ at the cytoplasmic side (but not orless toward K+) and, thus, allows an exchange withvacuolar H+ even at the low cytoplasmic Na+ concen-tration of about 5 mM. This might not exclude the ideathat, in intact cells, K+ gradients exist across the tono-plast that allow EcNHX1 to act in the K+/H+ antiportmode and be stimulatable by LPC.

The in silico models of EcNHX and ScNHX antipor-ters still await validation by site-directed mutationsand/or direct binding assays. Therefore, the suggestivedockingdata donotfinally prove the bindingof LPC.Hereis clearly scope for forthcoming studies. Nevertheless,

Figure 11. Recognition of LPC in the yeast Na+/H+

antiporter ScNHX1. LPC (in color) is supposed to in-teract through its phosphate moiety with Lys-242, His-244, and Asn-310 and the quaternary nitrogen withGlu-238. The fatty acid fits well to the hydrophobic sidechains of Phe-120 and Leu-450. The hydroxy group ofLPC is detected by Gln-453 as well as by Lys-242.


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the existence at EcNHX1 of a precise recognition site forLPC indicates a structural peculiarity not seen in thehomologs: this molecule appears to be selected by a 3-fold interaction (i.e. phosphate to Lys, quaternarynitrogen to Glu, and -OH to Thr and Ser). Each of thethree non-LPC-stimulated antiporters of E. californicalacks at least one of these potential sites of interaction.Thus, the risk of a misfit in the in silico procedures ofmodeling and docking (see “Materials and Methods”)appears low, although not negligible. Based on the 3Dmodels, it appears that only a few sequence deviationsfrom the other EcNHXproteins are required to create anLPC recognition site in EcNHX1, notably the replace-ment of Glu-269 by Gln (Fig. 9).Despite the evolutionarydistance betweenE. californica

and yeast, both organisms harbor Na+/H+ antiportersthat are activated by LPC (Fig. 7) and likely containspecific recognition sites for this signal molecule in theneighborhood of the catalytic site (compare Figs. 9 and11). ScNHX1marks the phylogenetic origin of the NHXfamily (Chanroj et al., 2012), and its stimulation by LPCmight thus indicate an original property of the NHXprogenitor protein. It is tempting to speculate that thesusceptibility to LPC was maintained or reinvented inone NHX antiporter of E. californica because of its ad-vantageous function in the LPC/DpH signal path: theability to selectively express the biosynthesis of phyto-alexins (i.e. independent of the hypersensitive celldeath) might cause a positive selection pressure. Thisidea would be compatible with a faster evolution rateof EcNHX1, as suggested by the phylogenetic tree inFigure 3B.The vacuolar NHX antiporters of plants are long

known as crucial players in cellular pH regulation andion homeostasis (Apse et al., 2003; Pardo et al., 2006).The complementation of physiological experiments bygenetic knockout techniques (Casey et al., 2010; Bassilet al., 2011; Barragán et al., 2012) and the discovery ofendosomal isoforms of NHX antiporters allowed abroader understanding of how these elementary func-tions are instrumental to a variety of growth and devel-opmental processes. Acting coordinately with vacuolarand endosomal proton pumps, NHX antiporters controlpH-dependent steps in organellar development, endo-somal trafficking, cell expansion, and responses to abioticstress (for review, see Bassil et al., 2012). In contrast withthis progress, not much is known about the control ofNHX-encoded Na+/H+ antiporters at the activity level.Their regulation by phosphorylation is deduced fromphosphoprotein screens but not finally proven (Endleret al., 2009). Clear evidence exists for the regulation ofNa+/K+ selectivity at the C terminus of AtNHX1, whichextrudes into the vacuolar lumen. Here, the calmodulin-like protein AtCaM15 binds in a pH- and Ca2+-dependentmanner, thereby inhibiting theNa+/H+, but not theK+/H+,antiporter activity (Yamaguchi et al., 2005)To our knowledge, genuine, low-molecular effectors

that control Na+/H+ exchange activities at plant intra-cellular membranes are not yet reported. The activationby LPC, as demonstrated in this study, points to a novel

function of NHX-encoded antiporters in pathogen de-fense and phytoalexin biosynthesis.

It appears worthwhile to test a variety of plantNa+/H+ antiporters for this particular property in orderto reveal potential roles in the transmission of elicitor-like signals and determinants of their molecular evolu-tion. A recentfinding thatmightmotivate such research isthe activation of monoterpenoid indole alkaloid biosyn-thesis in Catharanthus roseus cells by both LPC and yeastelicitor (Heinze et al., 2015).

MATERIALS AND METHODS

Plant and Yeast Cultures

Submerged growing cell cultures of Eschscholzia californica, establishedpreviously from upper root tissue of greenhouse plants (according to Angelovaet al. [2010]), weremaintained in Linsmayer and Skoogmedium in a 9-d growthcycle in rotary shakers (for details, see Viehweger et al., 2002). Cells weretransferred to new medium by filtration with mild suction through a nylonmesh of 50 mm2, and the cell density was kept at 50 mg fresh weight mL21, if notindicated otherwise. For biolistic transformation, callus cultures were used afterpregrowing 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 Schumacheret al. [1987]) and purified by ultrafiltration, FPLC, and SDS-PAGE. It containsabout 40% Man (Heinze et al., 2007). Dosage refers to the dry weight of thecrude elicitor preparation.

The yeast strain BY4741 and the related Dnhx mutant 4290 (nhx1::kanMX4)were obtained from the Euroscarf strain collection (http://euroscarf.de). Yeastwere grown in 1% yeast extract, 2% peptone, and 2% Glc or synthetic minimalmedium (0.67% yeast nitrogen base without amino acids) mixed with aminoacid/base supplement according to Sherman et al. (1986). The carbon sourcesGlc and Gal were added to a final concentration of 2%. For selection of trans-formants, the corresponding amino acid or base was omitted.

Fluorescence Assay of Benzophenanthridine Alkaloids

Elicited cell cultures of E. californica accumulate mainly dihydrobenzo-phenanthridines (about 80%) and benzophenanthridines (about 20%). Theywere quantified by their fluorescence at excitation 360 nm/emission 460 nm(dihydroalkaloids) and excitation 490 nm/emission 570 nm (benzophenan-thridines) using authentic dihydrosanguinarine and sanguinarine as referencecompounds. The assay is described in detail by Angelova et al. (2010). In brief,500-mL samples werewithdrawn from cell suspensions, thoroughlymixedwith500 mL of methanol containing 0.3 M KOH, extracted bymild shaking (20 min at40°C), and centrifuged (10 min at 13,000 rpm). A total of 150 mL of supernatantwas mixed with 15 mL of 5 N H2SO4 and transferred to black quartz microtiterplates, and the fluorescence was read in the Microplate Fluorescence ReaderFLX 800 (BioTek).

Activity of Vacuolar Na+/H+ Antiporters

Permeabilization of the Plasma Membrane for Micromoleculesin Plant and Yeast Cells

This procedure takes advantage of the higher flexibility against osmoticpressure of the tonoplast compared with the plasmamembrane and is based onmicroscopic studies of Viehweger et al. (2002). Cells loaded with the pH indi-cator 5-carboxyfluorescein (see below) were incubated for 15 min in hypertonicmedium 1 (300mMKCl, 20mMK-HEPES, and 5mM reduced glutathione [GSH],pH adjusted to 7.4 with KOH), followed by a further 15 min in the slightlyhypotonic medium 2 (100 mM KCl, 20 mM K-HEPES, and 5 mM GSH, pH ad-justed to 7.4 with KOH), and finally resuspended in the near-isotonic mainte-nance medium (100 mM KCl, 50 mM MOPS/1,3-bis(tris[hydroxymethyl]methylamino) propane, 5 mM GSH, 5 mM NaCl, 0.5 mM sodium citrate,0.5 mM Na2HPO4, 2 mM ascorbic acid, 10 mM KCN, and 0.1% [w/v] bovineserum albumin, pH adjusted to 7.4 with KOH). The enhanced permeability of




http://euroscarf.de


the plasma membrane to micromolecules (the above procedure generatespores that allow the permeation of fluorescent dextrans up to 10 kD; Viehwegeret al., 2002) was tested by adding 75 nM propidium iodine. This fluorescentcation stains double-stranded DNA and cytoskeleton of permeable cells, whilethe intactness of the tonoplast is indicated by the vacuolar accumulation of5-carboxyfluorescein (Fig. 2). Yeast cells were permeabilized by the same pro-cedure 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 indicator59-carboxyfluorescein, which is liberated from 59-carboxyfluorescein diacetateacetoxymethyl ester during the incubation of intact cells. It was shown earlierthat acetoxymethyl esters are split in the cytoplasm, whereas the vacuole lacksthis esterase but contains other deacylating esterases that cleave the absorbed59-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 carboxyfluoresceindiacetate acetoxymethyl ester plus 100 mM eserine (to inhibit extracellularcleavage of the fluorescein esters). Incubation was terminated if at least 90% ofthe intracellular fluorescence was accumulated in the vacuoles (30–60 min), asconfirmed by microscopy of test samples. The carboxyfluorescein diacetate-loaded cells were immediately subjected to the selective permeabilization ofthe plasma membrane (see above). Of the resulting cell suspension, 100-mLaliquots were pipetted onto a quartz microtiter plate with light-shielded com-partments, supplied with the indicated effectors, and mounted in the Micro-plate Fluorescence Reader FLX 800 (BioTek). pH-dependent fluorescence wasassayed by excitation ratioing, based on simultaneous measurements at exci-tation 4356 20 nm (channel 1), excitation 4856 20 nm (channel 2), and emission520 6 20 nm (both channels). The emission intensity was ratioed (channel 2 tochannel 1) and converted into pH using a calibration graph obtained withsimilarly treated cells that were suspended in nutrient solutions containing40 mM sodium-MES buffers to fix the external pH at eight different values. Tofacilitate the equilibration of external and vacuolar pH in the calibration ex-periments, each medium contained 5 mM pivalic acid (in the pH range 4–5.5) or80 mM methylamine (in the pH range 5–7.5).

Proton efflux from the vacuole was quantified as the change in H+ concen-tration between two measurements (at time intervals of 0.75 to 2 min) andratioed to the direct driving force (i.e. the difference between the pH of thevacuole at the first time point and the external medium; the latter was kept atpH 7.4 throughout the experiment).

Routine tests with cell suspensions treated by the above permeabilizationprocedure and subsequent pH assay confirmed essential properties of thevacuolar H+ pool, such as ATP-fueled acidification and bafilomycin-triggeredalkalinization, similar to the in situ vacuoles investigated earlier in single cells(Viehweger et al., 2002).

Cloning and Sequencing of NHX Genes

Standard procedureswere followed if not indicated in detail, and kit systemswere used as advised by the manufacturers.

To search for activeNHXgenes, total RNAwas isolated fromE. californica cellcultures (RNeasy PlantMini Kit; Qiagen) and used to generate double-strandedcDNA (RevertAid M-MulV Reverse Transcriptase Kit; Thermo Scientific). ThecDNA served as a template for a series of PCRs with primers derived fromconserved regions of plant NHX genes (searched in the NCBI nonredundantlibrary). The resulting amplimers were sequenced and used to design primersfor new PCRs, including sequences that extended from overlapping areas.PCRs were run for about 30 cycles under conditions recommended by themanufacturer of each polymerase used (mostly Taq polymerase [Fermentas] orPhusion High-Fidelity DNA-Polymerase II [Finnzyme]). In parallel, incompleteNHX cDNAs were subjected to 39 and 59 RACE-PCR with the Marathon cDNAAmplification Kit (Clontech). These procedures led to a stepwise disclosure offour ORFs with high homology to plant NHX antiporters.

For the detection of NHX mRNAs by RT-PCR, cDNA was obtained fromRNA of transformed and wild-type cells as described above and used as atemplate in PCRs with gene-specific primers (Supplemental Table S1) that weredesigned from the divergent sequences in the C termini of the EcNHX anti-porters (Fig. 3A). Each primer combination was tested at optimized meltingtemperature and different dilutions for a potential cross-amplification of un-desired NHX cDNAs. No such PCR products were found with different tem-plates, as proved by sequencing. The amplified cDNA bands were separated by

DNA agarose electrophoresis, ethidium stained, and quantified by fluorescencedensitometry. 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 thetargeted degradation of EcNHXmRNAswere selected from the known cDNAs(Fig. 3A), amplified by PCR with gene-specific primers (Supplemental TableS1), and cloned in an expression vector that was finally delivered to culturedcells by biolistic gene transfer. Using Gateway cloning (Invitrogen TOPOcloning procedure) and following the manufacturer’s advice, the target se-quence was first introduced into the entry vector pCR8/GW/TOPO andtransferred to the RNAi destination vector pK7GWIWG2(II) by LR Clonase II.Positive bacterial clones, resulting from the replacement of the vector’s lethalccdB gene by the target sequence, were selected, and the desired new vector wasisolated using the QIAprep Spin Miniprep Kit (Qiagen). As confirmed by DNAsequencing, the isolated vector contained the target sequence twice, in oppositedirections, separated by an intron and each flanked by an attR2 site. The map ofpK7GWIWG2(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 biolisticbombardment of E. californica wild-type cells by the Bio-Rad PDS-1000/HeParticle Delivery System, following a recently published protocol (HeinzeandRoos, 2013). The stably transformed cell lineswere grown over at least three9-d passages on selection medium containing 100 mg mL21 paromomycin andlater maintained by growth cycles in medium with 50 mg mL21 paromomycin.The presence in the transgenic plant cells of DNA encoding the intendeddouble-stranded RNA sequences was confirmed by PCR with genomic DNA(extracted by the cetyl-trimethyl-ammonium bromide (CTAB) method) andspecific primers that amplified sequences extending from a flanking region ofthe 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 clonedinto the pBluescript II vector (In-Fusion HD Cloning Kit; Clontech), therebyattaching the restriction sites XbaI (59) and EcoRI (39). The ORFs of EcNHX2,EcNHX3, and EcNHX4 received the restriction sites XbaI (59) and XhoI (39) viaPCR andwere cloned into the pJET1.2/blunt vector (CloneJet PCR Cloning Kit;Thermo Scientific). The primers used are listed in Supplemental Table S1. In asecond step, the genes were cloned into the yeast expression vector p416GAL1and isolated for further use in the transformation/complementation of yeast cells.

The yeast Dnhx null mutant 4290 (nhx1::kanMX4) was transformed with theexpression vector p416GAL1, carrying the ORFs of EcNHX1 to EcNHX4 (pre-pared as described above), according to Akada et al. (2000). Yeast culturestransformed with the empty p416GAL1 vector served as controls. Transformedyeast colonies were selected via the uracil prototrophy conferred by the URA3gene in the expression vector. The recombinant yeast strains obtained werefurther characterized by RT-PCR with primers that allowed the specific am-plification of EcNHX isoforms (Fig. 6, inset).

Molecular Modeling of NHX Antiporters

All 3Dmodels were established with the YASARA software, version 14.7.17(Krieger et al., 2009). The underlying algorithm selects potential templates notonly by their sequence identity to the target (which amounts here to about 20%)but includes similarities of secondary structures as predicted by PSI-BLAST andPSI-PRED (Jones,1999). By applying these criteria to the homology modeling ofEcNHX1 and EcNHX3, the bacterial Na+/H+ antiporter NapA (Lee et al., 2013;Protein Data Bank code 4BWZ) was found to be the best template proteinavailable, with sufficient resolution (3 Å). The comparison of this template withthe targets EcNHX1 and EcNHX3 yields expect values of 0.015 and 0.042, re-spectively (i.e. fairly below the minimum level of 0.5, which is required byYASARA’s homology-modeling procedure). The resulting models were posi-tively evaluated, as suggested by Z scores of 21.488 (EcNHX1) and 21.454(EcNHX3).

For themodeling of ScNHX1, four promising templates (eachwith an expectvalue below1e-19)were identified amongproteins of archaebacterial origin (e.g.MjNhaP1 [Paulino et al., 2014; Wöhlert et al., 2014]; Protein Data Bank codes4CZB, 4CZ8, 4CZA, and 4CZ9) and used to create a best-ranging hybrid modelthat was evaluated positively with a Z score of 21.595.


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http://www.uoguelph.ca/~jcolasan/pdfs/gateway_protocols_and_plasmids.pdf

http://www.uoguelph.ca/~jcolasan/pdfs/gateway_protocols_and_plasmids.pdf



The stereochemical quality of all models was finally checked with thePROCHECK software (Laskowski et al., 1993). In the cases of EcNHX1 andEcNHX3, 93.3% of all residues are located in the most favored area of theRamachandran plot, with two outliers. The model of ScNHX1 shows 90% of allresidues in most favored areas, with six outliers. All outliers are located in loopregions outside the binding site of LPC. The putative binding site of LPC wasidentified with the site-finder module in the MOE software, version 2013.08001(Chemical Computing Group). Subsequently, LPCwas docked to the active sitewith 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 toEcNHX4 have been deposited in GenBank with the following accession num-bers: KU156822 (EcNHX1), KU156823 (EcNHX2), KU156824 (EcNHX3), andKU156825 (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 benzophenanthri-dine alkaloids and RNAi-based procedures in E. californica cell cultures as wellas Gabriele Danders and Ursula Klokow for expert technical assistance.

Received October 6, 2015; accepted November 13, 2015; published November17, 2015.

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