Anastatica hierochuntica, an Arabidopsis Desert Relative, Is Tolerant to Multiple Abiotic Stresses

18 Pages·2017·3.82 MB·English

by  Simon Barak

fpls-07-01992 January12,2017 Time:16:14 #1
ORIGINALRESEARCH
published:17January2017
doi:10.3389/fpls.2016.01992
Anastatica hierochuntica, an
Arabidopsis Desert Relative, Is
Tolerant to Multiple Abiotic Stresses
and Exhibits Species-Specific and
Common Stress Tolerance Strategies
with Its Halophytic Relative, Eutrema
(Thellungiella) salsugineum
GilEshel,RuthShaked,YanaKazachkova,AsifKhan,AmirEppel,AroldoCisneros,
TaniaAcuna,YitzhakGutterman,NoemiTel-Zur,ShimonRachmilevitch,AaronFaitand
SimonBarak*
Editedby:
VasileiosFotopoulos, FrenchAssociatesInstituteforBiotechnologyandAgricultureofDrylands,JacobBlausteinInstitutesforDesertResearch,
CyprusUniversityofTechnology, Ben-GurionUniversityoftheNegev,SdeBoker,Israel
Cyprus
Reviewedby: The search for novel stress tolerance determinants has led to increasing interest in
LarsMatthiasVoll,
plants native to extreme environments – so called “extremophytes.” One successful
UniversityofErlangen-Nuremberg,
Germany strategyhasbeencomparativestudiesbetweenArabidopsisthalianaandextremophyte
JohnChandlerCushman, Brassicaceae relatives such as the halophyte Eutrema salsugineum located in areas
UniversityofNevada,Reno,USA
including cold, salty coastal regions of China. Here, we investigate stress tolerance
*Correspondence:
in the desert species, Anastatica hierochuntica (True Rose of Jericho), a member
SimonBarak
[email protected] of the poorly investigated lineage III Brassicaceae. We show that A. hierochuntica
has a genome approximately 4.5-fold larger than Arabidopsis, divided into 22
Specialtysection:
diploid chromosomes, and demonstrate that A. hierochuntica exhibits tolerance to
Thisarticlewassubmittedto
PlantPhysiology, heat, low N and salt stresses that are characteristic of its habitat. Taking salt
asectionofthejournal
tolerance as a case study, we show that A. hierochuntica shares common salt
FrontiersinPlantScience
tolerance mechanisms with E. salsugineum such as tight control of shoot Na+
Received:07September2016
Accepted:15December2016 accumulation and resilient photochemistry features. Furthermore, metabolic profiling
Published:17January2017 of E. salsugineum and A. hierochuntica shoots demonstrates that the extremophytes
Citation: exhibit both species-specific and common metabolic strategies to cope with salt
Eshel G,Shaked R,Kazachkova Y,
stress including constitutive up-regulation (under control and salt stress conditions)
Khan A,Eppel A,Cisneros A,
Acuna T,Gutterman Y,Tel-Zur N, of ascorbate and dehydroascorbate, two metabolites involved in ROS scavenging.
Rachmilevitch S,Fait AandBarak S
Accordingly, A. hierochuntica displays tolerance to methyl viologen-induced oxidative
(2017)Anastaticahierochuntica,an
ArabidopsisDesertRelative,Is stress suggesting that a highly active antioxidant system is essential to cope with
ToleranttoMultipleAbioticStresses multiple abiotic stresses. We suggest that A. hierochuntica presents an excellent
andExhibitsSpecies-Specific
extremophyte Arabidopsis relative model system for understanding plant survival in
andCommonStressTolerance
StrategieswithItsHalophyticRelative, harshdesertconditions.
Eutrema(Thellungiella)salsugineum.
Front.PlantSci.7:1992. Keywords:abioticstress,Arabidopsisrelatives,Brassicaceae,desertplants,Eutremasalsugineum,extremophile
doi:10.3389/fpls.2016.01992 plants,halophytes,extremophytes
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Esheletal. ExtremophyteBrassicaceaeStressTolerance
INTRODUCTION extremophytes from a range of different habitats. The Negev
desert in Southern Israel, part of the Sahara and Arabian
Abioticstressesdramaticallyreducecropyieldsleadingtohuge deserts belt, is characterized by a combination of temperature
economic losses and a threat to food security (Boyer, 1982; extremes, low water and nutrient availability, and high salinity
Bray et al., 2000; Cramer et al., 2011), a problem likely to be and radiation levels, and is considered an “arid” zone with an
exacerbated by climate change (Mittler and Blumwald, 2010; annualrainfallofbetween25and200mmthatisunpredictable
Lobell et al., 2011; Lesk et al., 2016). At the same time, the with high spatial and temporal variation (Gutterman, 2002;
worldwide human population is continuously growing; from 7 Ward, 2009). Temperatures can vary both daily and seasonally
billion in late 2011, it is expected to surpass 9 billion people between an absolute daily maximum and minimum of 46.8
by 2050 (United Nations, Department of Economic and Social and −3.6◦C, respectively1. These extreme conditions, together
Affairs, Population Division, 2011). Consequently, the demand with“islandsofsoilsalinity,”haveledtospecialmorphological,
forglobalcropproductionisexpectedtodoubleby2050(Tilman physiologicalandgeneticadaptationsthatallowplantstothrive.
etal.,2011).Yet,mostnewlandwiththepotentialforagriculture Thus,theNegevdesertisalikelybiogenetictreasureboxofgenes
is situated in harsh environments such as drylands and deserts, thatarerelatedtoabioticstressadaptations.
which cover about 41% of the world land areas (Millennium Wehavescreenedseveralofthe25extremophyteBrassicaceae
Ecosystem Assessment, 2005). Therefore, the improvement of species that inhabit the Negev for tolerance to multiple abiotic
crop yields under multiple stresses is vital to prevent losses stresses. One of these is Anastatica hierochuntica (True Rose of
wherecropsarecurrentlygrownandtocultivatethemonmore Jericho), a winter annual, Saharo-Arabian desert plant species
marginalland. first described by Linnaei (1753) that colonizes the uppermost,
The model plant, Arabidopsis thaliana has provided much driest zones of wadies or runnels (Friedman and Stein, 1980;
of our understanding of the molecular responses of plants to Friedman et al., 1981). A. hierochuntica is well-known due to
stress. However, Arabidopsis is a stress-sensitive plant and thus its hygrochasy seed dispersal mechanism whereby the plant
the search for novel stress tolerance mechanisms and genes dies, dries out and reduction in turgor pressure causes the
has led to increasing interest in naturally stress-tolerant plants branches to curl thereby protecting the seeds and preventing
native to extreme environments – so called “extremophytes” their dispersal (Friedman et al., 1978; Hegazy et al., 2006).
(John and Spangenberg, 2005; Amtmann, 2009; Dassanayake Once a serious rain event occurs causing wetting of the dead
et al., 2009; Smaoui et al., 2011; Gechev et al., 2012; Oh et al., plant,thebranchesunfoldandtheseedcapsules(siliculae)open.
2012; Bressan et al., 2013; Cheeseman, 2014; Flowers et al., Seedsarethendispersedbyraindropshittingtheopensiliculae
2015; Zhu et al., 2015). One successful strategy has been to (Gutterman, 2002; Hegazy et al., 2006). Thus, although dead,
study extremophyte Arabidopsis relatives (Brassicaceae) such A. hierochuntica has often been mistaken for a resurrection
as the metal hyperaccumulator Arabidopsis halleri (Hanikenne plant.
et al., 2008), and the halophytes, Schrenkiella parvula (formerly Here, we examine whether A. hierochuntica exhibits
Thellungiella parvula) and Eutrema salsugineum (formerly tolerance to heat, low nitrate and salt stresses associated
ThellungiellasalsugineaorThellungiellahalophila)(Volkovetal., with its desert habitat. Taking salt stress as a case study, we
2003; Inan et al., 2004; Orsini et al., 2010; Koch and German, determine whether A. hierochuntica possesses physiological
2013). In addition to being salt-tolerant (e.g., Inan et al., 2004; salt tolerance mechanisms that are similar to its halophytic
Tajietal.,2004;Kantetal.,2006),E.salsugineumisalsotolerant relative, E. salsugineum, and whether these two extremophytes
tolowsoilnitrogen(Kantetal.,2008),highboronlevels(Lamdan fromverydifferentenvironmentshaveevolvedcommonor/and
et al., 2012), low phosphate levels (Velasco et al., 2016), heat species-specificmetabolicstrategiesforcopingwithsaltstress.
stress (Higashi et al., 2013), and shows similar tolerance to
coldandfreezingstressasArabidopsis(Griffithetal.,2007;Lee
et al., 2012). A number of factors contribute to E. salsugineum MATERIALS AND METHODS
stresstoleranceincludingconstitutiveup-anddown-regulation
of stress tolerance genes and metabolites suggesting that Plant Material and Growth Conditions
E. salsugineum is “primed” for stress, gene copy number Anastaticahierochunticaseedswereoriginallycollectedfromthe
expansion of ion transport-related genes, sub-functionalization Negev Desert (455881(cid:48) N and 200751(cid:48) E), Israel. The original
and neo-functionalization of duplicated genes, biased codon seed was bulked at the Ben-Gurion University experimental
usage facilitating more efficient translation of proteins related fields in Midreshet Ben-Gurion, and seeds from this collection
to ion transportation, and the possible involvement of lineage- were further propagated under growth room conditions (16 h
specificgenes(Tajietal.,2004;Gongetal.,2005;Kantetal.,2006, light (150 µmol m−2 s−1)/8 h dark; 22◦C). Seeds from the F4
2008; Lugan et al., 2010; Oh et al., 2010, 2012, 2014; Sun et al., generation were used for experiments presented in the current
2010;Dassanayakeetal.,2011;Wuetal.,2012;Champignyetal., report.A.thaliana(Col-0),A.hierochunticaandE.salsugineum
2013;Kazachkovaetal.,2013;Yangetal.,2013). (Shandong ecotype) seeds were surface-sterilized with 50%
The various stresses to which an extremophyte exhibits commercial bleach for 5 min, and then rinsed four times
tolerancereflectthecombinationofstressescharacteristicofeach with sterile water. A. hierochuntica seeds were sown onto
specifichabitat.Thus,toobtainacomprehensiveunderstanding
ofhowplantscopewithmultiplestresses,itisvitaltoinvestigate 1http://www.ims.gov.il/IMSENG/All_Tahazit/homepage.htm
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Esheletal. ExtremophyteBrassicaceaeStressTolerance
nutrient agar plates containing MS (Murashige and Skoog, Growth, Pigment, Ion, and Chlorophyll
1962) medium, pH 5.7, 0.5 g L−1 MES, 2% (w/v) sucrose Fluorescence Measurements
and 0.8% (w/v) agar (Duchefa Biochemie), while Arabidopsis Shootdryweightswereobtainedbydryingsamplesat75◦Cfor
and E. salsugineum seeds were suspended in 0.12% agarose
3 days. For root elongation assays, root length was measured
prior to being sown on plates. Seeds were stratified at 4◦C
every24hfor4days,androotrelativegrowthratewascalculated
(Arabidopsis, 4 days; E. salsugineum, 7 days; A. hierochuntica,
foreachconsecutivedayaccordingtoHunt(1990).Foroxidative
1 day) and plates incubated in the growth room [16 h light
stress experiments, root elongation was calculated using the
(150 µmol m−2 s−1)/8 h dark; 22◦C]. Sowing of seeds from
formula (L -L )/L where L and L are root lengths at days 1
4 0 4 0 4
the three species was staggered so that germination occurred
or4,respectively,aftertransfertoMVplates.Forevaluatingtotal
atapproximatelythesametime.Forsoilexperiments,seedlings
leafarea,leavesweredetached,placedbetweentwotransparency
with fully expanded cotyledons were transferred from plates
films (Graphic Vision Media), and scanned. Leaf area was
to 7 cm × 7 cm × 8 cm pots containing Arabidopsis soil
calculatedwithImageJ1.43software2.Anthocyanincontentwas
(WeizmannInstituteofScience;70%finepeat[1–10mm],30% analyzedaccordingtoKantetal.(2006).Na+ andK+ ionswere
perlite 4) and irrigated to field capacity with 1 g L−1 of 20-
extracted as described by Kant et al. (2006) and ion contents
20-20 NPK + micronutrients solution (Haifa chemicals). Flats
determinedbyflamephotometry(Model410Flamephotometer,
containing pots were covered with plastic domes and placed in
SherwoodScientific,Ltd,UK).Chlorophyllandcarotenoidswere
thegrowthroom.After2days,domesweregraduallyremovedto
extracted in 100% methanol at a 1:10 ratio of tissue to solvent,
allowseedlinghardening.Eachday,platesorpotswereshuffled kept overnight at 4◦C in the dark, and measured according
to remove shelf position effects. Although for the purposes
toLichtenthaler(1987).Chlorophyllfluorescencewasmeasured
of comparative studies with Arabidopsis and E. salsugineum,
with a Mini-PAM device (Walz, Heinz Walz GmbH, Effeltrich,
light conditions in the growth room were below those usually
Germany) applying a rapid light curve (White and Critchley,
encounteredbyA.hierochuntica,SupplementaryFigureS1shows
1999).Non-photochemicalquenching(NPQ)andPSIIETRwere
that A. hierochuntica growth rates always remain lower than
calculatedaccordingtoBilgerandBjörkman(1990)andKralland
Arabidopsis even at higher light intensities. This suggests that
Edwards(1992),respectively.
the slower growth of A. hierochuntica relative to Arabidopsis
and E. salsugineum was not an effect of the growth room light
Chromosome Number and DNA Content
conditions.
Chromosome number analysis was performed on young shoot
Abiotic Stress Assays tipmeristemprotoplastsusingadrop-spreadtechniquefollowed
Forplate-basedinvitroanalyses,heatstress(45◦C)wasapplied by DAPI staining and bright-field microscopy according to
Anamthawat-Jonsson(2003).Forflowcytometryanalysisof2C
for various periods and plates transferred back to the growth
room for an additional 48 h. For salt stress, low NO − and DNA content, nuclear suspensions were prepared by chopping
3
60–100 mg of young shoot tip tissue in 1 ml of ice-cold nuclei
oxidative stresses, seedlings were first sown on control MS
isolation buffer (NIB; Dolezel et al., 1989) with the addition of
plates. Due to differing root growth rates, A. hierochuntica
5%polyvinylpirrolidonePVP-40.Afteradditionofafurther2ml
and Arabidopsis seedlings were transferred to stress treatment
NIB,sampleswereshakenfor1hfollowedbyfiltrationthrough
plates,1and5daysafterstratification,respectively.Fortheroot
50 µm nylon mesh and centrifugation at 1,900 rpm for 8 min.
elongation assay, 10 uniform seedlings were transferred to MS
The pellet containing the nuclei was resuspended in 1 ml ice-
vertical square plates containing various NaCl concentrations.
cold NIB supplemented with 50 µg m−1 of DNase-free RNase
To prevent any osmotic effects due to uptake of sucrose in
A and 2 µg ml−1 propidium iodide (Sigma-Aldrich Chemical,
the aerial tissues (MacGregor et al., 2008), the top of the agar
Co.,USA),vortexedgentlyforafewsecondsandthenincubated
was cut away before transfer of seedlings to ensure that shoots
were not in contact with the medium. For the low NO − and onicefor10min.Sampleswerefilteredthrough35µmmeshand
3
runonaBectonDickinsonFACSVantageSEequippedwitha640-
oxidative stress assays, uniform Arabidopsis or A. hierochuntica
nm dichroic long-pass filter, a 585/42-nm band-pass filter, and
seedlings were transferred for 5 days to vertical square plates
anair-cooledargon-ionlasertunedto15mWandoperatingat
containingMSmacroelements(minusammoniumnitrateforlow
488nm.Foreachsample,10,000nucleiwererecordedtocalculate
Nexperiments)andmicroelements,pH5.7(adjustedwithKOH),
0.5 g L−1 MES, 2% (w/v) sucrose, 0.8% (w/v) agar (Duchefa themeanpositionoftheG1 peakinthehistogram.Theratioof
Biochemie), and various concentrations of either KNO − or therelativefluorescencefromtheG0/G1peakpositionsforeach
3
sample was compared against the internal standard Raphanus
methylviologen(MV;Sigma,M-2254).
sativus cv. Saxa as a genome size reference (2C = 1.11 pg of
For soil experiments, salt treatments were applied to plants
DNA). Cytometer gain was adjusted so that the G /G peak of
with four true leaves (Arabidopsis and E. salsugineum) or two 0 1
true leaves (A. hierochuntica). Pots were irrigated with 1 g L−1 R.sativuswaspositionedonchannel1500.
of20-20-20NPK+micronutrientssolutionsupplementedwith 2CDNAcontentwascalculatedasfollows:
Sample 2C = (mean position of the G1 sample peak/mean
various NaCl concentrations. Salt treatments commenced with
positionoftheG1referencepeak)×reference2CDNA.
50% of the final salt concentration, and one week later, 100%
percentofeachsaltlevelwasapplied.Plantswereharvestedafter
anadditionalweek. 2http://rsbweb.nih.gov/ij/
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Esheletal. ExtremophyteBrassicaceaeStressTolerance
Metabolic Profiling the presence of 22 chromosomes (diploid) (Figure 2A).
Plants were grown in soil and subjected to salt stress as Flow cytometry revealed that the A. hierochuntica genome
described above. The fifth and sixth leaves of Arabidopsis and is approximately 4.5-fold larger than the Arabidopsis genome
E. salsugineum plants, and the third and fourth leaves of (Figures2B,C).Tovalidateourresults,wealsoperformedflow
A. hierochuntica were harvested, pooled, snap-frozen in liquid cytometry with E. salsugineum and showed that the genome of
nitrogen and stored at −80◦C. Four biological replicates were this species is approximately twice the size of the Arabidopsis
analyzed for each treatment and two independent experiments genome as previously reported by Inan et al. (2004). Thus,
werecarriedout.Metaboliteextractionwasperformedaccording A.hierochunticahasagenomesizeofroughly607Mbp.
toLisecetal.(2006)withminormodifications(Kazachkovaetal.,
2013). A. hierochuntica Exhibits Tolerance to
DerivatizationwasperformedaccordingtoLisecetal.(2006)
Abiotic Stresses Associated with Its
while separation, chromatogram evaluation and metabolite
Desert Habitat
identification/annotation were performed according to
Kazachkova et al. (2013). Metabolite relative abundance Anastatica hierochuntica plants can experience daily
was determined by normalizing the intensity of the peak of temperatures above 40◦C. Furthermore, the Negev desert is
each metabolite to the ribitol standard, yielding ‘response’ characterized by patchy soil nitrogen pools (Zaady, 2005).
values.Thesevalueswerenormalizedusinglog10transformation For instance, we measured soil NO3− levels ranging from
and analyzed statistically using MultiExperiment Viewer 0.4 to 4 mM (data not shown). We therefore tested whether
Version 3.1 software (Saeed et al., 2003). Two-way ANOVA A.hierochunticadisplaysgreatertolerancetoheatstressandlow
tests were performed between all species and paired species. NO3− levels in the growth medium, than Arabidopsis. In vitro-
To pinpoint metabolites that were significantly affected by grown seedlings (two-cotyledon stage) of both species survived
the salt treatment within each species (Figure 8), a one-way 1hofheatstress(45◦C)withanapproximate25%reductionin
ANOVA (Supplementary Table S3) followed by a Dunnett freshweight(FW)forbothspeciesbutnoeffectonchlorophyllor
test was performed. For principal component analysis (PCA), carotenoid content (Figures 3A–D; Supplementary FigureS2).
log transformed response data were used while hierarchical Two hours of heat stress severely affected Arabidopsis seedlings
10
clusteringwasperformeduponlinearresponsevaluesdividedby leaving them pale or bleached (Figure 3A). This effect was
themedianabundanceofeachmetabolite. reflectedina67%reductioninFW,anda45and54%reduction
in chlorophyll and carotenoid content, respectively, compared
to control seedlings. On the other hand, there was no further
reduction in the FW of A. hierochuntica seedlings compared to
RESULTS
1hheatstressandnoeffectonchlorophyllandcarotenoidlevels
comparedtothecontrol.Heatstressof3and4hcausedsevere
A. hierochuntica Morphology, Genome
stresstoArabidopsisseedlingsasmanifestbynofurthergrowth
Organization, and DNA Content
of seedlings and almost complete bleaching of cotyledons. In
In the wild, A. hierochuntica plants exhibit great variation in contrast, A. hierochuntica displayed considerably greater heat
developmentalspeedandbodysizerangingfrombetweenafew tolerance than Arabidopsis after 3 and 4 h of heat stress. For
mm to 15–20 cm in height, depending upon the availability of instance,4hheatstress,causeda50%dropinA.hierochuntica
soil moisture. Figure 1 shows various stages of development FW whereas Arabidopsis exhibited a 73% FW reduction.
of A. hierochuntica under our laboratory conditions. In their Furthermore, A. hierochuntica seedlings did not become
early stages of growth, plants are very uniform producing two bleached but maintained chlorophyll levels at approximately
cotyledons followed by two opposite pairs of obovate, densely 65%ofcontrollevelswithnoeffectofheatstressoncarotenoid
hirsute true leaves (Figures 1A–C). Bifurcation of the shoot content.
beginswiththethirdandfourthleavesseparatingintoindividual Anastatica hierochuntica also displayed tolerance to low N
branches. An axillary inflorescence is produced at the branch stresscomparedtoArabidopsis(Figure3E).Arabidopsisshowed
pointandformsadenseclusterofsub-sessile,whiteflowerswith a progressive, sharp reduction in FW as NO − levels were
3
four petals (Figures 1D,G,H). Repeated bifurcation proceeds reduced in nutrient agar, exhibiting a 75% reduction in FW
between subsequent pairs of more lanceolate, toothed, hirsute at 0.05 mM NO − (Figure 3F; Supplementary Figure S3). In
3
leaves,resultinginamulti-branched,sympodialshootstructure contrast, A. hierochuntica showed a muchmore moderate drop
that can vary greatly in overall three-dimensional architecture in FW as NO − levels were reduced, and FW only fell by
3
(Figures 1E,F). Fruits are hairy, two-winged siliculae each 25% at 0.05 mM NO −. The highly stressed condition of the
3
containingfourseeds(Figures1I–K). Arabidopsis seedlings at low NO − levels was manifest by the
3
It has been reported using acid carmine staining that production of progressively higher amounts of anthocyanins
the A. hierochuntica genome is organized into 11 (haploid) as NO − levels dropped (Figure 3G). On the other hand, no
3
chromosomes (Lifante et al., 1992). To confirm this finding, significant differences in A. hierochuntica anthocyanin content
wevisualizedA.hierochunticachromosomesusingaprotoplast- comparedtocontrolcouldbediscernedatanyNO −level.
3
dropping protocol (Anamthawat-Jonsson, 2003). Analysis of BecausetheNegevdesertpossessesislandsofhighsalinityand
4(cid:48), 6-diamidino-2-phenylindole (DAPI) staining clearly showed A.hierochunticaisalsofoundgrowingintheDeadSeavalley,we
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Esheletal. ExtremophyteBrassicaceaeStressTolerance
FIGURE1|GrowthanddevelopmentofAnastaticahierochuntica.A.hierochunticaplantswereraisedinthegrowthroom(seeMaterialsandMethods)and
photographedaftertransplantationtosoilatthefollowingtimes:(A)4days;(B)10days;(C)19days;(D)26days;(E)31days;(F)4months;(G)axillary
inflorescenceatbranchpointafter26days;(H)axillaryinflorescencewithdevelopingsiliculaeafter31days;(I)siliculaeon4month-oldplant;(J)drysilicula;
(K)A.hierochunticaseeds;(L)Arabidopsisseedsforcomparison.Scalebaris5mm.
testedwhetherA.hierochunticaexhibitsgreatertolerancetosalt A. hierochuntica Tightly Controls Na+
stressthanArabidopsis.AmuchgreaterreductioninArabidopsis Accumulation and Exhibits
growth was discerned as salt concentration increased than was
Photochemical Characteristics that Are
observed in A. hierochuntica (Figures 4A–C; Supplementary
Resilient to Salt Stress
FigureS4).Forinstance,at100and200mMNaCl,Arabidopsis
displayed a 55 and 77% reduction in FW, respectively, while One feature of natural salt tolerance is the ability of plants
A. hierochuntica FW was only reduced by 30 and 48% at the to tightly control Na+ accumulation (e.g., Kant et al., 2006).
respective iso-saline concentration (Figure 4A). Similarly, dry Under control conditions, A. hierochuntica possessed about 4.5
weight was affected to a lesser extent in A. hierochuntica than times as much Na+ in the shoots as Arabidopsis, similar to the
Arabidopsis at 100 and 200 mM NaCl (Figure 4B). Arabidopsis threefoldhigheramountofE.salsugineumshootNa+compared
leaf area was also affected to a greater extent by salt stress to Arabidopsis (Figure 5A; Kant et al., 2006). Both species
thanA.hierochuntica.Forexample,Arabidopsisexhibiteda74% accumulated increasing amounts of Na+ as salt concentrations
reductioninleafareaat200mMNaClwhereasA.hierochuntica increased, but Arabidopsis accumulated Na+ to a much greater
leafareaonlydroppedby49%(Figure4C).Furthermore,inan extent than A. hierochuntica (Figure 5B). Arabidopsis and
invitroplaterootelongationassay,Arabidopsisseedlingsshowed A. hierochuntica exhibited comparable shoot K+ levels under
aclearanddrasticsalt-mediateddose-responsereductioninroot control conditions and a similar reduction in K+ content
relativegrowthratesuchthatat200mMNaCl,rootelongation in response to salt stress (Figure 5C). Thus, under control
had virtually ceased (Figure 4D). On the other hand, 100 mM conditions, A. hierochuntica had a higher Na+/K+ ratio than
NaCl only had a mild effect on A. hierochuntica root relative Arabidopsis(Figure5D)butassoilsaltconcentrationsincreased,
growth rate while root elongation continued even at 200 mM Na+/K+ ratios rose to a much lower extent in A. hierochuntica
NaCl(Figure4E). (Figures5D,E).BothspeciesexhibitedsimilarFW/DWratiosat
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Esheletal. ExtremophyteBrassicaceaeStressTolerance
We recently reported that A. hierochuntica under control
conditions exhibits lower NPQ, a mechanism for dissipating
excess light energy, than Arabidopsis, and a relatively higher
photosystem II electron transport rate (PSII ETR) (Eppel
et al., 2014). In the present study, A. hierochuntica exhibited
an approximately 50% lower NPQ than Arabidopsis under
control conditions (Figure 5G), and while salt stress had no
effect on Arabidopsis NPQ, it caused a further 50% fall in
A.hierochunticaNPQ.Undercontrolconditions,PSIIETRwas
about 55% higher in A. hierochuntica compared to Arabidopsis
(Figure 5H). Salt stress caused a fall in PSII ETR in both
species but A. hierochuntica still maintained a 45% higher PSII
ETR in comparison to Arabidopsis. Thus, the NPQ and PSII
ETRdatasuggestthatA.hierochunticaphotochemistrydisplays
considerableresiliencetosaltstress.
Arabidopsis, E. salsugineum, and
A. hierochuntica Metabolic
Reprogramming in Response to Salt
Stress
Eutremasalsugineumexhibitsextremesalttolerance(Kantetal.,
2006; Kazachkova et al., 2013), which presented us with the
opportunityofcomparingthreerelatedspeciesoriginatingfrom
verydifferentenvironments,anddifferingintheirsalttolerance:
the salt-sensitive glycophyte Arabidopsis (temperate Columbia,
MT, USA), and the two extremophytes – moderately salt-
tolerant A. hierochuntica (hot, dry Negev desert), and highly
tolerantE.salsugineum(cold,saltycoastalareasoftheShandong
province,China).
Arabidopsis, E. salsugineum, and A. hierochuntica plants
growninsoilwereexposedtoincreasingconcentrationsofNaCl
(E.salsugineumwasnotexposedto50mMNaClduetothemild
effect of this concentration; Kazachkova et al., 2013), and the
relative abundanceof primary carbonand nitrogen metabolites
was determined for leaf tissue. To gain a global view of the
differencesbetweentheplantspeciesandacrosstreatments,PCA
wasemployed.Inspectionofthefirsttwoprinciplecomponents,
which account for 68% of the total variance within the data
set, allowed classification of samples by species and treatments
(Figure6).Thefirstprincipalcomponentaccountingfor48.2%
oftotalvarianceclearlyseparatedthemetabolismofArabidopsis
from its two extremophyte relatives (Figure 6A). Among the
FIGURE2|Anastaticahierochunticachromosomenumber(diploid)
metabolites most affecting the separation between species (in
andDNAcontent.(A)DAPI-stainedspreadofA.hierochuntica
chromosomes(2n=22)inyoungshoottipmeristems.(B)Flowcytometry decreasingorderofabsoluteeigenvectorvalueandconfirmedby
analysisof2CDNAcontentinArabidopsisthaliana,A.hierochunticaand two-way ANOVA) were raffinose, malate, galactinol, fumarate,
Eutremasalsugineum.(C)DNAcontentofthethreeBrassicaceaespecies phosphoric acid, and glycerate (Supplementary Tables S1 and
derivedfrom(B).Dataaremean(n=4)±SD.Theratiooftherelative
S2). Overall, the two-way ANOVA test for all three species
fluorescencefromtheG0/G1peakpositionsforeachsamplewascompared revealedthat25outof33metabolitesweresignificantlydifferent
againsttheinternalstandardRaphanussativuscv.Saxaasagenomesize
reference(2C=1.11pgofDNA).Cytometergainwasadjustedsothatthe between the species (Supplementary Table S2). Among these
G0/G1peakofR.sativuswaspositionedonchannel1500. 25 metabolites, 22 metabolites also displayed a significant
species×salt-treatmentinteraction.
The second principal component accounting for 19.8% of
allsaltconcentrations(Figure5F)suggestingthatdifferencesin totalvariancediscriminatedsamplesaccordingtosalttreatment.
ion contents was not due to variations in tissue water content. Among the metabolites most affecting separation between
Takentogether,ourionanalysisdatasuggestthatA.hierochuntica samples(indescendingorderofabsoluteeigenvectorvalueand
isabletotightlycontrolNa+accumulation. confirmedbytwo-wayANOVA)wereproline,malate,raffinose,
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FIGURE3|Effectofheatshock(A–D)andlowKNO3−concentrations(E–G)ongrowthparametersandpigmentcontentsofArabidopsis(At)and
A.hierochuntica(Ah).(A)PictureofseedlingsgrownonMSmedium,48hafterexposureto0,1,2,3or4hheatshock(45◦C).(B)ShootFW.(C)Totalchlorophyll.
(D)Totalcarotenoids.(E)Pictureofseedlingsgrownfor6daysonMSmediumsupplementedwiththeindicatedconcentrationsofKNO3−.(F)PlantFW.(G)Total
anthocyanincontent.DataaremeanofthreeandfourindependentexperimentsforlowNO3−andheat,respectively±SD.Eachindependentexperiment
comprisedfourreplicatescontainingca.10–15seedlings.BarswithdifferentlettersindicateasignificantdifferenceatP<0.05(TukeyHSDtest).FW,freshweight.
serine, fumarate, and myo-inositol. Overall, 20 metabolites that placed the two extremophytes on a separate clade from
were found to be significantly different between salt treatments Arabidopsis but separated E. salsugineum and A. hierochuntica
(Supplementary Table S2). Control and salt-treated Arabidopsis on two sub-clades (Figure 6C). PCA of the two extremophytes
sample groups were substantially spread out across the second alone clearly illustrated the species-specific metabolic response
component,whereasE.salsuginemandA.hierochunticasample tosaltstress(Figure6B).Themetabolitesthatprimarilyaffected
groupsweremorecondensed. the separation of the extremophytes (in decreasing order of
The complete separation of the metabolic profiles of absolute eigenvector value and confirmed by two-way ANOVA
Arabidopsis and the two extremophytes, and the partially test)were:proline,citrate,aspartate,raffinose,threonicacid,and
overlapping metabolic response of E. salsugineum and pyroglutamicacid(SupplementaryTablesS1andS2).
A. hierochuntica suggested both common and species-specific Examination of individual metabolites under control
responses of the extremophytes to salt stress (Figure 6A). This conditionsrevealedanoverallcomparablemetabolicphenotype
notionwassupportedbyhierarchicalclusteringanalysis(HCA) between the two extremophytes with most of the metabolites
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FIGURE4|TheeffectofsaltstressongrowthparametersofA.hierochunticaandArabidopsis.(A–C)Plantsgrownonsoilwereexposedtoincremental
increasesofNaClconcentration,andwereharvested1weekafterthefinalNaClconcentrationwasreached.(A)ShootFW.(B)ShootDW.(C)Rosetteleafarea.
(D,E)SeedlingsweregrownonMSmediuminverticalplateswithoutNaClandthentransferredtofreshverticalMSplatessupplementedwiththeindicatedNaCl
concentrations.(D)Arabidopsisrootrelativegrowthrate(RGR).(E)A.hierochunticarootRGR.Dataareexpressedasapercentageofcontrol(0mM)RGRonday1
aftertransfertosaltstress.Dataaremeanofthreetofourexperiments±SD.Eachindependentexperimentcomprisedfourreplicateplateswith10seedlingsper
plate.Forbothsoilandplateexperiments,barsortimepointswithdifferentlettersindicateasignificantdifferenceatP<0.05(TukeyHSDtest).FW,freshweight;
DW,dryweight.
exhibiting a significantly higher abundance in A. hierochuntica Inspection of the levels of metabolites in response to salt
andE.salsugineumcomparedtoArabidopsis(Figure7).Among stress revealed only a few common metabolite responses. All
the metabolites exhibiting differences between Arabidopsis three species exhibited increased levels of the osmoprotectants
and the two extremophytes under control conditions were: myo-inositol and proline in response to salt stress (Figure 8).
(i) the TCA cycle intermediates, citrate and malate that Notably, E. salsugineum exhibited greater proline content than
were significantly higher in the extremophytes, and fumarate Arabidopsis and A. hierochuntica at control and 100 mM NaCl.
which was significantly higher in Arabidopsis; (ii) the sugars, Highprolinelevelsareawell-characterizedfeatureofsoil-grown,
glucose and fructose; (iii) the hexose phosphates, fructose- well-fertilized E. salsugineum (Kant et al., 2006; Guevara et al.,
6-phosphate and glucose-6-phosphate; (iv) phosphoric acid; 2012; Kazachkova et al., 2013). In addition, glycine levels were
(v) the antioxidants, ascorbate and dehydroascorbate (Foyer reduced in response to stress in all three species although the
and Noctor, 2011). The levels of the metabolites indicated in actuallevelofglycinewashighestinArabidopsisatalltreatment
(ii) to (v) were all higher in the extremophytes. In addition, levelscomparedtotheextremophytes.
the osmoprotectant proline (Smirnoff and Cumbes, 1989) was Incontrasttothefewcommonmetabolicresponsesbetween
higher in the extremophyte plants under control conditions thethreespecies,aconsiderablenumberofmetabolitesexhibited
whiletheosmoprotectantsmyo-inositol(alsoaprecursorofthe an overall common response in both extremophytes that
osmoprotectant galactinol; Bohnert et al., 2006) and galactinol was different to that observed in Arabidopsis. For instance,
(alsoaprecursoroftheosmoprotectantraffinose;Tajietal.,2002; among amino acids, the level of alanine in Arabidopsis
Nishizawaetal.,2008)werehigherinArabidopsis.Interestingly, gradually declined in response to salt, while remaining
themajorityofmetabolitesdisplayedsignificantlylowerlevelsin constitutively high in the extremophytes and even rising
A.hierochunticacomparedtoE.salsugineum. slightly in A. hierochuntica at 200 mM NaCl. Aspartate was
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FIGURE5|EffectofNaClonNa+andK+contents(A–D)andphotochemistryparameters(E,F)ofArabidopsisandA.hierochunticashoots.Plantsgrownon
soilwereexposedtoincrementalincreasesofNaClconcentration.Theplantswereharvested1weekafterthefinalNaClconcentrationwasreached.(A)Absolute
Na+content.(B)RelativechangesinNa+contentcomparedtocontroltreatment.(C)K+content.(D)Na+/K+ratio.(E)RelativechangesinNa+/K+ratio
comparedtocontroltreatment.(F)ShootFW/DWratio.(G)Non-photochemicalquenching(NPQ).(H)PSIIelectrontransportrates(ETR).ValuesofNPQandPSII
ETRarepresentedfromplantsexposedtoalightintensityof1500PhotosyntheticPhotonFluxDensity(PPFD).Datarepresentmean(n=4)±SD.Eachreplicate
comprisedpooleddatafromsixindividualplants.Thedataarerepresentativeofsimilarresultsfromtwoindependentexperiments.Barswithdifferentlettersindicate
asignificantdifferenceatP<0.05(TukeyHSDtest).FW,freshweight;DW,dryweight.
barely detectable in Arabidopsis, yet increased substantially both Arabidopsis and A. hierochuntica but accumulated to
with salt concentration in both extremophyte species, but considerably higher levels in Arabidopsis at the highest salt
particularlyinE.salsugineum.Incontrast,thelevelsofthreonine concentration.Ontheotherhand,serineonlydisplayedaslight
increaseddramaticallyinArabidopsis,whileremainingrelatively salt-mediateddecreaseinE.salsugineum.Overallglyceratelevels
lower in the extremophytes, with a small but significant rise in were much lower in Arabidopsis than in the extremophytes.
A.hierochunticaathighersaltlevels.Serineandglycerate,which Furthermore, while glycerate exhibited no response to salt in
are intermediates of the photorespiratory cycle, also displayed Arabidopsis, it was maintained at a constantly higher level in
differences in accumulation between the extremophytes and A. hierochuntica and displayed a salt-mediated increase in E.
Arabidopsis. Serine content increased in response to salt in salsugineum.
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FIGURE6|Principalcomponentanalyses(PCA)andhierarchicalclusteringofmetabolicprofilesofArabidopsis(At),A.hierochuntica(Ah)and
E.salsugineum(Es)undercontrolandsaltstressconditions.ForPCA,metaboliterelativeabundancewasfirstnormalizedusinglog10transformationandthen
analyzedstatisticallyusingMultiExperimentViewerVersion3.1software(Saeedetal.,2003).Thevarianceexplainedbyeachcomponentisindicatedinbrackets.(A)
PCAcomparingallthreespecies.(B)PCAcomparingE.salsugineumandA.hierochuntica.(C)Hierarchicalclustering(HCA)ofallthreespecies.ForHCA,averages
ofabsolutevalues(n=4)foreachmetabolitenormalizedbytheribitolstandardweredividedbythemediannormalizedmetaboliteabundanceandarepresentedas
aheatmap.Forbothanalyses,dataarerepresentativeofsimilarresultsfromtwoindependentexperiments.
ThelevelsoftheTCAcycleintermediates,citrateandmalate, level of serine in E. salsugineum was significantly reduced by
were substantially higher in the extremophytes compared to salt stress whereas in A. hierochuntica serine content increased
Arabidopsis. However, whereas in E. salsugineum both citrate significantly. Pyroglutamate (possibly part of the glutathione
and malate levels increased significantly in response to salt, recycling pathway, an osmoprotectant and a Glu reservoir
citrate levels in A. hierochuntica increased significantly while (Ohkama-Ohtsu et al., 2008; Kumar and Bachhawat, 2012;
malateremainedataconstantlyhigherlevelthaninArabidopsis. Schreiber et al., 2012), fructose, and glycerophosphoglycerol all
Fumaratecontent,ontheotherhand,wassubstantiallyhigherin exhibitedconstitutivelyhigherlevelsinE.salsugineumcompared
Arabidopsis,decreasinginresponsetosaltstress,butremaining toA.hierochuntica.
constantly low in the extremophytes. The levels of other
A. hierochuntica is Tolerant to Severe
carboxylic acids, such as ascorbate and dehydroascorbate, were
constitutivelyhigherintheextremophytesthaninArabidopsis. Oxidative Stress
Differences in sugar contents between Arabidopsis and the The constitutively higher levels of the antioxidant compounds
extremophyteswerealsoobserved.Themoststrikingdifferences ascorbate and dehydroascorbate in A. hierochuntica (and
wereinthelevelsoftheosmoprotectantsgalactinolandraffinose E. salsugineum) compared to Arabidopsis (Figures7and8)
(Taji et al., 2002; Nishizawa et al., 2008). Both increased suggested that A. hierochuntica possesses a constitutively
dramatically in Arabidopsis, but were induced to a much lower active antioxidant system. Comparison of the response of
extent in the extremophytes. In addition, fructose-6-phosphate Arabidopsis and A. hierochuntica seedlings to ROS-forming
andglucose-6-phosphateaccumulatedtoagreaterextentinthe methyl viologen (MV) (Babbs et al., 1989; Fujii et al.,
extremophytes, being maintained at overall constant levels in 1990), demonstrated that A. hierochuntica seedlings exhibited
A.hierochuntica,whilesignificantlyreducedinE.salsugineumin remarkable tolerance to increasing concentrations of MV
responsetosalt. compared to Arabidopsis (Figure 9A). A. hierochuntica shoot
We also discerned species-specific responses that FW and root elongation decreased to a far lesser extent
differentiated between the extremophytes. For example, the than Arabidopsis as MV concentration rose. Furthermore,
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