Improved Tolerance to Various Abiotic Stresses in Transgenic Sweet Potato

14 Pages·2012·1.74 MB·English

Improved Tolerance to Various Abiotic Stresses in
Transgenic Sweet Potato (Ipomoeabatatas) Expressing
Spinach Betaine Aldehyde Dehydrogenase
Weijuan Fan1, Min Zhang2,3, Hongxia Zhang1, Peng Zhang1,2,3*
1NationalKeyLaboratoryofPlantMolecularGenetics,InstituteofPlantPhysiologyandEcology,ShanghaiInstitutesforBiologicalSciences,ChineseAcademyofSciences,
Shanghai,China,2Key Laboratory of SyntheticBiology, Instituteof PlantPhysiology and Ecology, Shanghai Institutes forBiologicalSciences,ChineseAcademyof
Sciences,Shanghai,China,3ShanghaiChenshanPlantScienceResearchCenter,ChineseAcademyofSciences,ShanghaiChenshanBotanicalGarden,Shanghai,China
Abstract
Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea
batatas),arootcropwithworldwideimportance.Theincreasedproductionofglycinebetaine(GB)improvesplanttolerance
to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield
productionunderunfavorableconditions.Thegeneencodingbetainealdehydedehydrogenase(BADH)isinvolvedinthe
biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic
stresstoleranceinplants.Thisstudyistoimprovesweetpotato,aGBaccumulator,resistanttomultipleabioticstressesby
promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet
potatocultivarSushu-2viaAgrobacterium-mediatedtransformation.TheoverexpressionofSoBADHinthetransgenicsweet
potatoimprovedtolerancetovariousabioticstresses,includingsalt,oxidativestress,andlowtemperature.Theincreased
BADHactivityandGBaccumulationinthetransgenicplantlinesundernormalandmultipleenvironmentalstressesresulted
inincreasedprotectionagainstcelldamagethroughthemaintenanceofcellmembraneintegrity,strongerphotosynthetic
activity,reducedreactiveoxygenspecies(ROS)production,andinductionoractivationofROSscavengingbytheincreased
activity offreeradical-scavenging enzymes.Theincreasedproline accumulation andsystemic upregulationofmany ROS-
scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-
scavengingsystemandprolinebiosynthesisviaanintegrativemechanism.Thisstudydemonstratesthattheenhancement
ofGBbiosynthesisinsweetpotatoisaneffectiveandfeasibleapproachtoimproveitstolerancetomultipleabioticstresses
withoutcausingphenotypicdefects.Thisstrategyfortraitimprovementinsweetpotatonotonlystabilizesyieldproduction
in normal soils in unpredictable climates but also provides a novel germplasm for sweet potato production on marginal
lands.
Citation:FanW,ZhangM,ZhangH,ZhangP(2012)ImprovedTolerancetoVariousAbioticStressesinTransgenicSweetPotato(Ipomoeabatatas)Expressing
SpinachBetaineAldehydeDehydrogenase.PLoSONE7(5):e37344.doi:10.1371/journal.pone.0037344
Editor:SunghunPark,KansasStateUniversity,UnitedStatesofAmerica
ReceivedJanuary11,2012;AcceptedApril20,2012;PublishedMay16,2012
Copyright:(cid:2)2012Fanetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbygrantsfromtheNationalHighTechnologyResearchandDevelopmentProgramofChina(No.2009AA10Z102),Shanghai
Municipal Afforestation & City Appearance and Environmental Sanitation Administration (G102410), Shanghai Science and Technology Committee
(No.09DZ2271800)andtheChineseAcademyofSciences(KSCX2-EW-J-12).TheShanghaiKeyLaboratoryofBio-EnergyCropsalsosupportedthework.The
fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction promising bioenergy crops, the sweet potato plays an important
role in the development of first-generation biofuels in China [7].
Sweet potato (Ipomoea batatas) is a major root crop that ranks
However,pests,viraldiseasesandenvironmentalstresses,e.g.low
seventh in annual production worldwide and is grown in more
temperature and drought, frequently restrict the increased
than100countriesasavaluablesourceoffood,animalfeed,and production of the sweet potato in many areas of the world [8].
industrial raw material [1,2,3]. Because of its tolerance to a wide New sweet potato varieties with enhanced tolerance to multiple
range of agroecological conditions, high yield potential, ease of abotic stressesare desirable.
cultivation, effective vegetative propagation and high nutritive
Although traditional breeding has significantly contributed to
value, the sweet potato is suitable for growth on marginal lands
trait improvement in sweet potato in the last several decades,
[2]. Recently, a strategy for producing energy from non-grain
producinganovelgermplasmwithdesirabletraitsremainsdifficult
biomassresourcesusingmarginallandstominimizethepotential
due to the high levels of male sterility, self and interspecific
impactofbioenergyproductiononfoodsecuritywasdevelopedin
incompatibility, and hexaploid nature of the sweet potato, which
China[4].Arecentsurveyrevealedanareaof34millionhm2of
resultsinthestrongsegregationofhybridprogeniesandthelossof
marginallandavailableforgrowingbioenergycropsinChinaand,
manyvaluabletraits[9,10].Asanalternative,geneticengineering
potentially, 45 million metric tons of liquid biofuels may be
offersgreatpotentialforimprovingthesweetpotatoorgenerating
produced if 60% of this area isutilized [5,6]. As one of the most
useful breeding materials with improved traits, such as increased
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BADHImprovesStressToleranceinSweetPotato
starch content and nutritional value, virus and nematode (WT) plants. After GUS staining, the transgenic OE plants
resistanceandsalttolerance,bytheexpressionofnativeorforeign displayed GUS activities in their leaves, in contrast with WT
genes [11,12]. Recently, we established an efficient Agrobacterium (FigureS2),confirmingthestableintegrationandexpressionofthe
tumefaciens-mediated transformation system for several farmer- uidA gene. The integration of the pCSoBADH T-DNA into the
preferredcultivarsofsweetpotato[13],whichprovidedaroutine genome of the transformed plants was determined by PCR
tool for genetic improvement via transgenesis and mediated the analysis using HPT-specific primers (Table 1). Three single-
examination of the function of exogenous and endogenous genes integrated SoBADH transgenicsweet potato linesOE1, OE2and
in sweet potato. OE3 were verified in a Southern blot analysis by hybridizing
Improving yield production and stability under stressful EcoRI-digested genomic DNA samples with a SoBADH-specific
conditions is necessary to fulfill the food demand of the ever- probe, and no integration was detected in the WT plants (Figure
growing world population [14]. Besides the successful commer- S3A). The RT-PCR analysis detected the expression of mRNA
cialization of transgenic crops with many desirable traits, e.g., transcripts in all three OE lines but not in the WT line (Figure
resistance to insect pests, viral diseases or pesticides [15], an S3B), showing the expression of the SoBADH gene in those
improved tolerance to adverse environmental conditions has also transgenic plants. To further ascertain whether the SoBADH
been achieved [16–18]. For improving abiotic stress tolerance in overexpression is correlated with increased BADH enzyme
plants through genetic engineering, many studies have primarily activity, we measured the BADH activity and GB content in
focused on transcription factors for gene regulation [19,20] and solubleextractsfromtheleavesofsweetpotatoplants(Figure1A,
genesthatencodeiontransportproteins[21],compatibleorganic B).TheBADHactivityandaGBcontentof0.52 mmolg21were
solutes [22,23], antioxidants [24,25], heat-shock [26,27] and late detectedintheWTleaves,indicatingthatsweetpotatoisanatural
embryogenesis abundant proteins [28,29]. Although many genes accumulator of GB. Importantly, the BADH activity in the OE
associated with plant responses to abiotic stress have been lines was elevated, and the OE2 plants exhibited a 1.84-fold
identifiedandcharacterizedinArabidopsisandrice,theirfunctions increaseinactivityascomparedwiththatoftheWTplants.Asa
remain tobeverified inother crops. result,underthenormalgrowthconditions,theGBcontentinthe
Differentabioticstressfactorsmayinduceosmoticandoxidative leaves of the OE lines was significantly higher than that of WT
stressinplants,leadingtosimilarcellularadaptiveresponses,such (Figure1B).PlantlineOE2demonstratedthehighestGBcontent
astheaccumulationofcompatiblesolutesandtheaccelerationof at concentration of 2.56mmol g21, which is about a five-fold
reactive oxygen species-scavenging systems [30]. Compatible increase overthat of WT.
solutes, including proline, trehalose, and GB, have been detected When OE transgenic and WT plants were grown in the
in a wide variety of organisms [23]. GB is a fully N-methyl- greenhouse under normal conditions, no visible phenotypic
substituted derivative of glycine. This quaternary ammonium difference of their foliages was detected during the plant growth
compoundispresent inbacteria,animals,andhigherplants,and (Data not shown). After 5 months, the weights of storage roots
functions by stabilizing the quaternary structure of complex fromOEplantlinesshowedsignificantincreasecomparedtothose
proteins, membrane systems, enzymes, and photosynthetic ofWTplants.TheyieldsoftuberousrootsinthethreeOEplant
machinery, such as PSII complexes and RuBisCo [31–34]. The lineswere156.67,300and155.56 gplant21,respectively,whichis
exogenous application of GB confers tolerance to various abiotic significantly heavier than that of the WT line (47.78g plant21)
stresses in plants and increases their yield under salt conditions (Figure 1C).
[34,35]. In general, the GB biosynthetic pathway starts with
choline and progresses through a two-step dehydrogenation of Enhanced tolerance of OE transgenic plants to high
choline and oxygenation of betaine aldehyde catalyzed by CMO salinity stress
and BADH, respectively [33]. Plants overexpressing the genes GB is primarily found in plants growing in salt-affected areas,
responsible for GB synthesis, e.g., BADH, have an enhanced and previous transgenic studies have demonstrated enhanced
tolerance to a wide range of abiotic stresses, including salt, heat, tolerancetosaltstressinGBoverexpressinglines[41–42].Inour
drought, and chilling, by the increased accumulation of GB in study,afterexposureto200mMNaClfor16daysusing2-month-
plant cells[36–40]. old plants, a significant impact on plant growth was observed
In this report, we used Agrobacterium to transform embryogenic (Figure 2A). Before treatment, the transgenic plants showed
suspensions of the cultivar Sushu-2 and develop transgenic sweet phenotypes indistinguishable from the WT plants (Figure 2A,
potatoplantsexpressingtheBADHgenefromSpinaciaoleracea.The upper panel); however, after treatment, the WT plants showed
expressionoftheSoBADHgeneincreasedBADHactivityandGB severelyinhibitedgrowthwithyellowishleafcoloration(Figure2A
synthesis in these transgenic sweet potato plants, which subse- lowerpanel)anddramaticallyreducedfreshweightincontrastto
quently improved their tolerance to multiple abiotic stresses by thevigorousgrowthofOEplantlines(Figure2B).Wedetecteda
inductionoractivationROSscavengingandtheaccumulationof reduction in the fresh weight of the transgenic plant lines from
proline. 40g/plantto25–30g/plant(25%–37.5%reduction)ascompared
with40g/plantto10g/plantofreduction(74%)intheWT.The
Results fresh weights of trangenic plants were a minimum of 2 times
greater than WT plants under the stress treatment. The BADH
Molecular, biochemical and phenotypic characterization
activity and GB concentration in the leaves of the three OE
of the SoBADH transgenic sweet potato transgenic plant lines were significantly higher than those of WT
Transgenic sweet potato plants overexpressing SoBADH (P,0.05,Figure2C,D).Forexample,intheWT,theGBcontent
(termed OE) were successfully generated by the Agrobacterium- wasincreasedfive-foldfrom0.63 mmolg21FWto2.56mmolg21
mediatedtransformationofthebinaryvectorpCSoBADH(Figure FW after treatment. Upon treatment of the transgenic line OE2,
S1)intoembryogenicsuspensionculturesofthecultivarSushu-2. theGBcontentwasincreasedto5.41 mmolg21FWincomparison
Morethan10independenttransgenicplantlineswereestablished withthe2.5mmolg21FWoftheuntreatedsamples.Overall,the
andpropagatedinthegreenhouse.Mostofthesetransgenicplant transgenicplant linesshoweda33.2%to111.3%increaseinGB
lines were phenotypically indistinguishable from the wild-type content as compared with the WT after salt treatment. These
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BADHImprovesStressToleranceinSweetPotato
Table1. Phenotypic andprimary molecularscreening ofthe transgenic sweetpotatolinesfrom transformedcultivar Sushu-2.
Line Rootingtest* Shootphenotype PCR GUSstaining Insertionnumber
OE1 Rooted normal positive stained 1
OE2 Rooted normal positive stained 1
OE3 Rooted normal positive stained 1
OE4 Rooted normal positive stained 2
OE5 Rooted normal positive stained 1
OE6 Rooted normal positive stained 1
OE7 Rooted normal positive stained 2
OE8 Rooted normal positive stained 3
OE9 Rooted normal positive stained 2
OE10 Rooted dwarf NT** NT NT
Sushu-2 - normal - Non-stained -
Notes:
*Arootingtestwasperformedbyculturingshootsonbasicmediumsupplementedwith10mgl21hygromycinandrecordedaftertwoweeks;
**Nottested.
doi:10.1371/journal.pone.0037344.t001
results indicate that the enhanced tolerance to salt stress in the Undernormalconditions,therewasnosignificantdifferencein
transgenic sweet potato is due to the increased accumulation of the proline content in the leaves of the transgenic OE lines and
GB inplants. WT (Figure 3E). After 16 days of NaCl treatment, the proline
The growth retardation observed in many plants under salt content in the WT plants increased from 330mg g21 FW to
stress is often associated with a decrease in their photosynthetic 990mg g21 FW, which is a 3-fold difference. In the OE lines,
capacity [43]. Under salt stress, the OE lines maintained a high however,thefoldchangesofprolinecontentintheleaveswas4.46
level of chlorophyll content (11.6% to 13.9% reduction), while a to6.14,whichismuchhigherthanthatoftheWT.Forexample,
dramaticdecreaseof58.8%wasdetectedintheWT(Figure2E). the proline contents of OE2 leaves were increased from 340mg
To further evaluate the effects of stress-induced damage to g21 FW before the treatment to 2091mg g21 FW after the
photosystemII(PSII),themaximalefficiencyofPSIIphotochem- treatment (Figure 3E).
istry (Fv/Fm) was studied. Without salinity stress, there was little In order to distinguish phenotypic differences between trans-
difference in the Fv/Fm ratio among these lines. After stress genic and non-transgenic plants in the greenhouse, e.g. tuberous
treatment,thetransgenicOElinesmaintainedan86–90%Fv/Fm rootproduction,theplantsweretreatedwith150mMNaClfor6
ratio as compared with the 76% ratio maintained in WT weeks.TheOEplantsdemonstratedavigorousgrowthwithmore
(Figure 2F). These results indicate that the photosystem of OE and bigger leaves in comparison with those of WT (Figure 4A,
transgenic plants is less affected than that of the non-transgenic rightupperpanel).Besidesmorefibrousroots,theOEplantsalso
WT plants during stress. developed storage roots. No any tuberous roots were observed in
Recent studies have demonstrated that elevated ROS inhibits theWTplants(Figure4A,leftlowerpanel).AmongtheOElines,
the repair of photoinhibited PSII in vivo [43]. Therefore, we OE2 performed the best with the strongest foliage and biggest
postulated that the accelerated repair of salt-enhanced photo- storage root development (Figure 3E, right lower panel). After
inhibition induced by the accumulation of GB in vivo may be exposureto150mMNaClfor6weeks,thetreatmentresultedina
associatedwiththeelevatedROSscavengingintransgenicplants. significantaccumulationofNa+intheleavesandroots.However,
ThesaltstressstimulatedanevidentincreaseofH O production no significant difference was found in the levels of Na+ between
2 2
in the leaves of the four sweet potato lines (Figure 3A, B). WT and transgenic plants in both control and treated plants on
However,asignificantdecreaseinH O production,with61.8%, thebasisof leaf androotdry weight(Figure 4B, C).
2 2
23.9% and 54.1% of the levels in WT, was observed in the
transgenic lines OE1, OE2 and OE3, respectively (P,0.05) Improved tolerance to MV-mediated oxidative stress
(Figure 3B). The SOD activity, an important indicator for ROS Upon treatment with 200mM MV, oxidative damage was
scavenging, in these plant lines was significantly increased after observedinalltreated2-month-oldplants.Ascomparedwiththe
NaCl treatment, and the OE lines showed much higher activity WT plants, which showed severe leaf damage, the three OE
than WT, with a 22.9% increase in WT and a 36.5% to 40.5% transgenicplantsshowedreduceddamage(Figure5A).Consistent
increaseintheOElines(Figure3C).Thisdifferenceindicatedthat with the reduced damage, a significantly reduced ion leakage in
the expression of the SoBADH gene might protect the synthesis theOEtransgeniclineswasdetected(Figure5B).TheGBcontents
and/or activation of this enzyme, which results in less accumu- inthethreeOEplantlinesafter48hofMVtreatmentwere1.71,
lationofROSandcelldamageinthetransgenicplants.Similarto 3.4 and 2.1mmol g21 FW (Figure 5C), respectively, which is
H O , NaCl treatment substantially increased the levels of significantly higher than that of the treated WT (0.95 mmol g21
2 2
malonaldehyde (MDA) in both the OE and WT lines FW).During thetimecourseof MVtreatment,theprolinelevels
(Figure 3D), but much less MDA accumulation was detected in were continuously increased both in the WT and transgenic OE
the OE plants (Figure 3D), indicating decrease in lipid peroxida- lines. However, the OE lines accumulated a significantly higher
tion undersalt stressin thetransgeniclines. amount of proline than the WT. At 48h, the proline content in
theWTreached837mgg21FW,whereastheprolineconcentra-
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BADHImprovesStressToleranceinSweetPotato
respectively. In contrast, the OE lines showed a more reduced
accumulationofMDAandH O .Forexample,thelevelofMDA
2 2
andH O intheOE2plantswas58.3 nmolg21FWand3.7mM,
2 2
respectively (Figure 4D, E). The OE2 plants also exhibited a
29.3%increaseinSODactivityascomparedwithWTplantsafter
48h ofMV treatment (Figure 5F).
Enhanced tolerance to cold stress
To assess the effects of SoBADH expression on cold stress
toleranceinsoil-grownplants,2-month-oldplantswereincubated
for 18h at 4uC and transferred to 25uC for recovery. When the
WTandOEplantswereexposedtocoldfor18h,theleavesofthe
WTplantsshowedseverewiltinganddownwardcurling,whereas
those of the OE plants were only slightly affected (Figure 6A).
Furthermore,aftera4 hrecovery,theOEplantswerealmostfully
recovered to the initial phenotype, whereas the WT plants still
exhibited wilting in the leaves (Figure 6A). In addition to
phenotypic changes, the levels of electrolyte leakage were also
moreincreasedintheWTthaninthetransgeniclines(Figure6B).
IncreasedGBexpressionwasdetectedintheOElines(Figure6C),
reaching3.04mmolg21FW,whichwasmorethan2-foldgreater
than that of WT. The low temperature stress also stimulated the
production of MDA and H O in WT leaves (Figure 6F). The
2 2
levels of MDA in the transgenic lines OE1, OE2 and OE3 only
reached to 65.4%, 38.7% and 81.2% of WT, respectively
(Figure 6D). The H O content in these OE plants was
2 2
accumulated to 53.4%, 38.3%, and 50% of WT, respectively,
after18hofcoldstress(Figure6E).Inresponsetocoldstress,the
SODactivitywasalwayshigherinthetransgeniclinesthaninWT
(Figure6F).Lowtemperaturesinducedmoreprolineaccumulation
in leaves of the OE lines than those of the WT. After 18h, the
proline content in the leaves of the OE2 plants had increased to
272%(1361mgg21FW)oftheWT(499mgg21FW)(Figure6G).
Expression profiling of GB biosynthetic and ROS-
scavenging genes
To investigate the impact of SoBADH expression on the gene
transcription of native GB biosynthetic genes and stress response
pathways,theexpressionofselectedgenes(TableS1)wasanalyzed
during the abiotic stress treatments. The most interesting results
were obtained from the prevalence of genes encoding the ROS-
scavenging enzymes and the genes encoding functions related to
the D1 protein and phosphoribulokinase (PRKase). During GB
biosynthesis,SoBADHexpressionwassignificantlyincreasedinthe
transgenicOElinesinalltreatmentconditionsincontrastwithno
changes in the level of CMO gene expression (Figure S4).
Importantly, a systemic upregulation of the ROS-scavenging
Figure 1. BADH accumulation and root biomass of wild-type enzymes genes, including SOD, catalase (CAT), ascorbate
andtheSoBADHtransgenicplants.AandBshowtheBADHactivity peroxidase (APX), monodehydroascorbate reductase gene
andGBcontentintheleaves,respectively;Cshowstherootbiomass. (MDHAR), glutathione peroxidase gene (GPX), glutathione
OE1–OE3,SoBADHoverexpressingtransgeniclines;WT,wild-typeplant.
reductase gene (GR), dehydroascorbate reductase gene (DHAR)
Valuesrepresentthemean6SD(n=9).Asterisksindicateasignificant
and peroxidase gene (POD), was detected (Figure 7). The
differencefromthatofWTat*P,0.05or**P,0.01byt-test.
doi:10.1371/journal.pone.0037344.g001 transcript levels of the PRK and psbA genes, which encode
phosphoribulokinase(PRKase)andD1protein,respectively,were
tionintheOE2plantswas1640mgg21FW,whichisatwo-fold not significantly changed in the WT and OE lines under any
condition.
difference (Figure 5G). Prior to MV treatment, there was no
obvious difference of MDA and H O contents between the
2 2
Discussion
transgenic lines and WT (Figure 5D, E). After treatment, the
MADandH O weredecreasedintheOElinesascomparedwith
2 2 This study demonstrates that the expression of the spinach
WT.InthetimecourseofMVtreatment,thelevelsofMDAand
(Spinacia oleracea L.) BADH in the sweet potato increases BADH
H O were elevated in the WT. At 48h, the MDA and H O
2 2 2 2 activity and GB biosynthesis in vivo, consequently leading to an
contents in the WT reached 150.3 nmol g21 FW and 72.3mM, enhanced tolerance of the transgenic sweet potato to various
respectively, which is an increase of 14.4 and 87.3 times, abiotic stresses. The increased accumulation of endogenous GB
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BADHImprovesStressToleranceinSweetPotato
Figure 2. Phenotypic and physiological analyses of theSoBADHtransgenicplantsunder 200mM NaCl stress for 16 days. A, the
phenotypicchangesbefore(upperpanel)andafterNaCltreatment(lowerpanel);B,freshweightoftransgenicandWTplantsafterNaCltreatment;C,
BADHactivitiesintransgenicandWTplantsbeforeandafterNaCltreatment;D,contentsofGBintheleavesoftransgenicandWTplantsafterNaCl
treatment.E,chlorophyllcontentintheleavesoftransgenicandWTplantsafterNaCltreatment;F,changesofmaximumfluorescenceratios(Fv/Fm)
intransgenicandWTplantsuponNaClstress.OE1–OE3,independent,SoBADHoverexpressingtransgeniclines;WT,wild-typeplant.Valuesrepresent
themean6SD(n=9).AsterisksindicateasignificantdifferencefromthatofWTat*P,0.05or**P,0.01byt-test.
doi:10.1371/journal.pone.0037344.g002
levels also demonstrated an enhanced tolerance, which is OElines,morethan3-foldincreaseintheweightofstorageroots
consistent with previous studies of the heterologous expression of under normal conditions (Figure 1C) and the tuberous root
bacterial choline dehydrogenase (betA), choline oxidase (codA) or development under the salt stress (Figure 4A) revealed that the
the plant BADH gene [31,34,42,44,45]. The elevated GB presence of the BADH transgene resulted in acceleration of the
biosynthesis in sweet potato also improves yield and promote plant growth, similar totheresults reported inYang et al.[48].
storagerootformation.Theseresultsindicatethattheengineering Under stress conditions, sweet potato plants instinctively
of enhanced GB biosynthesis in the sweet potato is a feasible synthesize and accumulate more GB, as evidenced in our study
approach toimprovingitstolerance tomultiple abiotic stresses. (a 5-fold increase of GB content in WT under the salt stress,
Manyapproacheshavebeenusedtoengineerstresstolerancein Figure 1B, 2D), indicating that the sweet potato is a GB
plantswithlimitedsuccess[46].Onemajorproblemisduetothe accumulator. The expression of CMO or BADH by RT-PCR
undesirable or unfavorable phenotypic changes, such as growth analysis (Figure S4) also substantiated the hypothesis. A series of
retardation and decrease in yield. So far positive effects of GB physiological studies have proven that GB most likely maintains
accumulationintransgenicplantshavebeenreported,evenwhen theosmoticbalancebetweentheintracellularandtheextracellular
grown under non-stress conditions [23,44]. Even under the environment under salt and drought stress [23,34,49]. The levels
stressful conditions, the performance of transgenic plants was of GB accumulation in different species are variable, and for
betterthanun-transformedcontrols,providinggreatpotentialfor certainplants,levelsofupto40mmolg21freshweightofGBhave
agriculturalapplication[23,34,47].Asevidencedinourtransgenic been reported under stress conditions [23,49]. Under the stress
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BADHImprovesStressToleranceinSweetPotato
Figure 3. TheSoBADHtransgenic plants showed improved ROS scavenging and proline contents under 200mM salt stress. A,
detectionofsaltstress-inducedHO productionbyDABstainingintheleavesofthetransgenicandWTplants;B–C,H O andMDAcontentsinthe
2 2 2 2
leavesofthetransgenicandWTplants;D,changesofSODactivityintransgenicandWTplantsafterNaCltreatment.E,prolinecontentsintheleaves
ofthetransgenicandWTplants.OE1–OE3,SoBADHoverexpressingtransgeniclines;WT,wild-typeplant.Valuesrepresentthemean6SD(n=9).
AsterisksindicateasignificantdifferencefromthatofWTat*P,0.05or**P,0.01byt-test.
doi:10.1371/journal.pone.0037344.g003
conditions, transgenic OE lines accumulate up to 5.6 mmol g21 burst of ROS, resulting in oxidative stress (Figure 8). ROS have
FW GB, a minimum of 2 times higher that of WT. In the been implicated in all types of stresses, and if not scavenged
transgenic sweet potato, such levels of GB could hardly be sufficiently, programmed cell death might occur [50,51]. On the
attributedtotheosmoticadjustmenttotheexternalenvironment, otherside,ifscavengingtoomuchROSmaynegativelyaffectthe
especially salinity stress. Nevertheless, a series of studies using signal transduction. Therefore, the balance of ROS generation
transgenic plants have demonstrated that GB, even at low levels, and scavenging is essential for stress response in plants. Despite
couldconfertransgenicplantswithanincreasedtolerancetocold, GB-mediated enhancement of stress tolerance in many plants, its
freezing,heat,photoinhibition,droughtandsaltstress[23,32,34]. mechanistic influence on stress signaling and perception remains
Mechanistically, GB stabilizes macromolecular activity and unclear because of a lack of direct evidence to support GB
membrane integrity [32]. Indeed, a reduced ion leakage and participation in antioxidative defense systems. Nevertheless, the
MDA content was detected in the leaf cells of the SoBADH increasedROSscavengingcapacitymediatedbyGBfunctionsdid
transgenic sweet potato (Figures 3D; 5B, D; 6B, D) in our study, not negatively affect the signal transduction, as reported by
suggesting an improved cell membrane homeostasis [31]. There- Kathuria H (2009) [52]. They used microarray-based transcrip-
fore,itcanbeassumedthatGBmightprotectthecellmembrane tome to analyses codA transgenic rice and GB accumulation
from stress-induced injuries, likely through alleviating oxidative unravelled altered expression of many genes involved in stress
stress (Figure 8). responses,signaltransduction,generegulation,hormonesignalling
Abioticstresses,suchassalinityandlowtemperature,whichare and cellular metabolisms. Studies have demonstrated that GB
knowntodisturbredoxhomeostasisinplantcells,couldinducea alone does not have antioxidative activity in vitro [53]. Thus, its
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BADHImprovesStressToleranceinSweetPotato
Figure4.ThegrowthandNa+levelsofSoBADHtransgenicplantsunder150mMsaltstresstreatmentfor6weeks.A,transgenicOE
linesshoweddistinguishabledifferencesfromWTonfoliageandrootgrowthafter150mMsaltstress.B–C,Na+levelinleavesandrootsofWTand
transgenicplants.Valuesrepresentthemean6SD(n=9).AsterisksindicateasignificantdifferencefromthatofWTat*P,0.05or**P,0.01byt-
test.
doi:10.1371/journal.pone.0037344.g004
ROS-scavengingfunctionmustbeindirect,e.g.,viatheinduction required in proline biosynthesis [56] and ROS scavenging [57],
ofthesynthesisoractivationofROS-defensesystems,asillustrated mightaffecttheredoxhomeostasisandfacilitateprolineaccumu-
in our proposed model (Figure 8). Such a scenario has been lation toactivate theROS-scavenging system (Figure 8).
demonstratedbothinplantsaftertheexogenousapplicationofGB It is important to maintain a stronger ROS-scavenging ability
and in the GB over-produced transgenic plants expressing GB- understressconditionstoalleviatetheinducedoxidativedamage,
biosyntheticenzymes[34].Forexample,transgenictomatoplants especially in plant leaves where photosynthesis is dramatically
expressing the choline oxidase encoded by the codA gene are impacted. Plants under abiotic stress have evolved a defense
targeted to chloroplasts, demonstrating an enhanced tolerance system against oxidative stress by increasing the activity of ROS-
against MV-mediated oxidative stress [54]. More profound scavenging enzymes [58]. Although the oxidative stress was
antioxidant enzyme activity had been evidenced in GB-overex- enhanced in the stressed leaves of the sweet potato, as reflected
pressing transgenic tomato under stress conditions [55]. Impor- intheincreasedH O content,wedetectedsignificantlylessH O
2 2 2 2
tantly, proline, a typical inert compatible osmolyte that protects in the OE transgenic lines as compared with that of the WT
subcellular structures and macromolecules under osmotic stress (Figure 3B, 5E, 6E). Consistent with this phenomenon, increased
[22,56], was more accumulated in the OE lines (Figure 3E, 5G, SOD expression and activity were detected in the OE lines. In
6G). In GB biosynthesis, the production of NAD(P)H, which is parallel, the expression of other important ROS-scavenging
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BADHImprovesStressToleranceinSweetPotato
Figure5.PhenotypicandphysiologicalanalysesintheSoBADHtransgenicplantsunderMVtreatment.A,phenotypicchangesbefore
(upperpanel)andafter(lowerpanel)MVtreatment(phototakenafter5days);B,decreasedionleakingintheMV-treatedleafdiscsoftheOEplants
thanthatofWTduringthetimecourse;C,contentsofGBintheleavesoftransgenicandWTplantsat48hafterMVtreatment;D–E,MDAandH O
2 2
contentsintheleavesofthetransgenicandWTplantsat24hand48hafterMVtreatment;F,changesinSODactivityinthetransgenicandWT
plantsat24hand48hafterMVtreatment.G,prolinecontentsintheleavesofthetransgenicandWTplantsat24hand48hafterthetreatment.
Valuesrepresentthemean6SD(n=9).AsterisksindicateasignificantdifferencefromthatofWTat*P,0.05or**P,0.01byt-test.
doi:10.1371/journal.pone.0037344.g005
enzymes,includingCAT,APX,MDHAR,GPX,GR,DHARand such as the oxygen-evolving PSII complex [32,59]. Previous
POD,was alsoupregulated atthetranscriptionallevel(Figure7), studiessuggestthatthemaineffectofROSistheinhibitionofthe
suggesting that the enhanced tolerance of the transgenic sweet repair of photodamaged PSII by the suppression of de novo
potato plants to abiotic stress is mainly due to improved ROS protein synthesis; the primary sites of photodamage are the
scavenging (Figure 8). Similar results were also observed in oxygen-evolving complex and the D1 proteins [60]. The rate of
transgenic plants transformed with the codA gene [36]. Thus, in repair of photodamaged PSII is severely depressed by various
sweetpotatoplants,itislikelythatGBinducesanH O -mediated typesofstress,suchasoxidativestress[43,61,62],salt[63,64]and
2 2
antioxidant system, which includes the enhanced transcriptional low temperature [62], with subsequent increases in the extent of
expression andactivity of theROS-scavenging enzymes. photoinhibition. The effect conferred by GB has been shown to
IthasbeenreportedthatGBprotectsthecellsfromstressesby protect themachinery required forthedegradation andsynthesis
stabilizing the quaternary structures of complex proteins like of the D1 protein under stress exerted by high-salt conditions
antioxidantenzymesandbiomembranesofotherfunctionalunits, [65,66].Inaddition,thechloroplast-targetedproductionofGBin
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BADHImprovesStressToleranceinSweetPotato
Figure6.PhenotypicandphysiologicalanalysesoftheSoBADHtransgenicplantsundercoldtreatment.A,phenotypicchangesbefore
(upperpanel)andafter(middlepanel)coldtreatment(4uCfor18h)andtherecoveredphenotype(lowerpanel);B,decreasedionleakinginthecold-
treatedleafdiscsofOEplantsthanthatofWT;C,contentsofGBintheleavesoftransgenicandWTplantsaftercoldtreatment;D–E,MDAandH O
2 2
contentsin the leaves of the transgenic and WT plants;F, changesof SOD activityin transgenic and WT plants after cold treatment.G, proline
contentsintheleavesofthetransgenicandWTplantsafterthetreatment.Valuesrepresentthemean6SD(n=9).Asterisksindicateasignificant
differencefromthatofWTat*P,0.05or**P,0.01byt-test.
doi:10.1371/journal.pone.0037344.g006
transgenictomatoplantsprotectsphotosyntheticmachinerymore suppose that the accumulation of GB in the transgenic sweet
efficiently than the cytosol-targeted production, showing the potatomightstabilizetheconformationofPRKaseandmaintain
accumulation of GB in chloroplasts ismore effective than that in theenzymeinafunctionallyactivestateundersaltstress,actingas
the cytosol for the protection of plants against abiotic stress [55]. a molecular chaperone.
Inourstudy,theOEplantsmaintainedhigherFv/Fmvaluesover A few reports have proposed the GB stabilizes the highly
the entire period of salt stress (Figure 2E). However, the results ordered structures of certain complex proteins to prevent
from the analysis by real-time PCR revealed that the transcrip- denaturation when plants or plant cells are exposed to stress
tional levels of the psbA and PRK genes, which encode the D1 conditions [59,67]. Based on our study,wepropose that elevated
protein and PRKase, respectively, were not significantly affected GBbiosynthesiscouldinduceorpromotetheexpressionofcertain
by the stresses or overexpression of GB in the plant cells. These stress-responsive genes, e.g., enzymes that scavenge ROS and
results suggest that, under stressful conditions, the functions of prolinebiosynthesis,andsubsequentlyreduceROSaccumulation
these genes might work or regulate at the protein level. We
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BADHImprovesStressToleranceinSweetPotato
Figure7.RelativeleveloftranscriptofROS-scavenginggenesinwild-type(WT)andSoBADHtransgenic(OE)plantsunderstresses.
Thetranscriptlevelofeachgeneintheleavesundernon-stressedconditionwasusedasareferenceforcomparison.APX,ascorbateperoxidase;
BADH,betainealdehydedehydrogenase;CAT,catalase;DHAR,dehydroascorbatereductase;GPX,glutathioneperoxidase;GR,glutathionereductase;
MDHAR,monodehydroascorbatereductase;POD,peroxidase;PSII,photosystemII;ROS,reactiveoxygenspecies;SOD,superoxidedismutase.Values
representthemean6SD(n=9).AsterisksindicateasignificantdifferencefromthatofWTat*P,0.05or**P,0.01byt-test.
doi:10.1371/journal.pone.0037344.g007
PLoSONE | www.plosone.org 10 May2012 | Volume 7 | Issue 5 | e37344