Transgeneration memory of stress in plants
Jean Molinier 1†,Gerhard Ries 1†,Cyril Zipfel 1†&Barbara Hohn 1
Owing to their ssile nature,plants are constantly expod to a multitude of environmental stress to which they react with a battery of respons.The result is plant tolerance to conditions such as excessive or inadequate light,water,salt and temperature,and resistance to pathogens.Not only is plant physiology known to change under abiotic or biotic stress,but changes in the genome have also been identified 1–5.However,it was not determined whether plants from successive generations of the original,stresd plants inherited the capacity for genomic change.Here we show that in Arabidopsis thaliana plants treated with short-wavelength radiation (ultraviolet-C)or flagellin (an elicitor of plant defences 6),somatic homologous recombination of a trans-genic reporter is incread in the treated population and the incread levels of homologous recombination persist in the subquent,untreated generations.The epigenetic trait of enhanced homologous recombination could be transmitted through both the maternal and the paternal crossing partner,and proved to be dominant.The increa of the hyper-recombina-tion state in generations subquent to the treated generation was independent of the prence of the transgenic allele (the recombi-nation substrate under consideration)in the treated plant.We conclude that environmental factors lead to incread ge
nomic flexibility even in successive,untreated generations,and may increa the potential for adaptation.
Plants are influenced by abiotic and biotic environmental factors on veral levels;apart from changes in plant physiology and the mounting of resistance respons,the dynamics of the genome can also be altered.Examples include the activation of transposable elements by abiotic and biotic stress conditions 7–9,induction of mutations by chemical and physical agents 10,and enhancement of homologous recombination by elevated temperatures 11or ultraviolet-B (UV-B)(ref.2).Especially interesting is the genomic flexibility shown by plant genomes in respon to pathogen attack 3,4,7.Whenever possible,such changes were monitored at the level of the quence of affected genes.The influence the changes have in evolutionary terms,however,remained poorly understood,becau most changes were detected in somatic tissue and not considered in further generations.In plants,the reproductive cell-lineage emerges from somatic tissue late in development 12,thus some genomic changes acquired during the life of a plant can be transmitted to the next generation.Indeed,with progeny of UV-B-or pathogen-treated plants,the frequency of occurrence of genetically fixed mutation (in this ca,homologous recombination)was reproducibly elevated 2,4.The degree of genomic change in the offspring of the stresd population was expected to return
to the basal level.We show here that incread levels of homologous recombination persist for veral generations in the lineage from the original parent plants that were expod to stress,including ultraviolet radiation or flagellin.
We measured the rate of homologous recombination in the untreated offspring of plants expod to conditions of environmental
damnitstress.We ud A.thaliana plants harbouring b -glucuronida (GUS )-bad constructs in which truncated but overlapping parts of the gene allow quantification of somatic homologous recombina-tion.The results of this event are visualized as blue spots on a white background following histochemical staining of plants (Fig.1a,b).Previous molecular analys of the plant DNA confirmed that the blue spots,which reprent GUS activity,indeed symbolize bona fide recombination events 13,14.Using this assay,the influence of ultra-violet-C (UV-C)was tested in six independent transgenic lines that carried the recombination reporter in different relative orientations of the GUS quence fragments:‘GU ’and ‘US ’(ref.15).The basal levels of homologous recombination,indicated as numbers of recombination ctors per plant,varied among the six lines;the degrees of stimulation were also different,but in all cas the treatment with UV-C stimulated the level of homologous recombi-nation (Fig.1c).UV-C induction of homologous recombination together with variation betwee
n independent transgenic lines is consistent with previous reports 2.
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Figure 1|Somatic homologous recombination in UV-C-and flg22-treated plants.a ,Schematic reprentation of a recombination substrate ud for monitoring somatic homologous recombination (lines IC1and IC9).GUS ,b -glucuronida gene;Hpt ,hygromycin-resistance gene.Homologous region is shown in dark blue.b ,Recombination events (blue spots highlighted by black arrows)giving a measure of homologous
recombination frequency (HRF;e Methods)in line IC1after flg22
treatment.Scale bar,1mm;int,£3original magnification.c ,Somatic HRF in untreated and UV-C-treated S 0plants.Results are means ^
(n .50plants;t -test *P ,0.05).d ,Somatic HRF in either untreated plants,plants treated with flg22A.tum .,or treated with flg22.Results are means ^(n .40plants;t -test *P ,0.05).
1
Friedrich Miescher Institute for Biomedical Rearch,Maulbeerstras 66,CH-4058Bal,Switzerland.†Prent address:Institut de Biologie Mole
´culaire des Plantes,12Rue du Ge
´ne ´ral Zimmer,F-67084Strasbourg Cedex,France (J.M.);BioMedinvestor AG,Elisabethenstras 23,CH-4051Bal,Switzerland (G.R.);The Sainsbury Laboratory,John Innes Centre,Colney Lane,Norwich NR47UH,UK
(C.Z.).
Inoculation of homologous-recombination-tester plants with pathogens was previously shown to increa homologous recombi-nation frequencies3,4.Here we uflagellin,a bacterium-derived elicitor,to activate plant defences.In A.thaliana,perception of flagellin,the building block of the bacterialflagellum,occurs by recognition of the most conrved domain on theflagellin amino terminus,reprented by the peptideflg22(ref.16).Treatment of plants with this peptide has been shown to trigger resistance to pathogenic bacteria6,unlike treatment with the inactive analogue from Agrobacterium tumefaciens(flg22A.tum.).In all six transgenic lines analyd,we detected an increa of homologous recombina-tion on treatment withflg22,but not with the inactiveflg22A.tum. peptide(Fi
g.1d).Thus,flagellin reprents an agent mimicking a pathogen in inducing both a pathogenic respon6and elevated levels of homologous recombination.
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Progeny of UV-treated and lf-pollinated plants grown under non-stress conditions were analyd for homologous recombination. In all six lines,the frequency of homologous recombination was greater than that of plants from untreated progenitors(Fig.2a).As the location of the transgene in the different lines varies,the transgeneration effect of incread frequencies of somatic homologous recombination is most likely independent of genome position.Homologous recombi-nation frequencies in the S1lfed generation of treated plants were 2–4-fold higher than tho of S1plants derived from untreated parents.The effect was also independent of ecotype and orientation of the recombination substrate(e Methods for details),as lines 1406,1445,IC1and IC9were in the Columbia ecotype,whereas lines 11and651were in C24.Similarly,the progeny offlg22-andflg22 A.tum.-treated plants,and of untreated plants,were analyd for persistence of elevated levels of homologous recombination;again, the plants‘memorized’their previous exposure or reaction to the biologically active peptideflg22and exhibited a constitutively elevated level of somatic recombination(Fig.2b).This respon was specific,as treatment with the inactive peptide did not lead to the described transgeneration effect.
The basis for the described transgeneration effect must be epi-genetic becau the whole population changes its behaviour,whereas a mutation would affect only very few plants.Epigenetic change can be described as mitotically and/or meiotically heritable,potentially reversible chromatin alteration,which occurs in the abnce of change in the DNA quence.Heritable changes of epigenetic traits may last for veral generations.We tested whether the homologous-recombination-enhancing epigenetic change acquired through the environmental stimulus of ultraviolet radiation can be detected in generations succeeding S1.Somatic homologous recombination was persistently found to be incread up to the lfed S4progeny of plants that were treated with UV-C only in S0(Fig.3).The epigenetic change leading to enhanced homologous recombination frequencies is thus stable for at least four generations.
So far,data on homologous recombination in successive genera-tions following treatment were generated by analysing lf-pollinated plants.It was of interest to test whether the epigenetic changes could be preferentially transmitted through the male or the female germ-line.To test this,plants were irradiated with UV-C and crosd to untreated plants.In offspring of treated and lf-pollinated plants of line1406,the frequency of homologous recombination was about one blue spot per plant as oppod to untreated plants,which gave ri to about0.4spots per plant in the next gene
ration(Fig.4a). However,recombination in the progeny of plants from a cross where only one parent was UV-treated was as high as in the treated,lf-pollinated plants,irrespective of the direction of the cross.Using line 1445,a similar picture emerged(Fig.4a).Comparable results were obtained in an experiment in which only one crossing partner was treated withflagellin;again,recombination was elevated to a similar level in the offspring of UV-treated outcrosd and lfed plants(Fig. 4b).We conclude that the epigenetic‘memory’could be inherited through both gametes in a dominant manner.
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The epigenetic change revealed may be inscribed on the entire genome,on a particular locus,or on the transgene of the
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treated Figure2|Somatic HRF in S0lines and their respective S1progenies.
a,Somatic HRF in six different untreated S0lines(white bars)and in
aerialsuntreated S1plants(grey bars)derived from tho same S0lines treated with
UV-C(S0þUV)or untreated(S02UV).Results are
(n.40plants;t-test*P,0.05compared with S1plants from S02UV).
b,Somatic HRF in S1plants(lines1406and1445)from S0plants that were
tracy mcgrady
untreated,treated with the inactiveflagellin analogue(flg22A.tum.),or
treated with the activeflagellin(flg22).Results are (n.40
plants;t-test*P,0.05compared with S1treated withflg22A.tum
.
).
Figure3|Somatic HRF in S0plants and in the next four generations.S0
plants(line IC1)were either untreated or UV-treated.Somatic HRF was
measured in untreated S1,S2,S3and S4plants.Results are
(n.50plants;t-test*P,0.05compared with the corresponding S02UV
generation).
NATURE|Vol442|31August2006LETTERS
plants.Although thefirst two alternatives are difficult to address,we tested whether the irradiated plants have to contain the transgene in order to transmit their epigenetic status to the descending genera-tion.We therefore crosd irradiated or untreated A.thaliana wild-type plants lacking the transgene locus with treated or untreated transgenic plants and analyd the somatic homologous recombina-tion frequency of the offspring.When both parental plants were expod to UV-C,recombination was elevated by a factor of about four in comparison with untreated parents(Fig.4c,
columns1and2, 5and6).When an irradiated female transgenic plant was crosd to a non-irradiated male wild-type plant,homologous recombination was incread to a similar extent(column3).Therefore,the irra-diated plant does not require the prence of the transgene in the crossing partner in order to be dominant(this also confirms and extends the data shown above,in which only one parent was irradiated,but both were transgenic;Fig.4a).Column4in Fig.4c shows the reciprocal experiment:homologous recombination was measured in the offspring of plants in which only the non-transgenic crossing partner was expod to irradiation,whereas the other partner carried the transgene.Comparison to columns1,2and3 allows the conclusion that the epigenetic change,measured as incread potential to undergo homologous recombination,can be transmitted through crossing with an irradiated plant lacking the recombination-substrate locus.Therefore,irradiation does not directly change the epigenetic status of the transgene locus,and activation(whatever this‘activation’may be)of the recombination potential of the transgene locus can be accomplished in trans.Again, the outcome of this experiment is independent of the direction of performed cross:columns5–8show levels of homologous recom-bination measured in offspring of cross in which the female parent was non-transgenic and the pollen-donor carried the transgene.
Influences of the environment on the plant genome have been documented,and include the activatio
n of transposition in maize7,8and Solanaceae9;genomic rearrangement following changes in the nutrition offlax5and climatic conditions in Tradescantia17;and alterations in the frequency of homologous recombination following exposure of plants to a variety of agents2–4,11,18.The influences have been interpreted as‘genomic shock’(refs1,19,20).Here we demonstrated that environmental influences,specifically ultraviolet radiation and a bacterial elicitor,change theflexibility of the plant genome in somatic tissue of treated plants and in somatic tissue of their progeny.As the influences persist in the entire population of plants,the basis for the change is epigenetic rather than genetic. Plants carrying the transgene locus do not have to face the environ-mental challenges themlves in order to transmit the epigenetic change to the offspring;the stimulus for an increa of recombina-tion can be impod in trans by a single treated parent.It is unclear, however,whether this stimulation is exerted by activation of a component of the homologous recombination machinery or by rendering the recombination locus more accessible,for instance by chromatin restructuring.Transcriptome analys of S0and S1plants originating from untreated or treated(UV-C orflg22)plants did not reveal significant changes in gene expression(data not shown).This indicates that a global transcriptional stimulation of key players of the homologous recombination machinery may not be responsible for the obrved phenomenon.However,it cannot be excluded that this functional genomic approach failed to reveal subtle transcrip-tional changes or i
nduction of microRNAs responsible for the obrved transgenerational‘memory’effect.A mechanism rem-bling paramutation in which one allele changes the epigenetic state of the other can be excluded;the enhancement of recombination can be exerted by a stresd crossing partner that lacks the transgene.The demonstrated epigenetic change is heritable for at least four genera-tions.In this respect,it rembles epimutation such as that in Linaria vulgaris affectingfloral symmetry21,or that in A.thaliana, called SUPERMAN,also affectingflower development22.Becau
of
Figure4|Somatic HRF in either lf-pollinated or outcrosd plants.
a,Somatic HRF in offspring of either lf-pollinated untreated plants,
UV-treated plants,or plants in which one of the parents was UV-treated (n.40plants;t-test*P,0.05).b,Somatic HRF in offspring of either lf-pollinated untreated plants(white bar),flg22-treated plants(grey bars), or plants in which one of the parents wasflg22-treated(hatched bars;n.35plants;t-test*P,0.05).c,Somatic HRF in offspring of plants in which one parent was wild type(WT)and the other harboured the recombination substrate(n.40plants;t-test*P,0.05).White bar,both parents untreated;dark hatching,both parents UV-treated;light hatching,one parent UV-treated.Refer to the main text for the lines ud.All the results are
LETTERS NATURE|Vol442|31August2006
the genetic stability of the epigenetic change in recombination potential,it is conceivable that meiotic recombination is affected as well.
We have demonstrated an induced,epigenetic,heritable change of a molecularly defined and quantit
atively measured trait.However,an adaptive value of the obrved changes cannot easily be evaluated at this point.We propo that the environmental influences that lead to incread genomic dynamics even in successive,untreated genera-tions may increa the potential for adaptive evolution23.This work should help in elucidating the underlying molecular mechanism that caus the obrved transgenerational‘memory’.
METHODS
Plant material.Homozygous A.thaliana lines,IC1and IC9(ref.14),1406and 1445(ref.24)(ecotype Columbia),11and651(ref.25)(ecotype C24),each carrying a single copy of a recombination substrate were ud for monitoring somatic homologous recombination frequency(HRF).The recombination substrate consists of a non-functional chimaeric uidA(GUS)gene containing partially overlapping homologous quence(Fig.1a).Lines IC1and IC9carry an intermolecular recombination substrate;lines1406,1445,11and651carry an intramolecular recombination substrate.Wild-type A.thaliana plants(ecotype Columbia)were also ud for cross.S1plants are defined as the progeny of either untreated or treated S0plants.S2,S3and S4plants correspond to the subquent generations.
Growth conditions.For soil-cultured plants,eds were sown(2per pot)and put at48C in the dark for3days.The pots were transferred to a phytotron(70% humidity)and kept under a cycle of16-h-light(208C)and8-h-dark(168C).For in vitro culture,plants were germinated on GM medium(MS salts(Duchefa), 1%sucro,0.8%agar,pH5.8).Plants were grown in a culture chamber under a16-h-light(208C)and8-h-dark(168C)photoperiod.Three-week-old plants were transferred to soil and grown for ed production(lf-pollination) or cross.
Treatment with UV-C andflagellin.For all experiments,plants were grown in vitro on solid GM medium(MS salts(Duchefa),1%sucro,0.8%Agar-agar ultrapur(Merck),pH5.8)in a culture chamber under a16-h-light(208C)and 8-h-dark(168C)photoperiod for13days before being subjected to the specific preculture condition established for each treatment.For UV-C treatment,plants were transferred to large Petri dishes(160-mm diameter)containing solid GM medium,to a density of1plant per cm2.For the UV-C irradiation(254nm, 6kerg cm22),a Mineral light-lamp(UV-Products)was ud.For theflagellin treatment with either the active peptide(flg22)or the inactive peptide(flg22A. tum.),plants were subcultured for24h in300m l of liquid GM medium in24-cell plates.Flg22orflg22A.tum.(1m M)diluted in liquid GM medium was applied. Four days after each treatment,plants were transferred to soil and grown for ed production(lf-pollination)or cross.
大学英语精读第四册答案Monitoring of somatic homologous recombination events.For monitoring the somatic homologous recombination events,the histochemical GUS assay26 was performed on3-week-old in vitro germinated plants.The HRF corresponds to the average number of blue spots(equivalent to the number of homologous recombination events)per ,in a population.For each recombina-tion line,experiments were at least duplicated.The t-test was ud for statistical analys with P¼0.05.Note that somatic HRF can only be compared within experimental ries due to the specific culture conditions ud for different treatments:solid culture medium and liquid culture medium for UV-C and flg22,respectively.
Received29March;accepted29June2006.
Published online6August2006.
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Acknowledgements We acknowledge the critical analysis of the manuscript by O.M.Scheid,D.Schuermann,R.Jorgenn,U.Grossniklaus,D.Schuebeler,
L.Comai and T.Boller.We are grateful to the Novartis Rearch Foundation and the European Union project PLANTREC forfinancial support.
Author Information Reprints and permissions information is available at /reprints.Th
e authors declare no competingfinancial interests. Correspondence and requests for materials should be addresd to B.H. (barbara.hohn@fmi.ch).
NATURE|Vol442|31August2006LETTERS
