Current Biology18,656–660,May6,2008ª2008Elvier Ltd All rights rerved DOI10.1016/j.cub.2008.04.034
Report Plant DELLAs Restrain Growth and Promote
Survival of Adversity by Reducing
the Levels of Reactive Oxygen Species
Patrick Achard,1,*Jean-Pierre Renou,2Richard Berthome´,2 Nicholas P.Harberd,3and Pascal Genschik1,*
1Institut de Biologie Mole´culaire des Plantes
Conventionne´avec l’Universite´Louis Pasteur
67084Strasbourg
France
2Unite´de Recherche en Ge´nomique Ve´ge´tale
91057Evry cedex
France
3University of Oxford
Department of Plant Sciences
South Parks Road
Oxford OX13RB
United Kingdom
Summary
Plant growth is adaptively modulated in respon to envi-ronmental change.The phytohormone gibberellin(GA)pro-motes growth by stimulating destruction of the nuclear growth-repressing DELLA proteins[1–7],thus providing a mechanism for environmentally responsive growth regula-tion[8,9].Furthermore,DELLAs promote survival of adver environments[8].However,the relationship
between the survival and growth-regulatory mechanisms was previously unknown.Here,we show that both mechanisms are depen-dent upon control of the accumulation of reactive oxygen species(ROS).ROS are small molecules generated during development and in respon to stress that play diver roles as eukaryotic intracellular cond mesngers[10]. We show that Arabidopsis DELLAs cau ROS levels to remain low after either biotic or abiotic stress,thus delaying cell death and promoting tolerance.In esnce,stress-induced DELLA accumulation elevates the expression of genes encoding ROS-detoxification enzymes,thus reducing ROS levels.In accord with recent demonstrations that ROS control root cell expansion[11,12],we also show that DELLAs regulate root-hair growth via a ROS-dependent mechanism.We therefore propo that environmental vari-ability regulates DELLA activity[8]and that DELLAs in turn couple the downstream regulation of plant growth and stress tolerance through modulation of ROS levels. Results and Discussion
In adver environmental conditions,DELLAs accumulate and both restrain growth and promote plant survival[8].It was previously unclear whether the unknown mechanisms under-lying the two phenomena are distinct or coupled.We there-forefirst compared the relative contributions of individual DELLAs to the control of plant growth and to the survival of adversity.We measured growth(plant height),developmental rate(timing offloral transition),and stress tolerance(relative surviv
算工资al of salt-stresd plants)of Arabidopsis mutants lack-ing various single,double,triple,or quadruple combinations of DELLAs(GAI,RGA,RGL1,RGL2)in the gibberellin(GA)-deficient ga1-3background[7](Figure S1available online). Strikingly,we obrved a strong correlation between the rela-tive growth and developmental effects of DELLAs and the degree of salt-stress tolerance that they confer(r=20.96 and0.94,respectively),suggesting a common regulatory mechanism(Figure1A).
To reveal the molecular basis of this regulatory mechanism, we performed a transcriptome-profiling analysis with com-plete Arabidopsis microarray(CATMA)chips[13,14].Tran-script levels of the wild-type(WT),ga1-3,and ga1-3gai-t6 rga-t2rgl1-1rgl2-1(also called ga1-3quadruple-DELLA) were analyzed in respon to short exposure(30and60min) of plants to200mM NaCl(and controls without salt treatment). Statistical analysis of comparisons(e the Supplemental Data)revealed that2.2%of the24,576Arabidopsis genes reprented on the chip displayed DELLA-dependent changes in mRNA levels(either constitutively and/or salt induced;Table S1).In order to identify common t of genes who expres-sion was affected by various stress,we compared the expression profile of DELLA-regulated genes with publicly available microarray data(urgv.evry.inra.fr/CATdb)in which the effects of salt,mannitol,or the pathogen Erwinia amylovora were surveyed(Figure1B).Surprisingly,this analy-sis led to the identification of126genes(23.2%of all DELL
A-regulated genes)of which a wide range(more than one-third) of genes that respond to oxidative stress.Among tho,we found that the genes encoding the antioxidant systems such as the Cu/Zn superoxide dismutas(Cu/Zn-SOD),catalas, peroxidas,or glutathione S-transferas[15]were upregu-lated in plants that accumulate DELLAs(Table S2).In addition, most of the DELLA-regulated genes identified did not display constitutive changes but rather earlier induction or repression to salt,indicating that DELLAs nsitize plants to stress (Figure1B).Besides,salt or mannitol treatment enhances accumulation of DELLAs(via reduction in GA levels[8]) (Figure1C).Thus,ourfindings suggest that stress-induced DELLA accumulation activates a complex genetic regulation network to control in part the amount of ROS.
We next showed that DELLA-dependent regulation of‘‘anti-oxidant’’genes affects cellular ROS levels,by ester-loading 5-(and6)-carboxy-20,70-dichlorodihydrofluorescein diacetate (H2DCFDA),a ROS-nsitive dye with good intracellular reten-tion,into growing roots[16].In the abnce of salt stress,WT (Ler)and ga1-3quadruple-DELLA mutant roots exhibited higher basal levels of ROS than did ga1-3roots(Figure2A, Figure S2).Application of50mM NaCl incread ROS levels within WT roots and even more so within ga1-3quadruple-DELLA roots.In accordance,spy-3mutant roots(SPINDLY, a negative regulator of GA signaling[17])also exhibited higher levels of ROS than did
诺氟沙星胶囊WT(Col)roots in the prence of NaCl (Figure2B).In contrast,there was no detectable ROS accumu-lation within salt-treated ga1-3roots(Figure2A).Combined treatment of ga1-3with both GA and NaCl resulted in an increa in ROS levels,similar to that en with salt-treated WT roots(Figure S2A).Furthermore,a genetic analysis
*Correspondence:patrick.achard@ibmp-ulp.u-strasbg.fr(P.A.),pascal. genschik@ibmp-ulp.u-strasbg.fr(P.G.)
indicates that it is the combined effect of GAI and RGA that predominate in salt-activated ROS accumulation (Figure S2B).A comparable obrvation was obtained with diaminobenzidine (DAB,a hydrogen peroxide [H 2O 2]-nsitive dye)or nitroblue tetrazolium (NBT,a superoxide [O 22]-nsi-tive dye)in salt-treated leaves (Figures S3A and S3B).Thus,in line with the transcriptome data,DELLA activity reduces ROS levels of salt-treated plants.
The likely source of the ROS generated in the above exper-iments (Figure 2A)is an NADPH oxida [15].The NADPH-oxida products of the Arabidopsis AtrbohD and AtrbohF genes are known to produce ROS after pathogen attack or abscisic acid (ABA)treatment [18–20].After salt treatment,we found that AtrbohD ,but not AtrbohF ,is required for ROS production (Figure 2B;Figure S4A).Accordin
gly,application of diphenylene iodonium (DPI,inhibitor of flavoprotein activity such as NADPH oxidas [19])substantially prevented ROS accumulation in WT and ga1-3quadruple-DELLA mutant roots (Figure 2A).In addition,a loss-of-function AtrbohD mutation (atrbohD )abolished ROS accumulation of spy-3(in spy-3atrbohD ;Figure 2B).Interestingly,DELLAs do not modulate AtrbohD and AtrbohF transcript levels or NADPH-oxida activity in fractionated plasma membranes (Figures S4A and S4B).However,O 22generating activity in leaf discs was
found
Figure 1.DELLA-Dependent Controls of Plant Growth and Stress Tolerance Are Coupled (A)Pearson rank correlation coefficients (r )com-paring the relative roles of the DELLA proteins in plant growth (plant height at 6weeks;top panel)or developmental rate (floral transition;bottom panel)versus salt resistance (survival rate [8]).(B)Cluster of the 126DELLA-dependent differen-tially expresd genes in the ga1-3and ga1-3quadruple-DELLA mutant in the abnce and prence of salt treatment for 30and 60min as in-dicated that coregulate with tho of WT plants that have been treated with salt or mannitol or in-oculated with Erwinia amylovora .The color code depicts relative transcript levels on the CATMA microarray (nd,no difference).The complete list of the 126stress-induced DELLA-dependent co-regulated genes is provided in Table S2.
(C)Immunodetection of GFP-RGA (by an anti-body to GFP)in salt or mannitol-treated (and mock)14-day-old pRGA:GFP-RGA plants.PSTAIRE rves as loading
control.
Figure 2.DELLAs Restrain Salt-Induced ROS Accumulation (A)ROS accumulation (H 2DCFDA imaging)of WT (Ler),ga1-3,and ga1-3quadruple-DELLA mutant edling roots after salt treatment (and mock)alone or plus DPI as indicated.Numbers reprent quantification of H 2DCFDA staining from at least 20roots per genotype with imageJ in arbitrary units (mean 6SE).
(B)ROS accumulation (H 2DCFDA imaging)of WT (Col),spy-3,atrbohD ,atrbohF ,spy-3atrbohD ,and spy-3atrbohF ed-ling roots after salt treatment (and mock).
(C)Time cour of appearance for cell death H 2O 2-induced (2mM)in WT (Ler),ga1-3,and ga1-3quadruple-DELLA mu-tant edling roots,visualized by PI staining.Similar results were obtained in three independent experiments (with at least 20roots for each experiment).
DELLAs Restrain ROS Accumulation
657
to be more than 4-fold and 5-fold higher in WT and ga1-3qua-druple-DELLA mutant plants,respectively,compared with ga1-3plants (Figure S4C).In contrast,O 22production was found to be very similar upon potassium-cyanide treatment (KCN,inhibitor of Cu/Zn-SOD activity [21])or H 2O 2(inhibitor of both Cu/Zn-SOD and Fe-SOD activities [21]),suggesting that DELLAs control ROS accumulation via their effects on SOD activity.Arabidopsis Cu/Zn-SOD are encoded by three genes (CSD1,CSD2,and CSD3)[21]and,on the basis of our microarray data,CSD1and CSD2were downregulated in ga1-3quadruple-DELLA mutant (Table S2).Indeed,we con-firmed that the levels of both transcripts and proteins were respectively reduced in ga1-3quadruple-DELLA mutant and incread in ga1-3in comparison to WT plants (Figure S5).In conquence,SOD and catala activities were enhanced in ga1-3plants compared to WT plants (Figure S5).Thus,DEL-LAs restrain stress-induced ROS accumulation by acting on the ROS scavenging system.
ROS generation is correlated with plant cell death [15].We next investigated H 2O 2-induced cell death in WT,ga1-3,and ga1-3quadruple-DELLA mutant edling roots,by using propi-dium iodide (PI),a nucleic-acid stain that can only penetrate cells with damaged or leaking cell membranes [22].Whereas ga1-3quadruple-DELLA and WT roots began to exhibit cell death within 30min and 1hr of H 2O 2treatment,respectively,ga1-3roots did so only after 2hr (Figure 2C).It is noteworthy that H 2
O 2treatment had no effect on RGA stability and thus likely on DELLAs in general (Figure S6).Thus,DELLAs delay H 2O 2-induced cell death,thereby promoting stress tolerance [8].Necrotrophic pathogens often promote host cell death through generation of ROS [18,19].For example,the necrotro-phic fungal Botrytis cinerea caus vere dias in a wide range of plant species,promoting plant cell death through ROS generation [23].We therefore investigated the contribu-tion of DELLAs to pathogen-induced cell death.We found that 2days after inoculation with Botrytis ,ga1-3quadruple-DELLA mutant leaves displayed substantially higher cell-death rate than did Botrytis -inoculated WT control leaves (Figures 3A and 3D),the magnitude of the damages being proportional to the amount of H 2O 2accumulated (Figures 3B and 3C).In contrast,Botrytis -inoculated ga1-3leaves exhibited no dam-aged cell (Figures 3A and 3D)and no detectable H 2O 2accumu-lation (Figures 3B and 3C).Finally,as upon NaCl treatment,AtrbohD mutation abolished the accumulation in H 2O 2of spy-3in Botrytis -inoculated spy-3atrbohD leaves (Figures 3E and 3F).Thus,DELLAs promote survival of adversity by restraining ROS inducing cell death.上火原因
The results prented in Figure 1A suggest a common regu-latory mechanism for DELLA-dependent controls of plant growth and stress tolerance.ROS play diver roles in plant biology [11,15,19].In addition to their well-known involvement in stress tolerance,ROS function as cond mesngers in
ABA signaling in guard cells [20]and,like GA,control root cell growth [11,12,24].To determine whether DELLAs restrain growth and promotes survival of adversity via a common mechanism,we evaluated the contribution of ROS to GA-mediated root and root-hair growth.We found that the root growth of ga1-3edlings (which accumulate lower
basal
Figure 3.DELLAs Delay Botrytis -Induced H 2O 2Accumulation and Plant Cell Death
(A–D)Six-week-old WT (Ler),ga1-3,and ga1-3quadruple-DELLA mutant plants were inoculated with Botrytis cinerea spores at a concentration of 23105spores/ml.In (A)the picture was taken 2days postinoculation (dpi).Scale bars reprent 0.5cm.Numbers reprent diameter (mean in mm 6SE)for at least 30lesions per genotype.(B)and (C)show detection and quantification of DAB staining 1(B)and 2(C)dpi from at least 20leaves per genotype measured with imageJ in arbitrary units (mean 6SE).Scale bars reprent 0.5cm.(D)shows quantification of cell death from ten leaves per genotype at 0and 2dpi.Cell death is expresd as relative ion leakage (percentage of total ions 6SE).
(E and F)Six-week-old WT (Col),spy-3,atrbohD ,atrbohF ,spy-3atrbohD ,and spy-3atrbohF plants were inoculated with Botrytis cinerea spores at a con-centration of 23105spores/ml.Reprentative leaves (E)and DAB staining (F)2dpi with means (6SE)are as in (A)–(C).Scale bars reprent 0.5cm.
Current Biology Vol 18No 9658
levels of ROS than the WT,e Figure 2A)was more resistant to the inhibitory effect of DPI than that
of the WT (Figure 4A).In conquence,although ga1-3roots are shorter than tho of the WT [24],their length was almost identical upon 0.1m M DPI treatment (Figure 4A).In contrast,the root growth of ga1-3quadruple-DELLA mutant edlings was slightly but statistically more nsitive to DPI than that of the WT.For higher concentrations of DPI (more than 1m M),the cell elonga-tion characteristic of DELLA-deficient mutants was abolished,phenocopying the ga1-3mutant (Figure S7and Table S3).Moreover,we found that ga1-3edlings exhibited shorter root hairs and ga1-3quadruple-DELLA longer root hairs than tho of WT edlings (Figure 4B).Finally,the rhd2-1mutation (ROOT HAIR DEFECTIVE2[RHD2]gene encodes the NADPH oxida C [12])suppresd the root-hair growth of spy-3(in spy-3rhd2-1;Figure 4B).Thus,ROS contribute at least in part in the GA-mediated root cell growth.
Although it was previously clear that DELLAs are repressors of GA respons,the mechanism by which DELLAs mediate the effects was unclear.Whereas previous reports have suggested that GA might modulate ROS levels [25,26],we here show that two key DELLA-dependent GA respons
(stress survival and growth process)are regulated via a com-mon mechanism.DELLAs modulate ROS levels through the regulation of gene transcripts encoding for ROS detoxification enzymes.DELLAs thus repress ROS accumulation (and as a conquence ROS-induced cell death)
and hence enhance tolerance to both biotic and abiotic stress.DELLA-depen-dent modulation of ROS accumulation also contributes to the GA-mediated cell elongation that is an important driver of growth.The preci way in which GA-mediated regulation of ROS levels acts as a biological signal in plants remains unclear.Perhaps ROS act as cond mesngers and modu-late Ca 2+content,as in the stomatal guard cell or in the root hair [12,20,27],or perhaps they allow the cell wall to expand by decreasing the resistance of the wall to the pressure [11].Whatever the mechanism,it is clear that stress-related environ-mental regulation of GA signaling contributes to fine tuning of ROS levels,thereby regulating cell growth and stress tolerance.
Experimental Procedures
Plants and Growth Conditions
Arabidopsis thaliana lines ud in this study were derived either from the Landsberg erecta (Ler)(ga1-3,gai ,multiple-DELLA mutant lines)[8]or Columbia (Col)(spy-3,atrbohD ,atrbohF ,and rhd2-1)[12,17,20]ecotype.atrbohD spy-3,atrbohF spy-3,and rhd2-1spy-3double mutants were gen-erated by crossing of corresponding single-mutant line (polymera chain reaction [PCR]-bad screening and quencing were ud for confirma-tion).The plant height (measured at 6weeks)and bolting time wer
e deter-mined on a population of 30plants grown on soil in controlled environment chambers (16hr photoperiod;20 C).The salt-survival experiment per-formed on a population of at least 48plants for each of the 17genotypes was as previously described [8].The Rank correlation coefficients were calculated with the CORREL PEARSON function of Excel.For root-length determination,the lengths of 7-day-old roots of at least 30edlings (grown vertically on growth medium (GM)-agar plates [9])were measured 5days after transfer to DPI (concentration as indicated).The experiment was repeated twice.Root hairs were measured from 7-day-old edlings grown on half concentrated GM medium.Images of root were captured with a Nikon E800microscope.Root hairs that were in focus throughout their length were measured with Image J 1.33u (W.Rasband)software.For each line,at least 100root hairs were measured.
Transcriptome Studies
Microarray analysis was carried with the CATMA array [13,14],containing 24,576gene-specific tags from Arabidopsis thaliana .RNA samples from two independent biological replicates were ud for each comparison,with dye-swap technical replicates (i.e.,four hybridizations per compari-son).The labeling,hybridizations,and scanning were performed as previ-ously described [28].Normalization and statistical analysis were bad on two dye swaps [28].To determine differentially expresd gen
es,we performed a paired t test on the log ratios,assuming that the variance of the log ratios was the same for all genes.The raw p values were adjusted by the Bonferroni method,which controls the FWER [29].We considered as being differentially expresd the genes with a FWER at threshold of 5%.The Bonferroni method (with a type I error equal to 5%)keeps a strong control of the fal positives in a multiple-comparison context [29].Micro-array studies and statistical analys are described in more details in the Supplemental Data .
Data Deposition
Microarray data from this article were deposited at Gene Expression Omni-bus (bi.v/geo/,no.GSE8556)and at CATdb (urgv.evry.inra.fr/CATdb/;Project RS06-07_DELLA)according to the ‘‘Mini-mum Information about a Microarray Experiment’’standards.
Imaging
For experiments with H 2DCFDA,7-day-old edlings were treated with 10m M DPI (and controls)for 30min at 22 C and then incubated for 30min at 4 C in 10m M H 2DCFDA plus NaCl and/or DPI (and controls).Seedlings were then washed with 10mM MES,0.1mM KCl,and 0.1mM CaCl 2(pH 6.0)and left for 60min at 22 C before experimentation.Dye
excitation
Figure 4.DELLAs Modulate Root Cell Elongation via Their Effect in ROS Accumulation
(A)Mean (6SE)growth of primary roots of 7-day-old WT (Ler;blue),ga1-3(red),and ga1-3quadruple-DELLA mutant (yellow)edlings grown in the prence of DPI (concentration in m M as indicated).An asterisk indicates a significant difference between the WT and ga1-3quadruple-DELLA mutant (p <0.05).
(B)Reprentative 7-day-old WT (Ler),ga1-3and ga1-3quadruple-DELLA mutant roots (top panels)and WT (Col)spy-3,rhd2-1and spy-3rhd2-1mutant roots (bottom panels).Scale bars reprent 300m m.Numbers reprent length (mean 6SE)of mature root hairs.rhd2-1and rhd2-1spy-3roots develop very short root hairs and were thus not measured.
DELLAs Restrain ROS Accumulation 659
was at488nm;emitted light was detected at522nm.DAB staining was performed on fully expanded leaves treated with NaCl or inoculated with path-ogens as previously described[18].For the visualization of cell death,7-day-old edling roots treated with2mM H2O2were stained with10m g/ml
PI.For each imaging,at least20edlings or leaves(for each genotype) were analyzed.The experiments were repeated three times.Quantification of the staining(H2DCFDA and DAB staining)was performed with Image J 1.33u(W.Rasband)in arbitrary units(mean6standard error[SE]).The index of staining was calculated for each image as the average of the index of stained pixels measured in three points inside the stained area minus the average of three points outside the stained area.
Tests with Pathogens
Botrytis cinerea(23105spores per ml in potato dextro broth[Duchefa]) was inoculated by placement of5m l droplets onto6-week-old fully expanded leaves(8hr photoperiod;20 C)from at least30plants per geno-type.The experiment was repeated twice.
Ion Leakage
Cell death was quantified by ion leakage from rotte leaves into4ml of distilled water for3hr,measured with a conductivity meter(Horiba B173). Mean and SE were calculated from ten leaves per genotype.The experiment was repeated twice.
Supplemental Data
Additional Results,Additional Experimental Procedures,venfigures,and three tables are available at /cgi/content/ full/18/9/656/DC1/.
画荷花图片大全
Acknowledgments
We thank J.Peng for the multiple-DELLA mutant lines,Tp.Sun for the pRGA:GFP-RGA line and the antibody to RGA,L.Dolan for rhd2-1,N.E.Ols-zewski for spy-3,J.I.Schroeder for atrbohD and atrbohF,T.Heitz for the Botrytis cinerea strain,D.Kliebenstein for the antibody to CSD2,and L.Ta-connat for transcriptome.We gratefully acknowledge funding from the Cen-tre National de la Recherche Scientifique,the ANR grant JC07_189599,and the European Molecular Biology Organization Grant ALTF-414-2005.We thank L.Navarro,J.D.G.Jones,and L.J.Sweetlove for comments on the manuscript.
Received:January15,2008
Revid:March24,2008
Accepted:April9,2008
Published online:May1,2008
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