Hairy root exudates of allelopathic weed Chenopodium murale L.

ORIGINAL PAPERHairy root exudates of allelopathic weed Chenopodium murale L.induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlingsSlavica Dmitrovic´•Ana Simonovic ´•Nevena Mitic´•Jelena Savic ´•Aleksandar Cingel •Biljana Filipovic´•Slavica Ninkovic ´Received:13December 2013/Accepted:10July 2014/Published online:19July 2014ÓSpringer Science+Business Media Dordrecht 2014Abstract The effects of Chenopodium murale root exu-dates,applied as phytotoxic medias (PMs),were tested on Arabidopsis thaliana and Triticum aestivum .The effects of PMs,where wild-type roots (K),hairy roots derived from roots (R clones)or from cotyledons (C clones)were cultured,were different.K medium suppressed Arabidopsis germi-nation,while other PMs reduced root and leaf elongation and the number of rosette leaves.R media were more phytotoxic than C media.Treatment of Arabidopsis with R8down-regulated expression of core cell cycle genes:cyclin-dependent kinase (CDK)A1;1,four B-class CDKs,and cyclins CYCA3;1,CYCB2;4,CYCD4;2and CYCH1in root and shoot tips.Only CYCD2;1transcript was elevated in treated shoots,but down-regulated in roots.Wheat Ta -CDC2and Ta -CYCD2genes showed the same expression profiles as their Arabidopsis counterparts,CDKA1;1and CYCD2;1.PMs also caused increase of antioxidative enzyme activities in both plants.Exposure of Arabidopsis to PMs induced one catalase isoform,but repressed another,resulting in no net change of catalase activity.Wheat seedlings treated with PMs had catalase activity significantly elevated in all treat-ments,particularly in shoots.In both plants,PMs induced the activity of different peroxidase isozymes and total peroxi-dase activity.Both plants responded to phytotoxic treatments by induction of CuZn-superoxide dismutase.Thus,thephytotoxicity of C.murale root exudates is,at least partially,based on down-regulation of the cell cycle regulators and on generation of oxidative stress in the affected plants.We propose that C.murale root exudates should be considered as means of biological weed control.Keywords Allelopathy ÁAntioxidative enzymes ÁCyclin ÁCyclin-dependent kinase ÁHairy roots ÁOxidative stress Abbreviations 3-AT 3-Aminotriazole BMBasal mediumC9and C10C.murale hairy root clones derived from cotyledons and corresponding phytotoxic root growth media CAT CatalaseCDK Cyclin-dependent kinase CYC CyclinKPhytotoxic medium from wild-type C.murale root culture PM Phytotoxic medium POXPeroxidaseR1,R2,R3,R5and R8 C.murale hairy root clones derived from roots and corresponding phytotoxic root growth media ROS Reactive oxygen species SOD Superoxide dismutase b -MEb -MercaptoethanolIntroductionAllelopathy comprises chemical interactions among plants (or other sessile organisms,such are algae,fungi or corals),Electronic supplementary material The online version of this article (doi:10.1007/s10725-014-9959-z )contains supplementary material,which is available to authorized users.S.Dmitrovic´(&)ÁA.Simonovic ´ÁN.Mitic ´ÁJ.Savic ´ÁA.Cingel ÁB.Filipovic´ÁS.Ninkovic ´Department for Plant Physiology,Institute for BiologicalResearch ‘‘Sinisˇa Stankovic ´’’,University of Belgrade,Bul.Despota Stefana 142,11060Belgrade,Serbia e-mail:smile@ibiss.bg.ac.rsPlant Growth Regul (2015)75:365–382DOI 10.1007/s10725-014-9959-zwhere chemicals released by one plant affect the growth, survival and reproduction of plants in the vicinity.The secreted or volatile compounds,root exudates and chemi-cals released to the soil during tissue decay,which are involved in allelopathic interactions,are a subclass of secondary metabolites called allelochemicals.In natural plant communities,allelopathic interactions influence spe-cies distribution,abundance and survival,having a pro-found effect on evolution.From agronomical point, allelopathy has dual implications:detrimental,when weeds’allelochemicals affect the growth of crop plants (Qasem1993,1995;Nishida et al.2005;Batish et al. 2007a,b),or beneficial,as in the case of natural weed control(Fay and Duke1977;Soltys et al.2013).Plant responses to allelochemicals are diverse,but often resemble the responses to pathogens and herbicides,with secondary oxidative stress being a common signature (Golisz et al.2008).The oxidative stress caused by many allelochemicals(Ye et al.2006;Bogatek and Gniazdowska 2007;Golisz et al.2008)may be a consequence of direct involvement of the allelochemicals in the production of reactive oxygen species(ROS,Bais et al.2003),distur-bance of the cell membranes that induces lipid peroxida-tion,or disruption of the organelar electron-transport chains(Weir et al.2004).Namely,one of the best-char-acterized phytotoxic mechanisms is the inhibition of pho-tosynthesis(Yu et al.2003),often through interactions of allelochemicals with photosystem II(Weir et al.2004), while many allelochemicals target mitochondrial respira-tion and ATP synthesis(Weir et al.2004).As a conse-quence,an increased activity of the antioxidative enzymes upon treatment with allelochemicals has been reported in many cases(Yu et al.2003;Weir et al.2004;Ye et al. 2006;Ghareib et al.2010).In addition to causing oxidative stress,allelopathic interactions often involve disturbances in cell divisions in the target plants(Nishida et al.2005;Hallak et al.1999; Sa´nchez-Moreiras et al.2008;Zhang et al.2010;Soltys et al.2011).The allelochemicals may retard cell prolifer-ation through inhibition of DNA synthesis(Nishida et al. 2005),disturbances in cytoskeleton formation(Rudrappa et al.2007;Soltys et al.2011),or by down-regulation of the cell cycle-related genes(Zhang et al.2010).Unlike the cell cycle-related genes,expression of a number of genes increase in the affected tissues,as reported for Arabidopsis treated with(-)catechin from Centaurea maculosa(Bais et al.2003)or with buckwheat allelochemicals(Golisz et al.2008).Induced were genes involved in oxidative stress and the phenylpropanoid and terpenoid pathways (Bais et al.2003),as well as genes related to interaction with the environment,subcellular localization,proteins with binding function or cofactor requirement,cell rescue, defence and virulence,and metabolism(Golisz et al.2008).Although most plants produce allelochemicals,rela-tively few,like buckwheat(Golisz et al.2008),C.macul-osa(Bais et al.2003),black walnut and Chenopodium murale L.or nettle-leaved goosefoot(Chenopodiaceae), have strong allelopathic properties.C.murale is a medic-inal plant,widely used in folk medicine as a potherb, spinach,anthelmintic and laxative(Ibrahim et al.2007), while some isolatedflavonoids showed antihypertensive activity(Gohara and Elmazar1997).However,C.murale is also an allelopathic weed that thrives in a wide range of soil types infesting at least25crops world-wide(Holm et al.1997).Both its allelopathic and pharmaceutical potential C.murale owes to a number of secondary metabolites it produces,including alkaloids,essential oils, lipids,carbohydrates,saponins,coumarins(Ahmad et al. 2003and references therein;Ibrahim et al.2007),flavo-noids(Gohara and Elmazar1997;Ahmad et al.2003;I-brahim et al.2007;),flavonol glycosides(El-Sayed et al. 1999),sterols,steroidal glycosides(Ahmad et al.2003)and phenolic acids(Batish et al.2007a,b;Ghareib et al.2010; Mitic´et al.2012).Allelochemicals can be present in different plants parts i.e.in the leaves,bark,flowers,fruits or roots(Weir et al. 2004).C.murale and Amaranthus retroflexus dried shoot extracts,as well as residues incorporated in the soil, inhibited or delayed germination of cabbage,carrot,egg-plant,cauliflower,tomato and pepper and reduced shoot dry weights of all tested crops(Qasem1995).Allelopathy is often mediated by secondary metabolites released by the roots(Bais et al.2003).However,the study of plant–plant interactions mediated by roots is aggravated by the com-plexity of soil as a chemical and biological system that affects the availability of allelochemicals through their retention,transformation and transport(El-Khatib et al. 2004a),thus interfering with allelopathic interactions.The bioassays set to elucidate the phytotoxic properties of roots are commonly based on treating target plants with root extracts,but novel tools for sampling root exudates from soil-grown plants(Oburger et al.2013)are promising.C. murale root and shoot aqueous extracts reduced germina-tion and growth in wheat and barley(Qasem1993),as well as various growth parameters and biomass and metabolite production in several other tested species(El-Khatib et al. 2004b).Soils amended with C.murale decomposing tissue, as well as rhizosphere soil,also showed phytotoxic effects (El-Khatib et al.2004a;Batish et al.2007a,b),but in-depth understanding of these effects would require isolating and characterizing metabolites exuded from roots into the soil.The use of in vitro assay systems can overcome limi-tations in studying the root exudates-mediated allelopathy by eliminating soil as a complex factor.Batish et al. (2007a)have demonstrated that early growth of wheat reduced significantly in agar medium where C.muraleseedlings were previously growing.We have established C. murale wild-type root and hairy root in vitro liquid cul-tures,as a new tool for allelopathic assays,and showed that C.murale hairy roots,as an independent system,synthesize bioactive substances with inhibitory activity on wheat and lettuce germination and growth(Mitic´et al.2012).Hereby we present the study of allelopathic effects of C.murale hairy root exudates,applied in the form of root culture media on germination and seedling development of Arabidopsis thaliana.A.thaliana is not only a convenient research object that facilitates the study of genomic responses(Bais et al.2003),but also a very sensitive test species suitable for phytotoxic bioassays(Pennacchio et al. 2005).To elucidate some of the C.murale allelopathic mechanisms,the roots and shoots of in vitro grown wheat (Triticum aestivum L.)and Arabidopsis seedlings were treated with C.murale root and hairy root phytotoxic media(PMs)and the changes in the activities of antioxi-dative enzymes—catalases(CAT),peroxidases(POX)and superoxide dismutases(SOD),as well as the expression of the core cell cycle genes—several cyclin-dependent kina-ses(CDKs)and cyclins(CYCs)were investigated. Materials and methodsPlant materialSeeds of A.thaliana(L.)Heynh.ecotype Columbia(Col-8) were purchased from the European Arabidopsis Stock Centre and propagated in the greenhouse.The collected Arabidopsis seeds were surface sterilized in20%comer-cial bleach in ethanol(0.8%active chlorine)for1min and rinsed5times in sterile distilled water.Seeds of wheat(T. aestivum L.,cv.Natasˇa)were obtained from Agricultural Faculty,University of Novi Sad and were also sterilized,as described by Mitic´et al.(2012).C.murale root and hairy root cultures and their growth media(PMs)were obtained as described earlier(Mitic´et al.2012).Experimental designFor all experiments,the seeds or seedlings of the test plants,Arabidopsis and wheat,were grown on,or treated with either basal nutrition medium(BM),containing MS (Murashige and Skoog1962)salts and vitamins and30g/l sucrose or phytotoxic root growth media(PMs),where wild-type C.murale roots(used as control and labeled as K),or hairy roots derived from roots(transgenic clones R1, R2,R3,R5and R8)or from cotyledons(transgenic clones C9and C10)were propagated(Mitic´et al.2012).For evaluation of the C.murale media phytotoxicity on Arabidopsis germination and seedling development,the sterilized Arabidopsis seeds were placed on a double layer offilter paper wetted with4ml of either BM or different PMs.The seeds were stratified at4°C for3days and then transferred to growth chamber at25±2°C under16h light regime.The germination percentage,the longest root length,the longest leaf length,the number of leaves in a rosette and seedling fresh weight were measured after 3weeks.The percentage of inhibition was calculated as %=(1-T/C)9100,where T is parameter of treated variants and C is the parameter of the control variants (Mitic´et al.2012).The experiments were repeated three times,with50seeds in three replicates for each treatment.To study the effect of PMs on activities of antioxidative enzymes,total soluble proteins were extracted from whole Arabidopsis seedlings and from roots and shoots of wheat seedlings grown in vitro on different PMs for24and7days respectively.Seedlings grown on BM or K media were used as controls.The effect of C.murale root exudates on expression of cell cycle-related genes was studied only for R8medium, using BM as a control treatment.Sterilized Arabidopsis seeds were stratified at4°C in50ml BM in100ml Er-lenmayerflasks for3days,and germinated for7days at 25±2°C on rotary shaker at60rpm under16h light regime.Surface-sterilized wheat seeds were germinated for 7days in9-cm Petri dishes on a double layer offilter paper wetted with10ml BM at25±2°C under16h light regime.Both Arabidopsis and wheat seedlings were then transferred into Erlenmayerflasks(25perflask for Arabi-dopsis and15perflask for wheat)containing25ml of either R8or BM medium and incubated for15h on a rotary shaker at60rpm.Arabidopsis roots tips(&7mm) and shoots(hypocotyl,cotyledons and1–2pairs of leaves), as well as wheat root tips and shoot apexes without co-leoptiles(&5mm,both)were excised and transferred to Eppendorf tubes.The tissue samples(&100mg)were frozen in liquid nitrogen and kept at-70°C until RNA extraction.For each treatment,the RNA was isolated from three biological replicates.All the experiments were performed for three times.The mean values are presented infigures with SE.The data were subjected to one-way analysis of variance(ANOVA) with the STATGRAPHICS Centurion XVI.For analyzing the level of significance among the means,Fisher’s LSD test(germination and enzyme bioassays)and Tukey’s HSD test(expression of cell cycle genes)was performed.Protein extraction and electrophoresisTotal soluble proteins were isolated from&750mg of PM-treated Arabidopsis seedlings and wheat root and shoot tips by grinding the tissue in liquid nitrogen and extracting with 3ml of cold buffer containing50mM Tris pH8,1mMEDTA,30%glycerol,1.5%(w/v)polyvinylpolypyrroli-done,10mM dithiothreitol and1mM phenylmethylsul-fonylfluoride.The homogenates were cleared by centrifugation at12,000g for10min at4°C and the obtained crude extracts were used for enzyme activity assays.The proteins were quantified according to Bradford (1976)using BSA as standard.For the gel assays,soluble proteins were separated on 7%discontinuous nondenaturing polyacrylamide slab 16916cm gels with Tris–glycine system pH8.3(25mM Tris,192mM glycine)as running buffer,using Hoefer SE600unit(Amersham Biosciences,USA),at200V,at 4°C.For SOD and POX,the electrophoresis was run until the dye front reached the bottom of the gel(5–6h),while the CAT isoforms were separated for18h.The gels with developed enzyme activities were recorded by Gel-Doc TM EQ System(Life Science Research,Bio-Rad Co.).The band intensities were measured densitometrically using ImageQuant software(ver. 5.2,Molecular Dynamics, Sunnyvale,CA)and presented as a percent of the controls.Enzyme assaysThe CAT gel activity assay involved a preincubation of the gels in0.01%H2O2for25min,followed by a brief wash and incubation in a mixture of2%K3FeCN6and2% FeCl3until the appearance of white bands on a dark background,as described by Woodbury et al.(1971).We also tested the sensitivity of the visualized CAT activities to inhibitors b-mercaptoethanol(added directly into the crude protein extracts prior to native-PAGE,at40mM final concentration)and aminotriazole(5mM,included in gel staining solution),according to Prasad(1997).Total CAT activity was assayed spectrophotometrically by measuring the decrease in the H2O2absorbance at240nm (Aebi1984).The reaction mixture contained10l l of crude protein extract and0.2%H2O2in50mM Na–K-phosphate buffer pH7,in a total volume of1.5ml.The absorbance was recorded every20s over3min,at25°C,using Agi-lent8453spectrophotometer(Life Sciences,USA).The results were expressed as specific activity(U/mg),i.e.as mmol H2O2(e240=0.0436/mM cm)decomposed per min per mg of soluble proteins and presented as means of three measurements.To visualize the POX isozymes separated by native-PAGE,the gels were incubated with universal peroxidase substrates:pyrogallol(1,2,3-trihydroxybenzene,benzene-1,2,3-triol)for Arabidopsis and guaiacol for wheat.The staining solutions contained appropriate substrate and H2O2,10mM each,in100mM citrate buffer pH4(Rothe 2002),so the visualized isoforms are mostly acidic.Red-brown bands of tetrahydroguaiacol or purpurogallin appeared after&20min of staining.The reaction mixture for spectrophotometrical determination of total POX activity contained10l l of crude protein extract,20mM pyrogallol,10mM H2O2and50mM K-phosphate buffer pH6.5in a1.5ml volume.Peroxidases catalyze oxidation and polymerization of pyrogallol to red purpurogallin (e430=2.47/mM cm),so the activity was calculated from the kinetics of absorbance increase at430nm and pre-sented as mmol/min of produced purpurogallin per mg of total soluble proteins.The spec assays were performed in triplicates.The SOD isozymes were visualized by nitroblue tetra-zolium—riboflavin reaction according to Beauchamp and Fridovich(1971).The gels were soaked in50ml of reac-tion buffer(0.1M K-phosphate pH7.8)containing1mM EDTA,0.3mM NBT,0.08mM riboflavin and1.33mM TEMED for30min,in darkness.The gels were then rinsed and illuminated with36Wfluorescent lamp for15min to initiate the photochemical reaction.To identify the SOD isoforms,the gels were,prior to staining,incubated for 30min either in the reaction buffer or in the buffer con-taining1mM KCN,which specifically inhibits CuZn-SOD or5mM H2O2,which inhibits both CuZn-SOD and Fe-SOD,while the Mn-SOD is insensitive to these inhibitors (Michalski1996).Additionally,total SOD activity was determined by measuring the capacity of the enzyme extract to inhibit the photochemical reduction of nitroblue tetrazolium to blue formazan(Beyer and Fridovich1987). One ml of the reaction mixture contained100mM K-phosphate buffer pH7.8,0.1mM EDTA,12mM L-methionine,75l M NBT,2l M riboflavin and0–25l l of crude protein extract.Reaction mixture was exposed to illumination of a36Wfluorescent lamp for15min at 25°C.Absorbance was recorded at540nm using an ELISA microplate reader(LKB Vertriebs GmbH,Austria). Maximal color was obtained in irradiated reaction mixture without enzyme.One SOD unit was defined as the amount of enzyme which reduced the absorbance reading of sam-ples to50%in comparison with tubes lacking enzymes and expressed as units of enzyme activity(U)per mg of total soluble proteins.RNA isolation,RT-PCR and qRT-PCRTotal RNA was isolated from root and shoot tips of Arabidopsis and wheat seedlings treated with BM or R8 medium,using Spectrum Plant Total RNA Kit(Sigma-AldrichÒ).The genomic DNA contamination was removed by DNAse I(Fermentas).Reverse transcription was per-formed using Revert Aid First Strand cDNA Synthesis Kit (Fermentas),with oligo(dT)primers.The PCR mixtures contained cDNA corresponding to100ng total RNA, 1l M primers(primer sequences are listed in Supplemen-tary material1),2U of Fermentas Taq(EP0401)andstandard components according to Fermentas protocol in a 25l l volume.Primers specificity was confirmed by BLAST,electrophoretic sizing of the RT-PCR products, and by melting curve analysis.The amplification was carried out in a PEQ STAR thermal cycler,following amplification profile:initial denaturation(95°C/10min), followed by40cycles of denaturation(95°C/15s), annealing(54°C/30s)and extension(72°C/30s),with final extension(72°C/10min).The obtained amplicons were purified electrophoretically,extracted from gel using Gene JET TM Gel Extraction Kit(Fermentas),quantified, and serially diluted in a109–102copies/l l range to be used as standards for absolute qPCR quantification.qPCR reactions were set using12.5l l Maxima SYBR Green/ROX Master Mix(Fermentas),1l l templates (cDNA corresponding to100ng RNA,or standards),and 0.3l M primers in a25l l volume.The qPCR program was the same as for PCR,followed by melting curve analysis. The amplification was performed on Sequence Detection System ABI PRISM7000(Applied Biosystems Co.,Foster City,USA).The results were analyzed and presented using 7000System SDS Software.Constitutive expression of the Arabidopsis actin gene(ACTIN7,NM_121018)and wheat actin(ACT,AB181991)was confirmed in parallel.All reactions were carried out as biological triplicates. Results and discussionC.murale root and hairy root growth media reduce germination and retard development of Arabidopsis seedlingsGermination of Arabidopsis seeds,scored after24days, was completely inhibited by PM derived from wild-type C. murale roots(K),only mildly(up to21%)affected by treatment with R media where root-derived hairy roots were growing,and insensitive to cotyledon-derived hairy root C9and C10media(Table1;Fig.1).None of the BM components affected Arabidopsis germination.In the same experimental setup,the germination of wheat seeds was inhibited only12%by K medium,whereas R media were more inhibitory(Mitic´et al.2012).For example,R5 caused a55%inhibition of wheat germination,but did not affect Arabidopsis seeds at all.It can be argued that Arabidopsis and wheat seeds are sensitive to different C. murale allelochemicals,where Arabidopsis is particularly sensitive to some metabolite(s)produced by wild-type roots only,while wheat is susceptible to allelochemi-cal(s)more abundant in the hairy root cultures.It has been shown that C.murale allelochemicals are potent germina-tion inhibitors,affecting germination of15wheat cultivars, four barley cultivars(Qasem1993),cabbage,carrot,eggplant,pepper(Qasem1995),Melilotus indicus,Trifo-lium alexandrinum,Triticum pyramidal,cucumber and tomato(El-Khatib et al.2004a,b).However,Ghareib et al. (2010)showed that low concentrations of the C.murale acetone fraction,containing vanillic and p-hydroxybenzoic acids,stimulated the germination of tomato.We have also shown that concentrations of phenolic(caffeic,ferulic and p-coumaric)acids in C.murale PMs were too low to be responsible for phytotoxic effects(Mitic´et al.2012).Since the K media fully inhibited Arabidopsis germi-nation,the seedlings’growth parameters were obtained for R and C media only(Table1).The development of Arabidopsis seedlings was significantly inhibited by all PMs,with root elongation being the most sensitive process, inhibited on average by67%(from47%for C10-treat-ment to87%for R1),the leaf elongation and fresh weight gain were inhibited from29–72to34–77%,respectively, while the number of leaves in a rosette was least affected (35%on average,Table1).In experiments with barley and wheat,Qasem(1993)also demonstrated that root growth was inhibited more than shoot growth by C.murale extracts and leachates.The greater sensitivity of roots in comparison to shoots was recorded for some other allelo-pathic interactions as well(Nishida et al.2005).The tested PMs statistically differed in their phytotoxicity and could be arranged from the most to the least potent as: R1[R8[R3[R2=R5[C9[C10,implying stron-ger inhibitory effect of root-derived hairy roots exudats. Unlike the bioassays with Arabidopsis,where C9and C10 were ineffective in germination test and less effective in retarding growth in comparison to R-type media(Table1), the cotyledon-derived hairy root media C9and C11,used in bioassays with wheat,were more phytotoxic for seedling development than any of the tested R-media(Mitic´et al. 2012).This again underlines the difference between the two test species in their sensitivity to specific allelochem-icals present in the PMs.The difference between C-and R-media also implies that the explant type used for hairy root induction influences the metabolic activity and the production of allelopathic chemicals in the transformed cultures.With few exceptions,the test species that were shown to be sensitive to C.murale allelochemicals in germination assays,were also sensitive to the same chemicals at the early developmental stages(Qasem1993, 1995;El-Khatib et al.2004a,b).However,decayed resi-dues of C.murale delayed germination of cauliflower, marrow and tomato but increased their shoot dry weights (Qasem1995),while low concentrations of C.murale phenolic acids stimulated the germination and growth of tomato(Ghareib et al.2010).The most potent PMs,R1an R8,also caused depig-mentation of the tested seedlings(Fig.1).The treatment of several test plants with C.murale root and shoot aqueousextracts (El-Khatib et al.2004b ),as well as with rhizo-sphere and amended soil of C.murale (El-Khatib et al.2004a )also decreased the pigment content in the affected seedlings.Indeed,it is well-established that allelochemi-cals often target the photosynthetic apparatus,particularly photosystem II (Yu et al.2003;Weir et al.2004).Exposure of Arabidopsis and wheat seedlings to R8medium down-regulates the core cell cycle genes in shoot and root tipsSince PMs inhibit germination and leaf and root elongation of Arabidopsis (‘‘C.murale root and hairy root growth media reduce germination and retard development ofArabidopsis seedlings ’’section)and wheat (Mitic´et al.2012),a set of real-time experiments were conducted to elucidate whether this growth retardation is related to the cell cycle regulation.The cell cycle in Arabidopsis is regulated by nearly 200genes,including over 70so-called core cell cycle genes (cyclins,cyclin-dependent kinases,and their direct regulators),augmented with proteins of the anaphase promoting complex,genes involved in DNA replication or repair and other regulators (Menges et al.2005;Van Leene et al.2010).Hereby we have tested the effect of a potent PM,R8,on the expression of ten Arabidopsis and two wheat core cell cycle genes in seed-lings shoot and root tips.Generally,our expression data are consistent with the finding that most core cell cycle regu-lators do not show strong tissue specificity (Menges et al.2005),since comparable expression in shoot and root tips was found for all CDK and CYC genes,with exception of CYCB2;4and CYCD4;2,whose transcripts were about twofold more abundant in roots than in shoot tips (Figs.2,3).Treatment of A.thaliana seedling with R8invariably caused down-regulation of the CDKs in comparison to BM-treated control seedlings,ranging from -35%for CDKA1;1to -77%for CDKB1;1,which in most cases was statistically significant (Fig.2).Among the tested CDKs,only CDKA1;1,the sole member of its class which is constantly expressed to regulate the entrance to the cell cycle,as well as both the G1-to-S and G2-to-M transitions (Vandepoele et al.2002;Van Leene et al.2010),showed higher expression in shoots than in roots and stronger inhibition by R8in roots in comparison to shoots.Its wheat counterpart,Ta -CDC2,explained the same expression profile,with even stronger inhibition by phytotoxic med-ium,of -52%in shoots and -80%in roots.On the contrary,all of the B-class CDKs,that control the G2-to-M checkpoint and exhibit strong cell cycle regulation of expression (Menges et al.2005),had higher expression in roots,while their expression was more inhibited by R8medium in shoots than in roots (Fig.2).CDKs show class-T a b l e 1T h e e f f e c t o f 4-w e e k -o l d P M s o f C .m u r a l e w i l d -t y p e r o o t s (K )a n d h a i r y r o o t c l o n e s (R 1–R 3,R 5,R 8,C 9,a n d C 11)o n g e r m i n a t i o n a n d s e e d l i n g d e v e l o p m e n t o f A r a b i d o p s i sC l o n e sF i n a l g e r m i n a t i o n (%)I n h i b i t i o n (%)T h e l o n g e s t l e a f l e n g t h (m m )I n h i b i t i o n (%)N u m b e r o f l e a v e s i n a r o s e t t eI n h i b i t i o n (%)R o o t l e n g t h (c m )I n h i b i t i o n (%)S e e d l i n g f r e s h w e i g h t (m g )I n h i b i t i o n (%)B M99±0.0d10.2±0.1g10.2±0.1f7.9±0.1f23.7±0.1hK0±0.0a100––––––––R 178±0.3b 212.9±0.1a 725.4±0.1a 471.0±0.0a 875.4±0.1a 77R 290±0.2c95.1±0.1d507.3±0.1d e282.6±0.1c6710.8±0.2d54R 388±0.1c113.6±0.1c656.4±0.1b372.6±0.0c678.1±0.1c66R 5100±0.0d–5.2±0.0d497.2±0.1c d292.4±0.0c7011.7±0.2e51R 881±0.3b 183.1±0.1b 705.4±0.1a 472.1±0.0b 737.0±0.1b 70C 9100±0.0d –6.7±0.1e 347.1±0.1c 303.2±0.1d 5913.1±0.1f 45C 10100±0.0d–7.2±0.1f 297.4±0.0e 274.2±0.1e 4715.6±0.2d 34B M w a s u s e d a s a c o n t r o l t r e a t m e n t .T h e g e r m i n a t i o n p e r c e n t a g e a n d i n d i c a t e d g r o w t h p a r a m e t e r s w e r e r e c o r d e d 3w e e k s a f t e r t h e t r e a t m e n t .P e r c e n t a g e o f i n h i b i t i o n w a s c a l c u l a t e d a s d e s c r i b e d i n m a t e r i a l s a n d m e t h o d s .T h e r e s u l t s r e p r e s e n t t h e m e a n s ±S E f r o m t h r e e r e p l i c a t e s w i t h 70s e e d s p e r t r e a t m e n t .T h e v a l u e s w i t h t h e s a m e l e t t e r b e l o n g t o s t a t i s t i c a l l y h o m o g e n o u s g r o u p s (P B 0.05),a c c o r d i n g t o F i s h e r ’s L S D t e s t。