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Planta (2010) 231:1301–1309 1305EVect of NaHS on root B concentrationsFig. 2 EVect of H 2 S donor of NaHS on PME activity in cucumber rootunder B toxicity conditions. After pre-culture, cucumber <strong>seedlings</strong>were treated in 1/4 Hoagland’s nutrient solution containing 5 mM B,200 μM NaHS, 5 mM boric acid plus 200 μM NaHS for 48 h. The controlsolution (CK) was 1/4 Hoagland’s solution. Data are mean § SEof four replicates. Means with diVerent letters are signiWcantly diVerent(P < 0.05) with regard to treatmentsTo examine the eVect of NaHS on B accumulation in roots,we measured soluble B concentrations in cucumber rootsexposed to high B concentrations in the absence and presenceof H 2 S donor, NaHS. As shown in Fig. 4, soluble Bconcentrations in cucumber roots increased rapidly whenexposed to solutions containing 5 mM B. For instance, thesoluble B concentrations were increased from 0.34 to 6.4and 9.2 mM after 1 and 4 h of exposure to 5 mM B, respectively.The soluble B concentrations in roots became relativelyconstant, ranging from 7.5 to 9.8 mM, after exposureto high B solutions for up to 48 h. Addition of NaHS to thehigh concentration B solutions reduced root-soluble B contentsin roots after 1 and 4 h exposure to high B solutionsby approximately 20% (Fig. 4). There were no diVerencesin soluble B concentrations in roots exposed to high B solutionsin the absence and presence of NaHS after 8 h exposureto the high concentration B solutions.PME to the high B concentration at the transcriptional levelusing quantitative RT-PCR (q-PCR) technique were investigated.In Arabidopsis thaliana, PME is encoded by 67PME-related genes (Micheli 2001). In cucumber, fourgenes encoding PME were identiWed (CsPME1, CsPME2,CsPME3 and CsPME7) and their responses to high B concentrationin the absence and presence of NaHS were studied.Exposure of cucumber <strong>seedlings</strong> to high concentrationB solutions led to marked increases in transcripts for thefour PME genes, and the increases in PME transcripts displayedtransient characteristics such that the transcriptswere greater after 24 than 48 h exposure to high B solution.More importantly, we found that the high B concentrationinducedup-regulation of CsPMEs expression was signiWcantlyreduced by NaHS (Fig. 3). In contrast, NaHS had noeVect on CsPMEs expression for cucumber <strong>seedlings</strong>grown in the control solutions (Fig. 3). In addition toCsPME, the eVect of high B concentration and NaSH onthe expression of genes encoding expansins, which areimportant cell wall proteins responsible for cell elongation,was also investigated. Similar to CsPMEs, there were signiWcantincreases in CsExp1 and CsExp2 transcripts whenthe <strong>seedlings</strong> were challenged with high B concentration(Fig. 3). However, unlike the expression of CsPMEs, theenhanced expression of both CsExp1 and CsExp2 by high Bconcentration was greater after exposure to the high concentrationB solutions for 48 than 24 h (Fig. 3). Moreover,the up-regulated expression of CsExp1 and CsExp2 genesby high B concentration was substantially attenuated byNaHS (Fig. 3), whereas NaHS had no eVect on the transcriptsof CsExp1 and CsExp2 in the control solutions(Fig. 3).DiscussionInhibition of root elongation is one of the earliest and distinctsymptoms of plant B toxicity (Chantachume et al.1995; Reid et al. 2004). In the present study, we found thatexposure of cucumber <strong>seedlings</strong> to 5 mM B rapidly inhibitedroot elongation and led to root curvature (Fig. 1a). Wefurther demonstrated that the B toxicity-induced inhibitoryeVect on root elongation and changes in root morphologywere closely related to enhanced activity of PME due to upregulationof CsPMEs. More importantly, we found that anH 2 S donor (NaSH) can alleviate B toxicity syndromes suchthat NaSH abolished B-induced increase in PME activityand substantially alleviated the inhibitory eVect on rootelongation. Therefore, these observations indicate thatalteration of PME activity is likely to underlie B toxicityinducedinhibition of root elongation, and that H 2 S plays aregulatory role in alleviating the inhibitory eVect of B onroot elongation by targeting PME.A rapid inhibition of root elongation was observed onexposure of cucumber <strong>seedlings</strong> to 5 mM of B (Fig. 1).This Wnding is consistent with that reported in literature.For instance, Reid et al. (2004) demonstrated that root elongationwas inhibited by 70% after exposure of wheat <strong>seedlings</strong>to 10 mM B for 27 h. In addition to inhibition of rootelongation, we also found that cucumber roots exhibiteddistinct curvature when exposed to high B concentration(Fig. 1b). The high concentration B-induced root curvature,which is an interesting observation and warrants furtherinvestigation, may result from alterations in gravitropicproperties of roots by possibly disrupting distribution ofhormones such as auxin and ethylene in roots. Boron is an123


1306 Planta (2010) 231:1301–1309Fig. 3 EVect of boron toxicity on expression of PME and Exp ofcucumber root. After preculture, cucumber <strong>seedlings</strong> were treated in1/4 Hoagland’s nutrient solution containing 200 μM NaHS for 48 h.The control solution (CK) was 1/4 Hoagland’s solution. The relativemRNA level was normalized based on the mRNA in roots grown inCK solutions. Data are mean § SE of three replicatesimportant constituent of primary cell wall, and excess Bdisrupts the cell wall synthesis (Reid et al. 2004). Pectin isthe main component of plant cell wall. PME, which catalyzesthe demethylesteriWcation of cell wall polygalacturonans,is closely associated with cell wall developmentsand cell elongation (Pelloux et al. 2007). It has been shownthat up to 90% of the cellular B is localized in the cell wallfraction (Bevins and Lukaszewski 1998). Several reportsdemonstrate that cell wall development is disrupted by BdeWciency (Bevins and Lukaszewski 1998; O’Neill et al.2004). In contrast to B deWciency, little is known of theresponse of cell wall components to B toxicity. Ghanatiet al. (2002) reported that the contents of suberin and ligninin cultured tobacco cells are enhanced when challengedwith 10 mM B, leading to a stiVening of the cell wallmatrix. Pollen tube growth is inhibited by high external Bconcentrations (16 mM), and the inhibitory eVect mayresult from disruption of cross-link between B and pectinchains, leading to a rigidiWcation of the cell wall(Holdaway-Clarke et al. 2003). Similar explanations mayalso account for the inhibitory eVect of high B on root elongationin cucumber <strong>seedlings</strong>.In the present study, we demonstrated that B toxicityelicited an increase in PME activity and enhanced expressionof CsPMEs (Fig. 3). These results resemble the eVectof Al on root growth. For example, Yang et al. (2008)found that Al signiWcantly stimulates the PME activity ofrice roots, especially in Al-sensitive cultivar, and that thePME activity is negatively correlated with Al-induced inhibitionof root elongation. The enhanced PME activity123


Planta (2010) 231:1301–1309 1307B concentration in root (mM)14121086420ccabCK +S +B +B+Sc cabbb1 4 8 24 48Fig. 4 Root-soluble B concentrations in control (CK) and high B solutionsin the absence and presence of NaHS. Cucumber <strong>seedlings</strong> pregrownin the CK solutions were transferred to the treatment solutionscontaining 5 mM B for varying periods (1, 4, 8, 24, 48 h) in the absenceand presence of 200 μM of NaHS. Data are mean § SE of four replicates.Means with diVerent letters are signiWcantly diVerent (P < 0.05)with regard to treatmentswould cause a higher degree of demethylesteriWcation incell wall pectin under B toxicity, which in turn may stiVenthe cell wall by disrupting pectin gelation status, leading tothe observed root elongation.We also found that genes encoding expansins were upregulatedon exposure of cucumber <strong>seedlings</strong> to high concentrationB solutions (Fig. 3). Expansins are importantproteins that mediate primary wall loosening and activatethe secondary wall loosening factors, leading to turgordrivenwall extension (Cosgrove 2005). Boric acid andborate are capable of forming complexes with a number ofbiological compounds containing two hydroxyl groups incis-conWguration. The complex formed between borateesters and apiose residues of rhamnogalacturonan II (RG-II) plays an important role in controlling cell wall porosityand tensile strength (Ryden et al. 2003; O’Neill et al. 2004)and is involved in B deWciency-induced changes in cellwall structure (Matoh 1997; Ishii et al. 2001). In contrast toB deWciency, little is known of whether the RG-II complexis involved in the B toxicity syndrome. There has been areport demonstrating that genes encodings expansins aredown-regulated by B deprivation in Arabidopsis roots(Camacho-Cristóbal et al. 2008). In contrast to B deWciency,we found that there were increases in the transcriptsof CsExp1 and CsExp2 on exposure of cucumber <strong>seedlings</strong>to high B concentration. The up-regulation of CsExp1 andCsExp2 may alter expansin-dependent wall loosening, thuscontributing to the observed inhibition of root elongationinduced by high B concentration. The observations that Btoxicity up-regulated the expression of both CsPMEs andCsExps (Fig. 3) and inhibited root elongation (Fig. 1) maya ab bTreatment time (h)aabba asuggest that both PME and expansins are associated with Btoxicity in cucumber <strong>seedlings</strong>. The interactions betweenPME and expansins in modulation of root growth under Btoxicity remains unknown and warrants further investigation.Plants suVering from B toxicity often exhibit symptomsassociated with oxidative stress and membrane peroxidationas evidenced by enhanced accumulation of H 2 O 2 andMDA (Choi et al. 2007). In the present study, we found thatexposure of cucumber <strong>seedlings</strong> to 5 mM B signiWcantlyincreased H 2 O 2 contents in roots, but MDA contents werenot aVected by the treatment (cf. Table 1). The reportedeVect of excess B on H 2 O 2 and MDA contents in rootsvaries in literature, including increases in both H 2 O 2 andMDA contents (Gunes et al. 2006; Molassiotis et al. 2006;Cervilla et al. 2007), and no changes in H 2 O 2 (Karabal et al.2003) and MDA contents (Molassiotis et al. 2006; ArdÂcet al. 2009) in response to high B treatment. The discrepancyin changes in H 2 O 2 and MDA contents among diVerentstudies may result from diVerences in plant species,growth conditions, B concentrations used to treat plants andduration of treatment with high B concentration. The inhibitoryeVect of high B concentration on root elongation wasalleviated by NaSH, but there was no eVect of NaSH on thehigh B concentration-induced accumulation of H 2 O 2(Table 1). These Wndings suggest that accumulation ofH 2 O 2 may not be a cause for inhibition of root elongationinduced by high B concentrations.Previous studies have shown that H 2 S acts as a signalingmolecule in animal cells (Wang 2002; Lefer 2007). Giventhat plants can also produce H 2 S, it is informative to examinewhether H 2 S also plays a role in modulation of physiologicalprocesses in plants. In the present study, we foundthat treatment with H 2 S donor mitigated the B toxicityinducedinhibition of root elongation. More importantly, wefound that the high B concentration-induced increases inPME activity and transcripts of CsPMEs and CsEXPs werealso markedly reduced by the treatment with H 2 S donor(Fig. 3). These observations corroborate that changes inPMEs and expansins may underlie the B toxicity-inducedinhibition of root elongation, and that the ameliorativeeVect of exogenous H 2 S on inhibition of root elongationmay result from its action on PMEs and expanisns.Recently, it was demonstrated that H 2 S may act as an antioxidantto counteract oxidative stress induced by coppertoxicity (Zhang et al. 2008) and osmotic stress duringwheat seed germination and sweet potato seedling growth(Zhang et al. 2009). In contrast to these studies, we found Btoxicity-induced inhibition of root elongation was not correlatedwith the accumulation of H 2 O 2 and MDA contents.Thus, the H 2 S donor alleviated high B concentrationinducedinhibition of root elongation and curvature (Fig. 1),but did not aVect H 2 O 2 and MDA contents (Table 1).123


1308 Planta (2010) 231:1301–1309Therefore, the ameliorative role of H 2 S played in B toxicitysymptoms seems unlikely to result from its eVect on oxidativestress.There was a rapid accumulation of soluble B in roots onexposure of cucumber <strong>seedlings</strong> to high B solutions (Fig. 4).In barley, B concentrations in roots can be rapidly equilibratedwith the external B concentrations in the B-sensitivegenotype, while for the B-tolerant genotype root B concentrationsare lower than those in the external solutions due tooperation of a B eZux transporter (Hayes and Reid 2004;Reid 2007). In the present study, we found that B concentrationsin cucumber roots were greater than in the externalsolutions, suggesting lack of eVective B eZux transportersin cucumber plants used in the present study. As the alleviatingeVect of H 2 S on root elongation was observed after 24 hof exposure to high B solutions (Fig. 1), the observationsthat H 2 S donor only marginally inhibited B concentrations inroots in the Wrst 4 h of exposure to high B solutions (Fig. 4)indicate that the alleviating eVect of H 2 S on high B-inducedinhibition of root elongation is unlikely to directly resultfrom its impact on B accumulation in roots.In summary, we found that high external B concentrationssuppressed root elongation, stimulated PME activityand up-regulated expression of genes encoding PME andexpansins of cucumber <strong>seedlings</strong>. The inhibition of rootelongation, increases in PME activity and up-regulation ofPME and EXP expression were reversed by treatment withH 2 S donor. These Wndings highlight that H 2 S plays a regulatoryrole in mediation of root elongation under conditionsof B toxicity by possibly targeting cell wall-related PMEand expansins.Acknowledgments This work was supported by the National HighTechnology and Research Development Program of P. R. China (Grantno. 2007AA091705) and National Science Foundation of China(30788003). We thank the two anonymous reviewers for their constructivesuggestions.ReferencesAlexieva V, Sergiev I, Mapelli S, Karanov E (2001) The eVect ofdrought and ultraviolet radiation on growth and stress markers inpea and wheat. Plant Cell Environ 24:1337–1344ArdÂc M, Sekmen AH, Turkan I, Tokur S, Ozdemir F (2009) TheeVects of boron toxicity on root antioxidant systems of two chickpea(Cicer arietinum L.) cultivars. Plant Soil 314:99–108Bevins DG, Lukaszewski KM (1998) Boron in plant structure andfunction. Annu Rev Plant Biol 49:481–500Blamey FPC (2001) The role of the cell wall in aluminum toxicity. 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