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Journal of Plant Physiology 192 (2016) 56–63

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l h om epage: www.elsev ier .com/ locate / jp lph

hort communication

ermicompost humic acids modulate the accumulation andetabolism of ROS in rice plants

ndrés Calderín Garcíaa,∗, Leandro Azevedo Santosa, Luiz Gilberto Ambrósio de Souzaa,rlando Carlos Huertas Tavaresa, Everaldo Zontaa, Ernane Tarcisio Martins Gomesa,

osé Maria García-Minab, Ricardo Luis Louro Berbaraa

Federal Rural University of Rio de Janeiro (Universidade Federal Rural do Rio de Janeiro—UFRRJ), Department of Soil, Soil Biology Laboratory, Rodovia BR65 km 7, Seropédica, RJ 23890-000, BrazilDepartment of Environmental Biology, Agricultural Chemistry and Biology Group-CMI Roullier, Faculty of Sciences, University of Navarra, Spain

r t i c l e i n f o

rticle history:eceived 15 July 2015eceived in revised form 22 January 2016ccepted 22 January 2016vailable online 27 January 2016

eywords:eactive oxygen speciessmotic stressumic substances

a b s t r a c t

This work aims to determine the reactive oxygen species (ROS) accumulation, gene expression, anti-oxidant enzyme activity, and derived effects on membrane lipid peroxidation and certain stress markers(proline and malondialdehyde-MDA) in the roots of unstressed and PEG-stressed rice plants associatedwith vermicompost humic acid (VCHA) application. The results show that the application of VCHA tothe roots of unstressed rice plants caused a slight but significant increase in root ROS accumulationand the gene expression and activity of the major anti-oxidant enzymes (superoxide dismutase andperoxidase). This action did not have negative effects on root development, and an increase in both rootgrowth and root proliferation occurred. However, the root proline and MDA concentrations and the rootpermeability results indicate the development of a type of mild stress associated with VCHA application.When VCHA was applied to PEG-stressed plants, a clear alleviation of the inhibition in root development

linked to PEG-mediated osmotic stress was observed. This was associated with a reduction in root ROSproduction and anti-oxidant enzymatic activity caused by osmotic stress. This alleviation of stress causedby VCHA was also reflected as a reduction in the PEG-mediated concentration of MDA in the root as wellas root permeability. In summary, the beneficial action of VCHA on the root development of unstressedor PEG-stressed rice plants clearly involves the modulation of ROS accumulation in roots.

© 2016 Elsevier GmbH. All rights reserved.

. Introduction

The current understanding of the molecular and biochemicalechanisms involved in the beneficial effects of dissolved organic

atter—humic substances (DOM—HS) and vermicompost humic

cid (VCHA) on plant development is partial and fragmented (Calvot al., 2014). These mechanisms may involve the coordinated

Abbreviations: ROS, reactive oxygen species; DAB, 3,3′-diaminobenzidine; DAT,ays after transplanting; DOM, dissolved organic matter; DOM—HS, dissolvedrganic matter—humic substances; HA, humic acid; H2O2, hydrogen peroxide; NBT,-nitro tetrazolium; PEG, polyethylene glycol; P5CS, �-pyrroline-5-carboxylateynthase; VCHAs, vermicompost humic acids.∗ Corresponding author.

E-mail addresses: cg.andres@gmail.com, calderin@ufrrj.br (A.C. García),eoazevedo2001@yahoo.com.br (L.A. Santos), betdemolay@hotmail.comL.G.A. de Souza), ochtavares@gmail.com (O.C.H. Tavares), ezonta@ufrrj.brE. Zonta), ernane floresta@yahoo.com.br (E.T.M. Gomes), jgmina@timacagro.esJ.M. García-Mina), rberbara@hotmail.com (R.L.L. Berbara).

ttp://dx.doi.org/10.1016/j.jplph.2016.01.008176-1617/© 2016 Elsevier GmbH. All rights reserved.

actions of signalling pathways that are regulated by major plantregulators, such as auxin (Canellas et al., 2002; Mora et al., 2012;Muscolo et al., 2013; Trevisan et al., 2010), ethylene (Mora et al.,2012), nitric oxide (Mora et al., 2012; Zandonadi et al., 2010), andcytokinins (Mora et al., 2010), in addition to secondary messengers,such as reactive oxygen species (ROS) (Berbara and García, 2014;García et al., 2012a) and Ca2+ (Ramos et al., 2015; Trevisan et al.,2011).

Previous studies from our laboratory showed that the appli-cation of VCHA promoted both the production of ROS and theactivity of the primary enzymes involved in ROS metabolism in theroots of rice plants that were cultivated under normal conditionsor polyethylene glycol (PEG)-induced osmotic stress conditions(García et al., 2014). Furthermore, the effect of the VCHA washighly correlated with significant increases in the development

of the roots (García et al., 2012b; García et al., 2014). Moreover,several studies have described the important roles of ROS in thesignalling pathways (Miller et al., 2008, 2010; Mittler et al., 2004,2011) involved in the fine regulation of root growth and archi-

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ecture (Foreman et al., 2003; Samaj et al., 2004). Recent studiesighlighted the relevant roles of ROS in the control of cell prolif-ration and elongation in roots (Dunand et al., 2007; Tsukagoshit al., 2010). The signalling action of ROS was directly linked to theodulation of the accumulation and distribution of ROS in specific

oot regions; therefore, the concentration levels of ROS regulatedhe promotion or inhibition of root growth (Demidchik et al., 2007,009).

Many of the deleterious effects of osmotic stresses, caused byoth drought and salinity, are associated with an uncontrolled

ncrease in ROS accumulation in the roots and shoots. Therefore,he beneficial effect of VCHA on the development of plants sub-ected to osmotic stress may be linked to the effect of VCHA on the

odulation of ROS metabolism in roots.To explore this hypothesis, we investigated the effects of apply-

ng several concentrations of a well-characterized VCHA to theoots on the regulation of the concentrations of a diverse groupf ROS molecules in rice plants that were cultivated under normalonditions and PEG-induced osmotic stress. This investigation wasomplemented by studies on root permeability and the activity ofhe primary enzymes involved in ROS metabolism. Finally, the rootsnd certain root morphological features were evaluated to deter-ine the alleviation of the osmotic stress, which was mediated by

he application of VCHA.

. Materials and methods

.1. Plant material and growth conditions

The experiments performed to determine the effects of VCHAioactivity in rice plants (Oryza sativa L. cv. Nipponbare) wereonducted in growth chambers under the following conditions:ight cycle: 12:12 h (light:dark); photosynthetic photon flux:50 �m mol m−2 s−1; relative humidity: 70%; and temperature:8 ◦C/24 ◦C (day/night). The rice seeds were disinfected withodium hypochlorite (2%). Four days after the germination of theeeds, the seedlings received Hoagland’s solution (Hoagland andrnon, 1950), which was modified to ¼ of the total ionic strength.fter three days, the Hoagland solution was changed to ½ of the

otal ionic strength, and this was used throughout the remainder ofhe experiment. The experimental design was completely random-zed, using five plants per pot with ten replications. The statisticalnalyses were performed in Statgraphic Centurion XVI (Statpointechnologies, Inc., Warrenton, VA 20186, USA).

Two days after the acclimatization of the rice plants to the-ionic-strength nutrient solution, they were treated with eight

oncentrations of VCHA, i.e., 0, 5, 10, 15, 20, 40, 80 and 100 mg L−1 ofrganic carbon (the concentrations were prepared according to theuantity of carbon present in VCHA). The VCHA was dissolved in theutrient solution, and the pH was adjusted to 5.8. These solutionsere renewed after each assessment (Fig. S1).

These experiments were conducted to select the concentrationsf VCHA that stimulated the root systems. Based on the exper-mental results, three concentrations of VCHA were selected toontinue the study: 40 mg L−1 of organic carbon (the concentra-ion that caused the greatest stimulation of the roots), 20 mg L−1

f organic carbon (less than the highest concentration that causedtimulation) and 80 mg L−1 of organic carbon (the concentrationhat exceeded the maximum stimulation). The VCHA used in thistudy is characterized in García et al. (2014) (Figs. S2 and S3).

.2. Experiment on the induction of water stress in rice plants

The evaluations of plant behaviour in response to water stressere conducted using the experimental design described in the

hysiology 192 (2016) 56–63 57

previous section. We used four treatments in this experiment:control plants (without VCHA), plants treated with 40 mg L−1 oforganic carbon (VCHA40), plants with water stress induced by PEG-6000 (PEG), and plants with water stress induced by PEG-6000 andtreated with 40 mg L−1 of organic carbon (+VCHA + PEG).

Two days after the acclimatization of the plants to the nutrientsolution (½ total ionic strength), a subset of plants was transferredto pots, and the VCHA treatment (VCHA40) and the nutrient solu-tion containing PEG-6000 (15%) or the PEG-6000 (15%) and VCHAtreatment (VCHA40) were applied. The evaluations began two daysafter the treatments were imposed.

2.3. Determination of the root morphological parameters

Plants were removed six days after transplanting (6 DAT) andthe radicular parameters were quantified using a WinRhizo system(Regent Instruments Inc., Quebec, Canada). Then, the roots weredried in a drying oven at 105 ◦C until a constant weight was reached.

2.4. Quantification and histochemical determination of ROS

Hydrogen peroxide (H2O2) was visualized as a brown color thatwas caused by the polymerization of 3,3-diaminobenzidine (DAB)according to the methodology described in Ramel et al. (2009).The H2O2 content was quantified using the method described byKotchoni et al. (2006).

The superoxide anion (O2•−) was visualized using a blue solu-

tion of p-nitro tetrazolium (NBT) (3.5 mg mL−1) in phosphate buffer(10 mM) and NaN3 (10 mM) according to the method of Rao andDavis (1999), as described in Ramel et al. (2009). The O2

•− contentwas quantified using the method described in Ramel et al. (2009).

2.5. Enzymatic activity of peroxidase-POX (EC 1.11.1.7) andsuperoxide dismutase (SOD) (EC 1.15.1.1)

The enzyme extract was prepared according to Peixoto et al.(1999). POX (EC 1.11.1.7) activity was determined according tothe methodology described by Kar and Mishra (1976). The resultsare expressed in �mol min−1 mg protein−1 (Chance and Maehley,1955). SOD activity was determined according to the methodologydescribed by Beauchamp and Fridovich (1971), Giannopolitis andRies (1977) and Del Longo et al. (1993).

2.6. Protein extraction and native-PAGE profiling of SOD

An assay was conducted using three independent biologicalreplicates (40 seedlings each). Rice roots (0.3 g) were homog-enized in 900 �L of 100 mM potassium phosphate buffer (pH7.5) containing 1 mM of EDTA, 0.1% (v/v) Triton X-100, 2% (w/v)polyvinylpyrrolidone (PVP), 1 mM PMSF and 1X protease inhibitorcocktail (Sigma P9599) at 4 ◦C. The homogenate was centrifuged at13000 × g for 10 min at 4 ◦C. The supernatant was collected and cen-trifuged again as described above. The resultant protein extract wasstored at −80 ◦C. The protein content was measured as described byBradford (1976) using bovine serum albumin as a standard. Equalamounts of protein (20 �g) and glycerol (12.5%) were loaded onto10% TGX gel (BioRad). Electrophoresis was performed under nonde-naturing conditions (loading buffer and tris-glycine buffer withoutSDS) at 4 ◦C for 40 min at a constant current of 25 mA.

After electrophoresis, the gel was briefly washed three times inwater, and SOD gel activity was assayed according to Beauchampand Fridovich (1971) with modifications. The gel was incubated in a

solution containing 2.5 mM NBT for 15 min, followed by incubationin 50 mM potassium phosphate buffer (pH 7.8) containing 28 mMTEMED and 28 �M riboflavin for 15 min at 25 ◦C. SOD isoenzymeswere visualized after 10–20 min of light exposure as colorless

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ands on a purple-coloured background of insoluble formazan. Themages were captured with a ChemiDoc MP Imaging System (Bio-ad) using a white light plate that converts UV into white light.

The SOD isoenzymes were identified using 3 mM KCN or 5 mM2O2 in 50 mM potassium phosphate buffer (pH 7.8) before staining

or SOD activity. KCN is an inhibitor of only Cu/Zn-SOD and H2O2nhibits both Cu/Zn-SOD and Fe-SOD. The relative activity (rela-ive densitometry) of the main active band of the SOD isoenzymeCu/ZnSOD) was accessed using the Relative Quantity Tool of Imageab software (BioRad) with the control treatment as a reference.

.7. Activity of �-pyrroline-5-carboxylate synthase (P5CS) anduantification of the proline content

To determine the P5CS activity, the enzyme extract was pre-ared by homogenizing 0.5 g of vegetal tissue in a buffer mixtureonsisting of Tris–HCl (100 mM), MgCl2 (10 mM), EDTA (1 mM), �-ercaptoethanol (10 mM), dithiothreitol (4 mM), phenylmethyl-

ulfonyl fluoride (2 mM) and polyvinylpyrrolidone (2%). Theomogenate was centrifuged at 4 ◦C for 20 min at 20,000 × g, andhe extract was stored until evaluation for enzyme activity. Thectivity is expressed as U mg−1 (protein) (Chilson et al., 1992). Theroline content was measured according to methodology of Batest al. (1973).

.8. Lipid peroxidation and membrane permeability testelectrolyte leakage)

The MDA content was determined using the method describedy Dhindsa and Matowe (1981). The MDA content was calculatedccording to the formula MDA = 6.45 × (A532–A600) − 0.56 × A450. Aembrane permeability test (electrolyte leakage) was performed

ccording to Gui-Lian et al. (2009). The conductivity is expresseds �S cm−1 g−1 (root tissue).

.9. RNA extraction and real-time PCR

Samples were powdered in N2 and homogenized in a mixtureontaining 0.9 mL NTES buffer (0.2 M Tris–HCl, pH 8.0, 25 mM EDTA,.3 M NaCl, 2% SDS) and 0.7 mL of phenol: chloroform (1:1). Homog-nized samples were centrifuged at 12,000 × g for 20 min at 4 ◦C,nd each supernatant was transferred to a new tube. Total RNAas precipitated by adding 0.1 volume of 3 M sodium acetate (pH

.2) and 1 volume of cold isopropanol. The samples were subjectedo −80 ◦C for 1 h, after which they were centrifuged at 12,000 × gor 20 min. The pellets were suspended in 0.5 mL of H2ODEPC andrecipitated again by the addition of 1.0 mL of 4 M lithium chlo-ide (LiCl-DEPC). After centrifugation at 18,000 × g for 20 min, theellets were re-suspended in 0.5 mL of H2ODEPC, precipitated withwo volumes of ethanol for 1 h at −80 ◦C and washed with 70%thanol. The pellets were dried on ice for 10 min and dissolved in0 �L of H2ODEPC. The total RNA samples used for cDNA synthesisere treated with DNAse I (Invitrogen, Inc., Waltham, MA, USA)

y following the manufacturer’s instructions. Single-strand cDNAas synthesized using the High Capacity Reverse Transcription Kit

Applied Biosystems, Inc., Loughborough, UK).Real-time PCR reactions were performed in duplicate using the

ower SYBR® Green PCR Master Mix Kit (Applied Biosystems, Inc.)ccording to the manufacturer’s instructions. The specific primersere designed with Primer-BLAST (http://www.ncbi.nlm.nih.gov/

ools/primer-blast/), using the Refseq mRNA rice database. The ricectin and Elongation factor 1-� genes were used as endogenousontrols (Jain et al., 2006). Relative transcription was calculatedccording to Livak and Schmittgen (2001).

hysiology 192 (2016) 56–63

3. Results

The application of increasing concentrations of VCHA (20, 40and 80 mg L−1) to the roots of rice plants affected root proliferation(root number, length, and area) and dry mass; the magnitude ofthese effects depended on the VCHA concentration (Fig. S1). The40 mg L−1 VCHA treatment resulted in the greatest increase in theroot parameters compared with the control plants, whereas the20 and 80 mg L−1 VCHA treatments caused only a slight increaseand no increase, respectively (Fig. 1a). By contrast, the PEG treat-ment caused a clear decrease in both root growth and lateral rootproliferation compared with the control plants. The applicationof 40 mg L−1 VCHA to the PEG-treated plants partially alleviatedthe inhibition of root development caused by PEG; this was indi-cated by the significant increase in the number of roots producedcompared with the PEG-treated plants. However, the recovery ofthe PEG-treated plants in the presence of VCHA was not completebecause the root parameters remained lower than those of thecontrol plants (Fig. 1b).

The application of increasing concentrations of VCHA to rootscaused a limited but significant increase in the root productionof O2

•− and H2O2. However, this effect was not reflected in adose-response pattern. The increases in both O2

•− and H2O2 wereassociated with considerable increases in the activities of the SODand POX enzymes (Fig. 2a). The application of PEG caused a signifi-cant increase in the root accumulation of O2

•− and H2O2 comparedwith the control and VCHA-treated plants (Fig. 2b). However, theincreases in POX activities caused by the PEG treatment relativeto the control were significantly lower than those caused by theapplication of VCHA (Fig. 2b).

The activity of the SOD isoenzymes native gel indicated the samepattern but different intensities for each treatment (Fig. 2c). Thefirst band from the top was named Mn-SOD due to its resistanceto peroxide and low mobility. The second band was named Fe-SOD due to its intermediate mobility, sensitivity to peroxide andresistance to cyanide (Fig. 2c). The third and most dense band wasidentified as Cu/Zn-SOD due to its strong inhibition of both per-oxide and cyanide. The Cu/Zn-SOD band is described as the majorband in rice (Tanaka et al., 1999) and was not completely inhibitedby the peroxide and cyanide concentrations (Fig. 2c). The last twobands, which showed very low intensity, were named SOD4 andSOD5. These are probably secondary forms of Cu/Zn-SOD due to itshigher mobility in the gel (Fig. 2c). Using other varieties of rice, Xieet al. (2015) showed a more complex pattern of SOD isoenzymes inroots. Treatment with VCHA40 resulted in an increase in SOD activ-ity, confirming the regulatory effect of humic acid (HA) on oxidativemetabolism in plant roots (previously shown in Fig. 2a). It was alsoconfirmed that the VCHA40 + PEG treatment had an enzymatic pro-file similar to that of the control, confirming the restoration of SODactivity after the addition of HA to plants stressed with PEG.

These results are reflected in the intensity of the staining for bothO2

•− and H2O2 accumulation and distribution in the root tips. Forthe superoxide anions, an accumulation of these ROS was observedin the region of elongation of the roots (Fig. 3).

According to the membrane lipid peroxidation results, the appli-cation of different concentrations of VCHA caused an increasein membrane permeability that was associated with significantincreases in the MDA concentrations in the roots (Fig. 4a and b).The effects of the PEG treatment were also linked to increasesin both the MDA root concentration and root membrane perme-ability, which were more intense than those in the control andVCHA-treated plants (Fig. 4c and d). Consistent with the results of

the experiments described above, the 40 mg L−1 VCHA (VCHA40)treatment caused a limited but significant increase in the O2

•− andH2O2 root concentrations, in addition to the SOD and POX activi-ties, compared with the control plants (Fig. 2a). These effects were

A.C. García et al. / Journal of Plant Physiology 192 (2016) 56–63 59

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ig. 1. Root parameters of rice plants. (A) Experiments under normal conditions atean value ± ES (standard error) for ten replicates (n = 10). Different letters indica

ifferences.) in treatments between mean value, according to Tukey test (p < 0.05).

ssociated with concomitant increases in the root MDA concentra-ion and membrane permeability compared with the control plantsFig. 4a and b).

The application of VCHA40 to the PEG-treated plants decreasedhe root accumulation of O2

•− and H2O2− and was accompanied

y significant increases in the POX activities to levels similar tohose of the VCHA40-treated plants (Fig. 2b). These effects were alsoinked with concomitant decreases in the root MDA concentrationnd membrane permeability of the PEG-treated plants (Fig. 4c and). All of these effects were reflected in the staining for O2

•− and2O2 accumulation and distribution in the root tips. Similar to thebservations made in the HA application experiments, superoxidenions accumulated in the elongation region of the roots (Fig. 3and b).

Notably, VCHA application caused a slight dose-responsencrease in both the proline content and P5CS activity in the rootsFig. 5a). The plants treated with PEG had higher levels of proline

T. (B) Experiments under stress conditions after 2, 4 and 6 DAT. Bars represent thenificant difference (uppercase: 6 DAT, lowercase letters: 4 DAT, nd: no statistical

than the control plants, which is indicative of stress. The applica-tion of VCHA40 to the PEG-treated plants also tended to reduce theproline root concentration (Fig. 5b).

The cCu/Zn-SOD1 and cCu/Zn-SOD2 genes were induced by theHA while cCu/Zn-SOD2 was repressed by PEG (Fig. S5). Note thatamong the evaluated genes, cCu/Zn-SOD1 and cCu/Zn-SOD2 had thegreatest abundance of transcripts, which was revealed by lowervalues of �CT (Table S2). This result corroborates those observedin the isoenzymes gels, which showed the greatest activity inresponse to HA treatment and lower values in response to PEGtreatment (Fig. 2c). The pCu/ Zn-SOD gene did not change expres-sion after treatment with PEG and HA (Fig. S5). The MnSOD1.1 andFeSOD2 genes showed a slight increase in expression after treat-ment with HA (Fig. S5). The POX1 and P5CS1 genes did not change

expression after treatment with PEG and HA (Fig. S5), while theP5CR gene showed a slight increase in expression in response toHA.

60 A.C. García et al. / Journal of Plant Physiology 192 (2016) 56–63

Fig. 2. ROS content (O2•− and H2O2) and enzyme activity (SOD and POX) in the roots of rice plants 6 DAT under normal (A) and stress conditions (B). Bars represent the mean

value ± SE (standard error) for ten replicates (n = 5). Native gel SOD isoenzyme profile in the roots of rice seedlings of 6 DAT and relative densitometry of the main activeb pose a( lings

v

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and, Cu/ZnSOD (C). KCN and H2O2 inhibitors for SOD isoenzymes were used to proC). Bars represent averages ± SE of three independent biological replicates (40 seedalue, according to Tukey test (p < 0.05).

The SOD activity and real-time PCR suggest that the most impor-ant isoenzyme for the detoxification of O2

•− in rice roots is aytosolic Cu/Zn-SOD that increases expression and activity with theddition of HA (Figs. 2 c and S5). As shown in Table S2, the tran-cript abundance of the cytosolic forms of Cu/Zn-SOD showed highalues.

. Discussion

Recent studies conducted in our laboratory found that the rootpplication of VCHA facilitated the growth of rice plants undersmotic stress conditions (García et al., 2012a,b). Notably, the pro-ective action of VCHA in plants experiencing osmotic stress was

n identification for the active bands as described in materials and methods sectioneach). Different letters indicate significant difference in treatments between mean

associated with a significant ROS accumulation in the roots. Theimportant roles of ROS in the fine regulation of root cell prolifer-ation and elongation, and thus root growth and architecture, havebeen demonstrated in a number of studies (Foreman et al., 2003). Inthis framework, the alleviation of the deleterious effects of osmoticstress on rice development associated with the application of VCHAwas linked to the VCHA regulation of ROS metabolism in roots.

Our results support this hypothesis. The application of VCHAto roots results in a slight but significant increase in both the ROSproduction and the activities of the enzymes involved in ROS regu-

lation, such as SOD and POX (Fig. 2 and 3). This increase in the rootROS concentration was not associated with negative stress-relatedeffects on plant development but rather with the beneficial effect of

A.C. García et al. / Journal of Plant Physiology 192 (2016) 56–63 61

Fig. 3. Staining detection of ROS (O2•− and H2O2) in the roots under normal conditions (A) and under stress conditions (B) of rice plants at 6 DAT. Bar 1 mm. (C) control;

HA20: VCHA20; HA40: VCHA40; HA80: VCHA80.

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ig. 4. MDA content (A and C) and test of membrane stability (B and D) in the roxperiments under stress conditions. Different letters indicate significant differenalue ± SE (standard error) for ten replicates (n = 10).

ignificant increases in root growth (root number, length, area andry mass) (Fig. 1). However, some level of mild stress that resultedrom the application of VCHA to the roots could not be eliminated,s a slight but consistent dose-response increase in proline syn-hesis and concentration occurred in the roots treated with VCHAFig. 5). There was also a slight increase in the membrane lipideroxidation, which was reflected in the slight increases in both

he root MDA concentration and membrane permeability (Fig. 4).ome authors indicated that mild and transient stresses might trig-er protective mechanisms in plants, which might be beneficial forlant growth (Asli and Neumann, 2010). This pattern is in accor-

rice plants at 6 DAT. (A and B) Experiments under normal conditions. (C and D)tween mean values according to Tukey’s tests (p < 0.05). Bars represent the mean

dance with the regulation of the expression of genes involved inthe redox metabolism observed in the present study. Consequently,although our results did not allow us to confirm this benefit, thecombined analysis of all the quantified gene expressions, enzymeactivities and metabolites of the redox regulation pathways allowsan understanding of the role of ROS as mediators of the effects ofVCHA in plants. The results did suggest that the promotion of root

development by VCHA might be causally related to the increasein the ROS concentration in the roots. Moreover, it is possible thatthe positive effects of IAA and NO on the promoting effect of theHS on lateral root proliferation (Mora et al., 2012; Zandonadi et al.,

62 A.C. García et al. / Journal of Plant Physiology 192 (2016) 56–63

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010) might also involve ROS as signalling molecules, as has beenescribed for other metabolic and physiological events (Gill anduteja, 2010; He et al., 2012; Suzuki et al., 2012).

Notably, the application of VCHA to the roots of rice plantshat suffered from PEG-mediated osmotic stress alleviated some ofhe deleterious stress-related effects, such as those involving rootevelopment (Fig. 1). This alleviation was associated with signifi-ant decreases in ROS accumulation to levels similar to those of theCHA-treated and unstressed plants (Fig. 2). These VCHA effectsere also reflected in the reduction of root membrane lipid perox-

dation, as indicated by the reduction in the increases in the MDAoot concentration and membrane permeability caused by PEG-ediated osmotic stress (Fig. 4). The alleviation of stress caused

y the VCHA was also reflected in a reduction in the proline con-entration in the roots (Fig. 5). Therefore, these findings indicatehat the beneficial effect of VCHA on the PEG-stressed plants mayave resulted from a reduction in the ROS increase that was causedy PEG-induced stress. If the reduction in the ROS increase was theeason for this beneficial effect, then this effect must be mediatedy increases in the antioxidant enzymes SOD and POX followingCHA application. Our results are consistent with these mecha-isms because the PEG-treated plants that received HA showedignificant increases in both SOD and POX activities as well as thenduction of gene expression in comparison to the control and PEG-reated plants (Fig. 2). However, these results are also compatibleith a VCHA-mediated reduction of ROS in PEG-stressed plant rootserived from an alternative mechanism. Moreover, the recoveryf the root development parameters of the PEG-stressed plantsollowing the application of VCHA may also be linked to the ROSignalling pathways, as in the case of unstressed plants.

. Conclusions

Our results showed that VCHA application slightly increasedhe ROS production in roots, the induction of genes responsive tohe redox regulatory metabolism and the activity of the primaryntioxidant enzyme involved in the regulation and modulation ofOS metabolism. The effect that was mediated by the applicationf VCHA to the roots, which did not negatively affect root devel-pment, might result from a type of mild stress caused by thenteraction of the VCHA with the root cells. The results demonstratehat the alleviation of the deleterious effects of the PEG-inducedsmotic stress by the VCHA was accompanied by a significant

ecrease in ROS accumulation. This was likely linked to the increase

n root SOD-POX activities mediated by the application of HA. Col-ectively, these results strongly suggest that ROS play a major rolen the mechanism by which HA affects root development.

6 DAT. (A) Experiments under normal conditions. (B) Experiments under osmoticean values according to Tukey’s tests (p < 0.05). Bars represent the mean value ± SE

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

A.C.G. (sisFaperj: 2012028010) thanks Fundac ão de Amparo àPesquisa do Estado do Rio de Janeiro (FAPERJ) for his grant. A.C.G,R.L.L.B and J.M.G.M thank the National Counsel of Technological andScientific Development for the PDJ scholarship and funding throughthe project Science without Borders-PVE A060/2013. The authorsalso thank Coordenac ão de Aperfeic oamento de Pessoal de NívelSuperior project No. 46/2013, 215/13.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jplph.2016.01.008.

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