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Citral Induces Auxin and Ethylene-Mediated Malformations and Arrests Cell Division in Arabidopsis thaliana Roots E. Graña & T. Sotelo & C. Díaz-Tielas & F. Araniti & U. Krasuska & R. Bogatek & M. J. Reigosa & A. M. Sánchez-Moreiras Received: 5 October 2012 / Revised: 18 January 2013 / Accepted: 24 January 2013 # Springer Science+Business Media New York 2013 Abstract Citral is a linear monoterpene which is present, as a volatile component, in the essential oil of several different aromatic plants. Previous studies have demonstrated the abil- ity of citral to alter the mitotic microtubules of plant cells, especially at low concentrations. The changes to the micro- tubules may be due to the compound acting directly on the treated root and coleoptile cells or to indirect action through certain phytohormones. This study, performed in Arabidopsis thaliana, analysed the short-term effects of citral on the auxin content and mitotic cells, and the long-term effects of these alterations on root development and ethylene levels. The results of this study show that citral alters auxin content and cell division and has a strong long-term disorganising effect on cell ultra-structure in A. thaliana seedlings. Its effects on cell division, the thickening of the cell wall, the reduction in intercellular communication, and the absence of root hairs confirm that citral is a strong phytotoxic compound, which has persistent effects on root development. Keywords Terpenoid . Phytotoxicity . Mitotic index . Pectin content . Long-term effects . Mode of action Introduction It is well known that natural plant-derived compounds usu- ally have more than one mode of action on plant metabo- lism, and it is precisely the combination of the different effects at different levels that makes these compoundsactions on plant metabolism so effective, and at the same time, makes it so difficult to establish with precision their different modes of action (Morrissey, 2009). In general, the low molecular weight terpenes, such as monoterpenes and sesquiterpenes, are volatile compounds that are part of essential oils and give plants their characteristic scents. The effects of exogenous monoterpenes in plant me- tabolism include both benefits and damages, depending of it concentration in the tissues. Among the harmful effects, they act like cytotoxic compounds, causing inhibition of respiration and photosynthesis (Pauly et al., 1981), decrease of cell mem- brane permeability (Muller et al., 1969), depolarization of membrane potential (Maffei et al., 2001), induction of lipid peroxidation (Zunino and Zygadlo, 2004), or interference with the action of phytohormones (Ishii-Iwamoto et al., 2012; Rentzsch et al., 2012). Among the beneficial effects, mono- terpenoids can induce, for example, thermotolerance (Delfine et al., 2000), plant resistance against pathogens (Godard et al., 2008), or at low concentrations, enhance dormancy release (Rentzsch et al., 2012). Citral (3,7-dimethyl-2,6-octadienal) is a linear monoter- pene with two geometric isomersgeranial (trans-citral or citral A) and neral (cis-citral or citral B) (Dudai et al., 1999; E. Graña : C. Díaz-Tielas : M. J. Reigosa : A. M. Sánchez-Moreiras (*) Department of Plant Biology and Soil Science, University of Vigo, Campus Lagoas-Marcosende s/n, 36310 Vigo, Spain e-mail: [email protected] T. Sotelo Misión Biológica de Galicia (CSIC), PO Box 28, 36080 Pontevedra, Spain F. Araniti Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale (BIOMAA), Università Mediterranea di Reggio Calabria, Salita Melissari, Reggio Calabria, RC, Italy U. Krasuska : R. Bogatek Department of Plant Physiology, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland J Chem Ecol DOI 10.1007/s10886-013-0250-y

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Page 1: Citral Induces Auxin and Ethylene-Mediated Malformations ... · Citral Induces Auxin and Ethylene-Mediated Malformations and Arrests Cell Division in Arabidopsis thaliana Roots

Citral Induces Auxin and Ethylene-Mediated Malformationsand Arrests Cell Division in Arabidopsis thaliana Roots

E. Graña & T. Sotelo & C. Díaz-Tielas & F. Araniti & U. Krasuska &

R. Bogatek & M. J. Reigosa & A. M. Sánchez-Moreiras

Received: 5 October 2012 /Revised: 18 January 2013 /Accepted: 24 January 2013# Springer Science+Business Media New York 2013

Abstract Citral is a linear monoterpene which is present, as avolatile component, in the essential oil of several differentaromatic plants. Previous studies have demonstrated the abil-ity of citral to alter the mitotic microtubules of plant cells,especially at low concentrations. The changes to the micro-tubules may be due to the compound acting directly on thetreated root and coleoptile cells or to indirect action throughcertain phytohormones. This study, performed in Arabidopsisthaliana, analysed the short-term effects of citral on the auxincontent and mitotic cells, and the long-term effects of thesealterations on root development and ethylene levels. Theresults of this study show that citral alters auxin content andcell division and has a strong long-term disorganising effecton cell ultra-structure in A. thaliana seedlings. Its effects oncell division, the thickening of the cell wall, the reduction inintercellular communication, and the absence of root hairs

confirm that citral is a strong phytotoxic compound, whichhas persistent effects on root development.

Keywords Terpenoid . Phytotoxicity . Mitotic index .

Pectin content . Long-term effects . Mode of action

Introduction

It is well known that natural plant-derived compounds usu-ally have more than one mode of action on plant metabo-lism, and it is precisely the combination of the differenteffects at different levels that makes these compounds’actions on plant metabolism so effective, and at the sametime, makes it so difficult to establish with precision theirdifferent modes of action (Morrissey, 2009).

In general, the low molecular weight terpenes, such asmonoterpenes and sesquiterpenes, are volatile compounds thatare part of essential oils and give plants their characteristicscents. The effects of exogenous monoterpenes in plant me-tabolism include both benefits and damages, depending of itconcentration in the tissues. Among the harmful effects, theyact like cytotoxic compounds, causing inhibition of respirationand photosynthesis (Pauly et al., 1981), decrease of cell mem-brane permeability (Muller et al., 1969), depolarization ofmembrane potential (Maffei et al., 2001), induction of lipidperoxidation (Zunino and Zygadlo, 2004), or interferencewiththe action of phytohormones (Ishii-Iwamoto et al., 2012;Rentzsch et al., 2012). Among the beneficial effects, mono-terpenoids can induce, for example, thermotolerance (Delfineet al., 2000), plant resistance against pathogens (Godard et al.,2008), or at low concentrations, enhance dormancy release(Rentzsch et al., 2012).

Citral (3,7-dimethyl-2,6-octadienal) is a linear monoter-pene with two geometric isomers—geranial (trans-citral orcitral A) and neral (cis-citral or citral B) (Dudai et al., 1999;

E. Graña :C. Díaz-Tielas :M. J. Reigosa :A. M. Sánchez-Moreiras (*)Department of Plant Biology and Soil Science, University of Vigo,Campus Lagoas-Marcosende s/n,36310 Vigo, Spaine-mail: [email protected]

T. SoteloMisión Biológica de Galicia (CSIC), PO Box 28, 36080Pontevedra, Spain

F. AranitiDipartimento di Biotecnologie per il MonitoraggioAgro-Alimentare ed Ambientale (BIOMAA),Università Mediterranea di Reggio Calabria, Salita Melissari,Reggio Calabria, RC, Italy

U. Krasuska :R. BogatekDepartment of Plant Physiology,Warsaw University of Life Sciences-SGGW,Nowoursynowska 159,02-776 Warsaw, Poland

J Chem EcolDOI 10.1007/s10886-013-0250-y

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Djiordevic et al., 2008; Chaimovitsh et al., 2010), which ispresent, as a volatile component, in the essential oil ofseveral different aromatic plants (Djiordevic et al., 2008),such as Melissa officinalis and Cymbopogon citratus. Citralis used in the cosmetic, food, medical, and agricultureindustries as a bactericide, nematicide, fungicide, insecti-cide, antihistamine, and anti-tumor agent (Rodov et al.,1995; Inderjit and Mukerji, 2006; Abramson et al., 2007;Echeverrigaray et al., 2010; Farah et al., 2010; Somolinoset al., 2010).

In 1999, Dudai et al. ran experiments using several differ-ent essential oils, in order to test their potential use as bio-herbicides, and found that the essential oil from Cymbopogoncitratus resulted in a dose-dependent inhibition of wheat ger-mination and of radicle and coleoptiles elongation. Theseeffects were attributed to the presence of citral. Similarly, anice experiment ran by Chaimovitsh et al. (2010), demonstrat-ed the ability of citral to alter the microtubules of cotyledonsof Arabidopsis thaliana, while the actinic fibres remainedintact. In a recent study, the same group (Chaimovitsh et al.,2012) also found that the mitotic microtubules were morestrongly affected by citral in the short term than corticalmicrotubules, especially at low concentrations. The changesto the microtubules may be due to the compound actingdirectly on the treated cells or to indirect action through certainphytohormones. Since the effect of citral on microtubules isclear, it would be interesting to know how auxins interact onthe organization of microtubules (or vice versa).

This study, done with the model species Arabidopsisthaliana, therefore analyzed also the effect of citral on theauxin/ethylene balance in both radicles and shoots, becauseboth hormones have numerous points of interaction, bothphysiological and molecular levels. Their synergistic inter-actions are especially important in the regulation of radicleelongation, root hairs formation (Stepanova et al., 2007),and in the control of root growth (Pierik et al., 2009). It isalso known that natural compounds can change the activityof enzymes involved in auxins' biosynthetic pathway and intheir specific transport proteins, as has been demonstratedby Prasad and Subhashini (1994) with the allelochemicalmimosine, which reduces the activity of the IAA-oxidaseenzyme in Oryza sativa, and by Rahman et al., 2001 withchromosaponin I, a γ-pyronyl-triterpenoid saponin isolatedfrom peas and other leguminous plants, which interacts withthe AUX1 protein, changing the response of the roots ofArabidopsis to auxin and ethylene by controlling the uptakeof auxins (Rahman et al., 2001).

The role of auxins taking part in the control of ethylenelevels is essential in plant metabolism (Abeles, 1966), whichhas been shown to regulate the arrangement and reorienta-tion of microtubules in stress situations (Roberts et al., 1985;Shibaoka, 1994). This latter behavior has been found incitral-treated seedlings, suggesting that the early mode of

action of the terpenoid citral could be mediated byhormones.

Additionally, the capacity of plant metabolism to detox-ify, compartmentalize, and transform natural compoundsmakes it especially important to monitor not only the pri-mary, short-term effect, but also the secondary effects ofnatural products in the short, medium and long term, thustesting the persistence and effectiveness of the compound’sphytotoxic action on the target plants.

The synchronization of cell division and the orientation ofthe new wall are crucial for plant growth and development.The cell wall has important functions in plants: it gives themrigidity, gives the cells their shapes, mediates interactionsbetween cells, and provides a barrier to attacking pathogensand other harmful agents (Scheller et al., 2007).

The aim of this work was to analyze, over different lengthsof time, citral’s long-term effects on the ultrastructure of theroot meristem cells, the composition of their cell walls, the rateof cellular division, and the auxin and ethylene content in bothroots and shoots.

Methods and Materials

Growth Conditions Arabidopsis thaliana (L.) Heynh. eco-type Columbia (Col-0) seeds were sterilized and kept in0.1 % agar at 4 °C for 72 hr for the synchronization ofgermination. Synchronized seeds then were sown in squarePetri dishes (100×15 mm) at a concentration of 24 seeds perdish, in a plant agar medium with a mix of macro- andmicro-nutrients (Murashige-Skoog, Sigma-Aldrich) and a1 % sucrose supplement. Arabidopsis seedlings wereallowed to grow for 15 days in a vertical position at aconstant temperature of 22 °C±2 °C, a photoperiod of 8 hrof light (120 μmolm−2sec−1) and 16 hr of darkness, and arelative humidity of 55 %.

Dose–response Curve: The Phytotoxic Effect of Citral on A.thaliana Citral’s phytotoxic potential, on both the germinationand radicle growth of A. thaliana, was evaluated by addingdifferent concentrations of citral (0, 50, 100, 200, 400, 800, and1,200 μM) to the warm plant agar to avoid it volatilization(pH6), using ethanol at 0.1 % as the solvent (5 replicates perconcentration). After 15 days growth in the same medium, theaverage number of germinated seeds was calculated, and rootlength was measured. Dose response curves were used tocalculate the IC50 and IC80 concentrations (the concentrationsof citral which cause, respectively, 50 and 80 % inhibition),and also the LCIC values (lowest complete inhibition concen-tration), both for germination and growth (Dayan et al., 2000).These concentrations established a frame of reference for thefollowing trials. Finally, a morphological analysis of the rootswas made by using a Nikon SMZ 1500 stereo microscope. The

J Chem Ecol

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thickness of the radicle, the presence or absence of root hairs,and root growth were evaluated.

Ultra-structural Analysis: Electron Microscope Arabidopsisseedlings were grown, both in the absence of citral and inthe presence of the IC50 concentration. After 7 and 14 daysof growth, the root apices were harvested and submerged inglutaraldehyde (5 %) for 4 hr, washed, submerged for 3 hr inOsO4 (2 %), all prepared in 0.1 M cacodylate buffer pH7.2,and then in 10 % acetone with 2 % uranyl acetate for 1 hr.The roots were dehydrated in increasing acetone concentra-tions and then infiltrated in 100 % Spurr’s resin at 4 °C.Sections were cut semithin (0.7 μm) for optical microscopy,and ultrathin (50–70 nm) for electron microscopy. Ultrathinsections were stained with uranyl (2 %) for 30 min, and withlead citrate (as per Reynolds, 1963) for 12 min. They weremounted on copper grids (100- and 200-mesh), and ob-served by JEM-1010 (100kv) high contrast transmissionelectron microscope equipped with an Orius CCD cam-era and Digital Montage Plug-in and ‘Gatan DigitalMicrograph’ software. Semithin sections were viewedwith a Nikon Eclipse 800 optical microscope and a DigitalSight Nikon DS-U2 camera and NIS-Elements D 2.30 SP1software.

Pectin and Callose Staining Both stains were performed onfresh roots harvested 14 days after treatment having grownin plant agar with the IC0 and IC50 concentrations of citral.To stain the callose, the meristems were incubated for5 min in the dark in a 0.05 % solution of Aniline Blue(Sigma-Aldrich), in PBS (pH8.5) (modified from Khoand Baër, 1968). To stain the pectins, the meristemswere incubated for 30 min in the dark in a 0.05 %solution of Ruthenium Red (Sigma-Aldrich), in distilledwater (pH7.4) (modified from Jensen, 1962). Semithinsections of 7 and 14 days specimens also were stainedwith the same procedures, except that the concentrationof Ruthenium Red used was 0.1 %. The specimens wereviewed with a Nikon Eclipse 800 optical microscopeand a Digital Sight Nikon DS-U2 camera and NIS-Elements D 2.30 SP1 software. Image ProPlus imageanalysis software was used for the colorimetric analysisof the images.

Mitotic Indices After 7 days of growth, A. thaliana seed-lings were treated with a 2 mM Hydroxyurea (HU) solution(Cools et al., 2010) at pH6.0 for 24 hr, for synchronizationof cell division. After 24 hr, the HU was removed byrepeated washing with distilled water. The roots were thentreated with 4 ml of each of the following citral treatmentsolutions: IC0, IC50 and IC80 (using ethanol at 0.1 % as thesolvent). Root meristems were harvested 0, 2, 4, and 6 hrpost-treatment (Cools et al., 2010), fixed in a (6:3:1) mixture

of acetic acid, chloroform, and ethanol with traces of iron,and stored for 24 hr at −20 °C. After 24 hr, the traces of ironwere removed, the fixing mixture was replaced with freshfixing mixture and the specimens were once again stored atthe same temperature, for 5 days. The next step was hydro-lysis with HCl 1 N for 20 min at 60 °C, which alloweddispersion of cells and chromosomes. After hydrolysis,roots were recovered and kept for 2 hr in the darkness inSchiff reagent, to stain the chromosomes. Finally, the reac-tion was stopped with acetic acid. To observe the cellsundergoing division, each meristem was sectioned, mountedon slides with a drop of acetocarmine, fixed for 3 sec over aflame, and squashed with a cover slip (procedure modifiedfrom Armbruster et al., 1991). A total of 1000 cells werecounted in between 10 and 20 meristems per treatment.Specimens were viewed and photographed using a NikonEclipse 800 optical microscope (100× objective) with aDigital Sight Nikon DS-U2 camera and NIS-Elements D2.30 SP1 software.

Auxin Content Arabidopsis plants were growth for 14 daysin plant agar in the same conditions described in ‘Dose–response curves’. After that, plants were treated by coveringfor 5 or 10 hr with 8 ml/plate with control and citral IC50

treatments (both with 0.01 % ethanol in distilled water).Then, treatment was removed, and plants were harvestedseparating roots and shoots.

Levels of indoleacetic acid (IAA) were analysed byELISA according to Marcussen et al. (1989) and Elsorra etal. (2004) with some modifications. Roots and shoots ofthree independent replicates were separately homogenizedwith 80 % MeOH and 20 mg/L of butylated hydroxytoluene(BHT). The homogenized samples were incubated at −70 °Cfor 24 hr. Then, samples were centrifuged at 9,500×g for15 min, and the supernatant was collected. The pellet waswashed with 80 % MeOH, and the supernatant was added tothe first lot. Standard solution of IAAwas prepared in 80 %MeOH. The samples and the IAA standards were methylat-ed with a 2.0 M solution of trimethylsilyldiazomethane inhexane for 30 min at room temperature. The reaction wasstopped with 0.05 N acetic acid, and the mixture was left forMeOH evaporation. The pellets were resuspended in PBSbuffer. Standard IAA was used as a positive methylatedcontrol.

Microtiter plates were incubated with Rabbit Anti-MouseIgG (RAMIG; Sigma-Aldrich), (25 μg/ml prepared in 0.05 MNaHCO3, pH9.6) overnight at 4 °C. After removing theRAMIG, plates were covered with monoclonal Anti-Auxin(Sigma-Aldrich) (1 μg/ml in 50 mM TBS buffer pH7.8),incubated overnight at 4 °C, and washedwith deionized water.Then, samples, IAA standards, and positive control wereincubated with TBS buffer at 4 °C for 1 hr. After that, plateswere covered with IAA tracer (Agrisera; 3 μl/5 ml in TBS

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buffer with 0.1 % gelatine) for 3 hr at 4 °C. Plates werewashed with deionized water and incubated at 37 °C for 1 hrwith pNPP solution (para-Nitrophenylphosphate disodium;Sigma-Aldrich), prepared as 1 mg/ml in DEA solution (1 Mdiethanolamine with 0.5 mM MgCl2). The reaction wasstopped with 5 M KOH, and absorbance was read at 405 nmusing a Dynatech Microplate Reader MR5000. Results arepresented as % of control.

Ethylene Content Plants were grown and treated in thesame way described for auxin measurement. After har-vesting roots and shoots separately, samples were sub-merged in 8 ml glass vials containing 0.5 ml of distilledwater or in 0.5 ml of 1 mM ACC (1-aminocyclopropane-1-carboxylic acid, SIGMA), in order to estimate the realand the potential amount of ethylene. At the beginning ofincubation, lids remained opened for 30 min in order toeliminate ethylene produced by harvesting stress; then,with closed lids, samples were incubated for 3 hr in dark.Then, 1 ml of air from vials was injected into a gaschromatograph Hewlett Packard 5890 Series II providedwith a FID detector and a stainless steel column (6 fit×1.8 in. × 2.1 mm). All measurements were run intriplicate.

Statistical Analyses Following the Kolmogorov-Smirnovtest for non-normality and Levene’s test for heterocedas-ticity, statistical significance of differences among groupmeans was estimated by analysis of variance followed bythe least significant difference test for homocedastic data,and by Tamhane’s T2 test for heterocedastic data.

Results

Germination and Growth Bioassays

The dose response curves confirmed the phytotoxicity ofcitral to the test species, A. thaliana (Fig. 1). Althoughgermination was only slightly affected (from less than10 % respect to the control at citral 200 μM, to 58.47 % atthe highest concentration, 1,200 μM); root development wasstrongly inhibited by citral, and significant inhibition wasfound for 50 μM, the lowest concentration tested. The IC50

and IC80 concentrations were established at 194 and 311 μMof citral, respectively. The LCIC was reached at a concen-tration of 1,200 μM (Fig. 1).

Root Morphology in A. thaliana

Magnifier and optical microscopy images of control rootsshowed straight root apices with uniform root’s thicknessand appearance, and well-distinguishable parallel rows ofcells. Treatment with citral, however, resulted in a loss of thisstraightness: Wavy lines of cells were observed, resulting intwisted apices with irregular thickness (Fig. 2). However, forany of the tested concentrations, there was no spiral growth ofthe roots or loss of the gravitropism. Another important aspectwas the total absence of root hairs on citral-treated seedlings atthe 200 μM concentration.

These differences also were detected with the transmissionelectron microscope. While, 7 days after treatment, controlroots had rows of homogeneous cells with easily distinguish-able zones into which radicle tissue is subdivided, the apical

b Control 50 µM 100 µM 200 µM 400 µM 800 µM 1200 µM% Germination 100 101.69 95.76 90.68 85.59 63.56 41.53

Statistical significance a b (*) c (***) d (***) e (***) f (***) g (***)

c Control 50 µM 100 µM 200 µM 400 µM 800 µM 1200 µM% Root length 100 89.25 80.25 35.02 11.41 6.30 1.92

Statistical significance a b (*) c (***) d (***) e (***) f (***) g (***)

a

0 µM 200 µM 400 µM

Fig. 1 a Arabidopsis thaliana grown for 15 days on plant agar withdifferent concentrations of citral. b and c Data for germination (b) andradicle length (c) given as percentages with respect to the control, foreach concentration tested. The tables show the statistical differencesfound between treatments (represented by letters), and also with

respect to the control (represented by asterisks). One, two and threeasterisks indicate, respectively, significant differences (P≤0.05), (P≤0.01), (P≤0.001). Statistical differences were estimated by ANOVAwith LSD to homocedastic data, and Tamhane’s T2 to heterocedasticones

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meristems of the radicles treated with the IC50 concentrationof citral showed a high level of tissue disorganization, whichmade it difficult to distinguish the different regions of theradicle. In the apical zone, a thick band was seen, apparentlymade up of the remains of cells, which covered the calyptra(Fig. 3). The first line of cells appeared to be completelydeformed, with highly irregular walls that were considerablythickened, with a zigzag, multilamellar appearance (Fig. 4a).Despite the thickening of the wall, no major differences wereseen with the naked eye between the thickness of the controlradicles and those treated with citral. The cells in both thequiescent center of the meristematic region and the zones ofelongation and differentiation conserved this irregular mor-phology. Similarly, anomalies were seen in phragmoplastformation, which gave rise to erroneous planes of division(Fig. 4b), as well as to a reduction in the number of cells indivision with respect to the control. As a result, the daughter

cells were of different sizes, with non-uniform distribution oforganelles. Large intercellular deposits of an electrodense na-ture also were seen (Fig. 4c), which were principally located inthe zone corresponding to the middle lamella. The number ofplasmodesmata was reduced by 32 % with respect to thecontrol. They appeared thickened, and many of them wereunfinished, due to the thickening of the cell wall (mentionedabove) (Fig. 4d).

Fourteen days after treatment, the same tissue disorganiza-tion was once again observed in the meristems which had beentreated with citral. Many accumulations and aggregates ofvesicles arising from the trans-Golgi network were found, andone of the most characteristic aspects of the 14 days of treat-ment was the high number of secretion points found in the wall(Fig. 5a). This vesicular material was found accumulated in thezone of the middle lamella. Additionally, the many Golgicomplexes (200%more than in the control) were disorganized,

IC50 citral-treated root

Control root

Fig. 3 Transmission electronmicroscope (TEM) images ofroot apices of Arabidopsisthaliana grown for 7 dayswith the IC0 (control) andIC50 concentrations of citral

b

1 2 3

a

1 2 3

Fig. 2 a Root meristems ofArabidopsis thaliana under amagnifier (increased 13 times).b Semithin sections observedusing an optical microscope:1. Roots grown in 0 μMof citral (control); 2. Rootsgrown with citral IC50 for7 days; 3. Roots grownwith citral IC50 for 14 d(20× objective)

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Fig. 5 Transmission electronmicroscope images of rootstreated with citral IC50 for14 days. a Sites of activeexocytosis (arrows); b) Highlyvesiculated Golgi complex(vG); c) Broken mitochondria(bm); d) Two differentmitochondrial morphologies:low electrodense matrix andreduced number of crests (left),and condensed and dark matrixmitochondria (right); e)Distorted nuclei withfragmented chromatin; f)Autophagic vacuoles (av)

a b

c d

e f

Fig. 4 Transmission electronmicroscope (TEM) images ofArabidopsis thaliana rootsgrown in citral (IC50) for 7 d. a)Zig-zag walls; b) Irregularphragmoplast formation c)Electrodense deposits at thewalls d) Thickening of the wall,irregular plasmodesmata andslack and broken upamyloplasts. Abnormalphragmoplast (ap); electrodensedeposits (ed); irregular andincomplete plasmodesmata(arrows); e) and f) Micrographsfrom 7 days-old control plants

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with the ends of the cisternae highly vesiculated and a stimu-lated secretory activity (Fig. 5b). The plasmodesmata kept thesame morphology as at 7 days after treatment, and the numberof plasmodesmata was reduced to approximately 22 % of thecontrol. In the case of the mitochondria, there were 11 % morethan in the control, and some of them appeared to be broken(Fig. 5c,d). Lastly, the chromatin appeared to be partiallyfragmented, and on some occasions it was close to the periph-ery of the nucleus (Fig. 5e). Numerous autophagic vesicles alsowere detected in the treated cells (Fig. 5f). The most importantchanges detected are summarized in Table 1.

The pectin staining of the roots revealed highly signifi-cant differences between the control meristems and thosetreated with citral (Fig. 6). The intensity of the stain, andalso the area stained, increased significantly in the treatedseedlings. Seven days after treatment, the semithin rootsections showed increased pectin staining in the walls ofthe root’s more external cells. Fourteen days after treatment,the intensity of the staining was high, and large deposits ofpectin could be seen in the walls of all the root cells. Theresults in live, fresh roots were similar to those obtainedwith the semithin sections.

The callose staining, however, did not reveal significantdifferences between treated plants and the control 7 and14 days after treatment (data not shown).

When citral was applied for a few hours, there was a drasticfall in mitotic activity, especially for the IC80 concentration,which completely paralyzed cell division in all time periodsanalyzed (2, 4, and 6 hr of treatment). Additionally, with this

concentration, a clear malformation of nuclear morphologywas observed 6 hr after the treatment, with the appearance ofnumerous deformed nuclei, which showed a typical kidney-like shape (Fig. 7d). However, the IC50 concentration resultedin stimulation of the number of cells in metaphase found after2 hr, although as at 4 hr, the inhibition caused in the treatedradicles also was complete (Table 2). These results confirmthe reduction in the number of cells in division found in theimages of transmission electron microscope.

Hormonal Content

The auxin level also decreased significantly in the roots after5 hr of treatment, although it increased significantly in theaerial part (Fig. 8). The situation after 10 hr of treatment wasdifferent, since the auxin level in the roots increased, butdecreased in the shoots, although it was still greater than thatin the control. After 10 hr of treatment, similar levels ofauxin were found both in roots and shoots, and the differ-ences with respect to the control were highly significant forboth parts.

Concerning ethylene content (Fig. 9), after 5 hr treatment,there were no changes in the amounts in roots (measured viathe incubation of plant material in distilled water) with respectto the controls, while the detected amount in shoots wassignificantly lower. Regarding potential levels of ethylene,measured after the incubation of samples in aminocyclopro-pane carboxylyic acid (ACC), the ethylene precursor, therewere no changes relative to the control in either roots or

Table 1 Most significant differences by transmission electron microscopy (TEM) between control roots and roots grown for 7 and 14 d with theIC50 concentration (194 μM) of citral

IC50 CITRAL

7 days 14 days

Tissue organisation Chaotic tissue organisation throughout the whole of the apical zone. The cellsdo not show the typical polygonal morphology seen in the control

Cell wall Wavy or zigzag walls, thicker than in the control. Middle lamellaconsiderably thickened. Intercellular highly electro-dense deposits

Walls very thickened. Numeroussecretion zones close to the wall

Plasmodesmata Very thick. Presence of unfinished plasmodesmata

Represent 32 % of the control Represent 22 % of the control

Cell division Irregularly formed phragmoplast. Daughter cells of different sizes, withnon-uniform distribution of organelles. Few cells in division

Mitochondria 32 % more than the control 11 % more than the control

Golgi complexes Disorganised cisternae. Highly vesiculated anddisorganized cisternae.

Same number as in the control 214 % more than in the control

Amyloplasts Large, with a high number of granules of starch in their interiorsof ovoidal morphology, slack and poorly organised

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shoots. After 10 hr treatment, actual ethylene quantities weresignificantly higher than in the control in both analyzed parts,as were the potential amounts.

Discussion

This study has confirmed the phytotoxicity of the monoterpenecitral to the germination and growth of A. thaliana, and dem-onstrated the degree of morphological and structural disorgani-sation of the root meristems. Since citral causes proliferation inthe root cells to cease, with values of mitotic indices of almostzero before 4 hr, it seems clear that the inhibition of root growthis directly related to the inhibition of cell division in the treatedseedlings. This severe inhibition in the mitotic indices also wasfound for other monoterpenes, such as 1,8-cineole on thespecies Echinochloa cruss-galli and Cassia obtusifolia(Romagni et al., 2000), although, in the case of citral, thisinhibition was far stronger, and even became total. The peakof cells in metaphase found for the IC50 concentration after 2 hr

of treatment could be due to changes to the microtubules anddelays during chromosome segregation, increasing the percent-age of metaphases (Fusconi et al., 2007). Chaimovitsh et al.(2010, 2012) recently demonstrated that citral caused the dis-ruption of microtubules in roots and cotyledons, and observedthat this disruption only affected the microtubules in a dose-dependent manner, the mitotic microtubules being more affect-ed than the cortical microtubules (Chaimovitsh et al., 2012).

The effect on microtubules may be caused by differentplant metabolic mechanisms. Seedlings treated with citralshowed in the short term, after 5 hr, a strong inhibition ofpolar auxin transport. These results seems to be closelyrelated to the results of the ethylene content of roots andshoots, in which a steep reduction in the ethylene content ofthe aerial part was found after 5 hr, while root content didnot change at that time. After 10 hr, the ethylene levels aftercitral treatment increased significantly with respect to thecontrol in shoots and roots. The bibliography includes nu-merous documented interactions between auxins and ethyl-ene, at both the physiological and molecular levels. Of

a

c d

bFig. 6 Pectin staining withRuthenium Red. a) Staining onfresh tissue after 14 daystreatment; b) Staining onsemithin sections of control; c)Staining of IC50 citraltreated-roots after 7 daystreatment; d) Staining of IC50

citral treated-roots after14 days treatment

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particular note are their combined effects on the regulationof root elongation, formation of root hairs, and growth(Stepanova et al., 2007), in which the balance of theirconcentrations is critical. Additionally, it is well known thatauxin needs ethylene to be redistributed (Vandenbussche etal., 2003), and that changes in auxin transport system orsignalling elements also cause aberrant responses to ethyl-ene (Swarup et al., 2007). The interaction of these phyto-hormones with the microtubule arrangement becomesespecially important when studying the effects of citral oncells, since an increase in the ethylene content and a reduc-tion in auxin content, which was detected in this study inplants treated with citral, has previously been related toalterations to microtubules (Chaimovitsh et al., 2010,2012). Roberts et al. (1985) demonstrated that an increasein ethylene content would induce a longitudinal and obliquereorientation of the microtubules after 10 hr of treatment,which could be the cause of the change of microtubulearrangement when treated with citral, although additionalstudy is needed to confirm this relationship.

The major change to the auxin content found in thetreated roots also could play a role in the absence of roothairs, as root hair formation is sensitive to hormonal and

environmental factors (Schiefelbein, 2000), and the localpresence of auxin and expansins is required for growthand development of root hairs.

One of the characteristic changes in citral-treated meristemsis the large number of Golgi complexes producing high numb-ers of vesicles, and the numerous secretion zones found 14 daysafter treatment, which seem to indicate processes of cell detox-ification in the treated seedlings. The products of these pro-cesses may be being deposited at the walls (Inderjit andKaushik, 2005). It is also known that all the Golgi complexesof a same cell work in a synchronised way to produce pectin-rich mucilage, and the number of complexes per cell increasesdramatically when there is a need to produce it in large quan-tities. The structural changes induced by this organelle dependon the volume of mucilage to be produced (Young et al., 2008).Pectin staining demonstrated that the increase in the number ofGolgi complexes, their high degree of vesiculation, and thelarge number of secretion points found in citral-treated roots isrelated to the increase in the thickness of the middle lamella ofthese cells, which is made up mostly of pectin-type com-pounds. The biosynthesis of wall polysaccharides shows enor-mous plasticity to adapt to various environmental factors (Hiset al., 2001). It is known that different types of stress cause a

a b

c d

Fig. 7 Images of meristematiccells in Arabidopsis rootsincubated with a) Distilledwater for 4 hr (control); b)Distilled water for 6 hr(control); c) Citral IC50 for 6 hr;and d) Citral IC80 for 6 hr. Theblack arrows indicate cells indifferent stages of mitosis. Thewhite arrows indicate thepresence of deformed nuclei(100× objective)

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change to the balance of deposition of the different compo-nents, principally affecting the cellulose-xyloglucan network insuch a way that the reduction in cellulose content is compen-sated for by an increase in pectin content (Burton et al., 2000;Aouar et al., 2010). The accumulation of pectin may be mod-ifying the architecture of the citral-treated meristems and theassembly of the new walls. In bean and onion cells treated withdichlobenil (a commercial herbicide), structural anomalies havebeen found which were similar to those found after the treat-ment with citral: increased cellular disorganisation, reducedgrowth, irregular, thickened and multilamellar cell walls, andirregularities in phragmoplast formation. These anomalies havebeen attributed to the replacement of the cellulose-xyloglucannetwork by a thick layer of a pectic nature, as occurs in theradicles treated with citral (Vaughn et al., 1996; García-Anguloet al., 2009).

These irregularities in walls are closely related to the pres-ence of unfinished and considerably thickened plasmodesma-ta. Intercellular communication via plasmodesmata has anessential role during plant development and in the coordina-tion of physiological functions in adult plants (Ehlers andKollmann, 2001). The reduction observed in the number ofplasmodesmata, together with the fact that many of them donot establish a real “bridge ” between cells, can cause the lossof tissue integrity, in which the cells would respond autono-mously (Roberts and Oparka, 2003), thus originating abnor-mal development of the future plant.

All the observed changes were detected at 7 and 14 days oftreatment, which indicates that citral has a persistent effect onthe roots of Arabidopsis, and confirms that the effect found inthe short term on the microtubules (Chaimovitsh et al., 2012)and on the cell cycle also has a long-term effect, without thecells having the opportunity to recover suitable growth anddevelopment.

Taken together, the results of this study show that citralalters auxin content and cell division and has a strong long-term disorganizing effect on cell ultra-structure in A. thali-ana seedlings. Many of these are consistent withprogrammed cell death (Zhu and Rost, 2000; Yao et al.,2004; Reape and McCabe, 2010; Hogg et al., 2011), such asfragmented chromatin, cytoplasm vesiculation, and detoxi-fying and autophagy processes. Its effects on cell division,the thickening of the cell wall, the reduction in intercellularcommunication, and the absence of root hairs suggest thatcitral is a promising phytotoxic compound. The analyses notonly demonstrate this compound’s long-term phytotoxiccapability, but also the presence of important secondaryeffects as a consequence of the accumulated toxicity andthe processes of detoxification.

***

***

***

**

**

-50

50

150

250

350

450

Citral 5 h Citral 10 h Citral 5 h Citral 10 h

% of ppm ethylene*g DW-1*h-1

respect to the control

Roots

Shoots

ACCH2O

Fig. 9 Ethylene level in IC50 citral-treated roots and shoots, both 5 and10 hr after treatment and incubated in distilled water (real level) or inACC (potential level). Data are given as percentage of the control. One,two and three asterisks indicate, respectively, significant differences(P≤0.05), P (p≤0.01), (P≤0.001). Statistical differences were estimat-ed by ANOVA with LSD to homocedastic data, and Tamhane’s T2 toheterocedastic ones

Table 2 Number of mitotic cells counted on a minimum of 3 meristems,counting at least 1000 cells in each; in synchronized apical meristems ofcontrol, IC50 and IC80-treated roots after different treatment times T0

(0 hr); T1 (2 hr); T2 (4 hr), and T3 (6 hr)

Control IC50 Citral IC80 Citral

T0 0.074 0.074 0.074

(a) (a) n.s. (a) n.s.

T1 0.75 0.996 0

(a) (b) *** (c) ***

T2 2.28 0 0

(a) (b) *** (b) ***

T3 2.07 0.093 0

(a) (b) *** (b) ***

The letters indicate the differences between treatments, while theasterisks represent significant differences compared to the control(* significant differences (P≤0.05), ** (P≤0.01), *** (P≤0.001); datawere analyzed with ANOVAwith LSD test, N=3)

***

***

***

***

0

50

100

150

200

250

5 h 10 h

)lortnoceht

fo%(level

nixuA

Roots Shoots

Fig. 8 Auxin level in IC50 citral-treated roots and shoots, both 5 and 10 hrafter treatment. Data are given as percentage of the control. One, two andthree asterisks indicate, respectively, significant differences (P≤0.05),(P≤0.01 (P≤0.001). Homocesdastic data were analyzed by ANOVAwithLSD, and the heterocedastic ones with Tamhane’s T2

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Acknowledgments The authors especially thank Jesús Méndez andInés Pazos from the Central Research Services of the University ofVigo (CACTI) for technical assistance on all the microscopic analyses.This research was supported by the Regional Government of Galiciathrough Project No. 10PXIB310261PR and a grant to Elisa Graña, andby the Spanish Ministry of Science and Technology AGL2010-17885through a grant to Carla Díaz.

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