Proteomic characterization of copper stress response inCannabis sativa roots

RESEARCH ARTICLE

Proteomic characterization of copper stress response in

Cannabis sativa roots

Elisa Bona, Francesco Marsano, Maria Cavaletto* and Graziella Berta*

Department of Environmental and Life Sciences, University of Piemonte Orientale “A. Avogadro”,Alessandria, Italy

Cannabis sativa is an annual herb with very high biomass and capability to absorb and accumu-late heavy metals in roots and shoots; it is therefore a good candidate for phytoremediation ofsoils contaminated with metals. Copper is an essential micronutrient for all living organisms, itparticipates as an important redox component in cellular electron transport chains; but is ex-tremely toxic to plants at high concentrations. The aim of this work was to investigate coppereffects on the root proteome of C. sativa, whose genome is still unsequenced. Copper stressinduced the suppression of two proteins, the down-regulation of seven proteins, while five pro-teins were up-regulated. The resulting differences in protein expression pattern were indicativeof a plant adaptation to chronic stress and were directed to the reestablishment of the cellular andredox homeostasis.

Received: September 21, 2006Revised: November 28, 2006

Accepted: December 21, 2006

Keywords:

Cannabis sativa / Copper / Heavy metal / MS/MS / Root

Proteomics 2007, 7, 1121–1130 1121

1 Introduction

Copper is an essential micronutrient for all living organisms.Since copper can readily gain and loose an electron, it acts asa cofactor of many enzymatic systems, some of them devotedto counteract oxidative stress. It is also an important compo-nent of electron transfer in the mitochondrial respiratorychain and in chloroplasts, where it is incorporated into plas-tocyanin [1, 2]. Yet, copper is a heavy metal and the redoxproperty that makes copper an essential element, also con-tributes to its inherent toxicity at high concentrations [3]. Itcan generate reactive oxygen species (ROS) such as super-oxide and hydroxyl radicals via Haber–Weiss and Fentonreactions that damage cellular components (proteins, aminoacids, nucleic acids, membrane lipids), interfere with cellular

transport processes, and produce changes in the concentra-tions of essential metabolites [1, 2, 4]. Cellular damage canoccur from both copper deficiency and toxicity, so cells elab-orate a fine control of the free copper level, mostly byexpressing highly efficient metal-chelating proteins [5].

Copper contamination of soil is often caused by indus-trial sources, repeated use of metal-enriched fertilizers,organic manures, and wastewaters. In recent years, phyto-remediation, the use of plants for extraction of heavy metalsfrom contaminated soils, has emerged as a promisingmethod for remediation of polluted soil [6–8]. In thisapproach, plants capable of accumulating and toleratinghigh levels of metals, termed hyperaccumulators, are grownin contaminated soil [9, 10]. Plants for phytoremediationmay be annual herbs, usually with a low or null economicvalue and very little biomass; the best choice is to employcrops of commercial interest in phytoremediation activities[11]. Among plants producing a high aboveground biomass,Cannabis sativa is widely employed in many types of non-food industries. It is a multiple-use plant providing rawmaterial for the production of natural fiber, insulating board,rope, oil, varnish, and paper. It is also a good candidate for

Correspondence: Dr. Maria Cavaletto, Department of Environ-mental and Life Sciences, University of Piemonte Orientale “A.Avogadro”, Via Bellini 25/G, 15100 Alessandria, ItalyE-mail: [email protected]: 1390131360390

Abbreviations: ANOVA, analysis of variance; FDH, formate dehy-drogenase; GRP, glycine rich RNA binding protein; rpL12, riboso-mal protein L12; SOD, superoxide dismutase * These authors contributed equally to the work.

DOI 10.1002/pmic.200600712

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1122 E. Bona et al. Proteomics 2007, 7, 1121–1130

soil phytoremediation as it is a tall plant with about 1 m deeproots that grow fast and easily in dense stands [12]. Further-more, it has been shown that C. sativa is a tolerant organismthat has evolved mechanisms allowing it to cope with highmetal (Cd and Ni) concentration in soil [10]; it has also beenshown that C. sativa tolerates and in part accumulates copperin the whole plant [13]. Despite these morphological andphysiological studies, C. sativa has not been investigated at aproteomic level, due to its unsequenced genome, with theexception of a single paper reporting the comparison offlower and gland 2-DE maps for the analysis of cannabinoidsbiosynthesis [14]. In general, there is a scanty knowledge onplant response to heavy metal stress at the proteomic level.Maize root and shoot proteomes have been investigated uponthe exposure to arsenic toxicity [15]. The proteome of Arabi-dopsis thaliana cells have been evaluated for the detection ofthe molecular and cellular changes elicited by Cd exposure[16], while in another work the copper-binding proteins ofArabidopsis roots have been explored by metal affinity chro-matography and MS [17]. The proteomic profiles of thehyperaccumulator Thlaspi caerulescens, exposed to Zn andCd, have been recently explored [18]; the response to Zn hasalso been evaluated in the proteome of rice [19] and themodulation of Cd stress by mycorrhizal symbiosis has beenanalyzed in pea roots [20].

The aim of this work was to investigate the copper mo-lecular effects on the root proteome of C. sativa plants, sinceroots are the primary sites of metal-ion uptake from thecontaminated soils. We focused our attention on the differ-entially expressed proteins to better understand C. sativadefense system and its correlation with the morphologicalparameters.

2 Materials and methods

2.1 Plant material and growth conditions

Seeds of C. sativa Var. Felina 34 were washed with deionizedwater and germinated in controlled conditions (247C, 16 hday/8 h night, 150 mmol?m22?s21 light irradiance). Homo-genous seedlings were transplanted into individual potscontaining 2 kg of loam (Agral, Agrimont, Bolzano, Italy),quartz sand, and gravel mix (9:5:1). Five pots were addedwith a 150-ppm solution of CuSO4?5H2O and five wereadded with sterile water. Plants were grown in the controlledenvironment, described above, and harvested 6 wk aftertransplanting. All plants received weekly 400 mL of LongAshton solution [21].

2.2 Measurement of leaf and root parameters

The photosystem II activity was measured at 5 and 6 wk ofgrowth using Handy-PEA (Hansatech instruments, UK), acontinuous-excitation type chlorophyll fluorescence analy-zer; it utilizes the continuous excitation fluorescence meas-

urement principle. High time resolution (100 KHz initialsampling rate) electronics and saturating levels of illumina-tion allow accurate monitoring of the time-dependent fluo-rescence-induction kinetics observed when a sample isstrongly illuminated after a dark adaptation time (in ourexperiment 30 min) (Kautsky effect). We measured: Fo, cor-responding to the fluorescence level when plastoquinoneelectron acceptor pool (Qa) is fully oxidized; Fm, fluorescencelevel when Qa is transiently fully reduced; Fv, variable fluo-rescence (Fm 2 Fo); Fv/Fm, maximum quantum efficiency ofphotosystem II. The Fv/Fm parameter was used as leaf stressparameter to value the leaf stress induced by copper treat-ment, Fv/Fm values of 0.80–0.82 are considered as normalvalues for plants not stressed [22], lower values of Fv/Fm

indicate photosynthetic damage. Moreover, after harvesting,root and shoot fresh weights, stem length, and diameterwere measured and samples stored at –807C for proteomicanalysis. Root and leaf morphometric analyses were per-formed on parallel set of plants, with Win-RHIZO Pro V.2002c (Regent Instruments, Quebec, Canada), an interactivescanner-based image analysis system [23]. Before analysis,roots were preserved in 70% ethanol. The following parame-ters were measured: total root length, total root surface area,total root projected area, total root volume, leaf area, andnumber of tips.

2.3 Copper content in plant organs

For inductively coupled plasma–MS (ICP-MS) analysis, plantsamples were carefully washed with tap water and thenrinsed with distilled water, separated in shoots and roots, andoven-dried at 807C for 3 days. Then they were ground withpestle and mortar; 0.5 g of each sample was digested in 6 mLof 65% nitric acid (Sigma–Aldrich) using a MARS 5 micro-wave oven (CEM, Matthews, North Carolina, USA).

Total copper concentration was determined after diges-tion according to Standard Methods 17 edition. Digestedsamples were diluted with purified water (MilliQ reagentgrade) and then analyzed by ICP-OES (IRIS Advantage ICAPserie DUO HR, Thermo Jarrell Ash, Franklin, USA) andICP-MS (Plasma QUAD 3, VG Elemental Europe, Cedex,France). Certified standards of analyzed metal (1573a) andacid blanks were run with all sample series for quality con-trol. The copper concentration was given as ppm (ppm, mg/kg).

2.4 Protein extraction and solubilization

Proteins were extracted according to Bestel-Corre et al. [24],with some modifications. For each sample, 2 g of C. sativaroots were ground with liquid nitrogen and homogenized in10 mL of 0.5 M Tris-HCl, pH 7.5 lysis buffer, containing0.7 M sucrose, 50 mM EDTA, 0.1 M KCl, 10 mM thiourea,2 mM PMSF/DMSO, and 2% v/v b-mercaptoethanol. Tenmilliliters of Tris-HCl pH 8.8 saturated phenols were added.After mixing for 45 min the phenolic phase was separated by


Proteomics 2007, 7, 1121–1130 Plant Proteomics 1123

centrifugation and rinsed with another 10 mL of lysis buffer.After centrifugation, the phenolic phase was separated forthe second time. Proteins were precipitated overnight at–207C after adding five volumes of methanol containing0.1 M ammonium acetate.

The pellet recovered by centrifugation was rinsed withcold methanol and acetone, dried under nitrogen gas, andresuspended in 600 mL of solubilization buffer containing9 M urea, 4% w/v CHAPS, 0.5% Triton X-100, 20 mM DTT,and 2% v/v IPG Buffer (GE Healthcare, Cologno Monzese(MI), Italy).

Protein content of the sample was quantified by themethod of Bradford [25].

2.5 2-DE

IEF was performed on IPG strips in an IPG-Phor unit (GEHealthcare Bio-Sciences). For semipreparative separations,500 mg of protein extracts were mixed with a rehydrationbuffer (8 M urea, 4% w/v CHAPS, 18 mM DTT, 0.5% 3–10IPG Buffer) and focused at 60 kV?h at 207C on precast 13 cmlinear pH 3–10 strips. The second dimension was carried outwith a Protean II xi system (BioRad); 12% gels were run at107C under constant amperage (30 mA). Gels were CBB-stained. 2-DE standards (BioRad) were coelectrophoresedwith the samples.

2.6 2-D gel analysis

The gels were scanned in a GS 710 densitometer (BioRad).The gel images were recorded and computationally analyzedusing PDQuest 7.3.1 (BioRad).

The intensity of each protein spot was normalized rela-tive to the total abundance of all valid spots. After normal-ization and background subtraction, a matchset was createdfor both control gels (five replicates) and Cu-treated gels (fivereplicates). The differential expression analysis was per-formed comparing the quantity of matched spots in the Cu-treated versus the control gels. The program created a quan-titative table with all normalized optical spot densities thatallowed us to perform an analysis of variance (ANOVA) todetect statistical differences between the quantitation of thesame spot in all replicates. We performed ANOVA usingStatView 4.5 (Abacus Concepts, Berkeley, CA, USA) andp�0.05 was adopted as the level of significance.

2.7 In-gel digestion

For MS analysis, spots of interest were cut from the gel anddestained overnight with a solution of 25 mM ammoniumbicarbonate and 50% ACN. The proteins were digested in-gelwith trypsin (Roche, Segrate, Milano, Italy), as described byHellmann et al. [26].

2.8 MS/MS analysis

MS/MS analysis of the digested peptide was performedusing either a QSTAR XL hybrid quadrupole-TOF instru-ment (Applied Biosystems, Foster City, CA, USA) coupledwith LC Packings Ultimate nanoflow LC system (Dionex,Amsterdam, The Netherlands) or an LCQ Thermo (San Jose,CA, USA) IT mass spectrometer fitted with a nano-ESIsource.

The tryptic peptides mixture was desalted and con-centrated in ZipTipC18 devices (Millipore, Bedford, MA,USA), then the eluted mixture was dried in speed vac andreconstituted in 0.1% TFA/5% ACN in water for the separa-tion in nano-HPLC. Using the FAMOS autosampler (Dio-nex) and the SWITCHOS loading pump (Dionex), peptideswere preconcentrated into the sample trap cartridge. Pep-tides separation was achieved on a nanoscale PepMap col-umn C18 (15 cm675 mm id, 3 mm, 100 Å) at a flow rate of200 nL/min, with the following linear binary gradient pro-file: 5% B at 0 min to 40% B by 30 min, 40–95% for 7 min,then 95% B for 5 min, finally returned to 5% B by 5 min andheld at 5% B for 60 min. Solvent A was 1% formic acid, 5%ACN; solvent B was 0.8% formic acid, 95% ACN. NanoscaleESI was performed using a Protana interface and nanoelec-trospray needles set to 1.6 kV (New Objective, Woburn, MA).The QSTAR XL operated in the data-dependent mode, wherethe top three peaks in every full MS scan were subjected toMS/MS analysis. The dynamic exclusion feature of the Ana-lyst QS software (Applied Biosystems) was enabled, with thefollowing settings: exclusion mass width: 6 3 m/z, exclusionduration: 60 s.

For the LCQ Thermo IT analysis, the tryptic peptidemixture was eluted from ZipTipC18 with 60% methanol plus1% formic acid, then 4.5 mL were introduced into a gold-coated borosilicate capillary (Proxeon Biosystems, Odense,Denmark) and analyzed in an LCQ IT mass spectrometerfitted with a nano-ESI source. The capillary voltage was set to46 V and spray voltage to 1.8 kV. The most stable signalsfrom the full-scan mass spectrum were trapped and frag-mented by low-energy CID, with normalized collision energyranging from 22 to 24%.

The remaining sample was derivatized in-tip by additionof acetic anhydride and returned to the instrument for spec-tra acquisition, as described in [27, 28]; spectra data weremanually interpreted.

2.9 Cross-species protein identification

Since C. sativa genome is still unsequenced, a limited num-ber of protein sequences are included in NCBI database.Proteins were identified through peptide fragmentation(MS/MS) data by de novo sequencing using either softwaresto obtain automated descrambling of mass spectra by com-puter, or by manual interpretation of the data after the deri-vatization procedure.



In some cases, identification was made difficult sincehomologous genes and their corresponding proteins retain alower percent identity, when organisms become more phy-logenetically distant.

Once confirmed, LC-MS/MS data were searched accord-ing to the procedure of Liska and Shevchenko using MSBlast [29], by partially aligning the analyzed proteins fromC. sativa to a database sequence from related organisms.Spectra data were searched against NCBInr and Swiss-Protdatabases using the ProID program (Applied Biosystems)available on the QSTAR instrument and peptides hits withscores better than 600 in ProID were considered significant.Alternatively, in the case of the N-acetylation derivatization,spectral data were manually interpreted and the peptidesequences were submitted to the web homology-tool of theProtein Prospector package.

3 Results

3.1 Plant biomass and development

The threshold of C. sativa tolerance to copper was tested withdifferent concentrations of copper sulfate (from 500 to150 ppm); 150 ppm was chosen, since at higher concentra-tions dramatic toxicity was observed.

Seedlings were of the same size when transplanted; after6 wk, the control plants were uniformly grown and weresignificantly higher than the treated plants; root and shootdry weights were significantly lower in the treated plants(Table 1). Copper-treated plants did not show evident symp-toms of chlorosis in shoots. Other analyses, carried out onphotosystem apparatus, showed no effect on photosystem IIactivity induced by the used copper concentration. As shownin Fig. 1, copper content increased with the treatment in bothshoots and roots, its concentration in shoots was two-foldincreased with respect to controls, while it was eight-foldincreased in treated roots; this kind of accumulation is inagreement with our findings showing that copper damage is

Figure 1. Copper concentration in shoot and root, expressed inppm (mg/kg dry weight). Equal letters (a, a) indicate no signifi-cant statistical differences and different letters (a, b) indicate sig-nificant statistical differences. Averages of five replicates (6SEM) are shown for control- and Cu-treated plants.

Table 1. Leaf and root parameters

Parameter Control Cu150 ppm

Fv/Fma) 0.830 6 0.003 0.818 6 0.008

Stem length (cm) 61.84 6 3.51 44.41 6 3.38b)

Root dry weight (g) 1.48 6 0.22 0.93 6 0.08b)

Shoot dry weight (g) 3.86 6 0.23 2.76 6 0.05b)

Leaf area (cm2) 1679.56 6 70.02 1148.16 6 159.73b)

Total root length (cm) 16 079.31 6 4194.99 7031.62 6 1269.54Total surface area (cm2) 3355.18 6 754.21 1547.19 6 225.00Root volume (cm3) 57.97 6 10.20 27.53 6 3.10b)

Number of tips 32 928 6 9382.8 12 489.6 6 3319.8

a) Maximum quantum efficiency of photosystem II.b) A significant statistical values, ANOVA test, p�0.05.

Figure 2. Hemp root digital images made with Win Rhizo soft-ware. On the left (A) a control root is shown and on the right (B) aCu-treated root.

preferentially localized at the root level. Our approach hasbeen directed to evaluate proteomic changes in the C. sativaroot system which underwent growth inhibition and resultedto be highly affected by copper at the morphological level, asshown in Fig. 2.

3.2 2-DE functional analysis of copper-treated root

proteins

In Fig. 3 the Coomassie reference map of C. sativa root pro-teins is shown, where nearly 300 spots were reproduciblyseparated. Among them, 20 spots were cross-species identi-fied and are listed in Table 2. For each protein, the percentageof residue identities with the A. thaliana homolog has beendetermined with the BLAST program (http://www.expa-sy.org) and the similarity searching tool Proteomes Fasta(http://www.ebi.ac.uk/fasta33/proteomes.html#) [30, 31].The boxed areas A (40–80 kDa molecular weight proteins), B(low molecular weight proteins), and C (basic proteins) arezoomed in Fig. 4, where the Cu treated- and the control-mapsare compared. In order to highlight the copper effect, image



Table 2. Identification of C. sativa root proteins

Spot Precursorion massm/z

Sequence Protein Mr (kDa)/pITheoretical

AC number (gi NCBI)and referenceorganism

% Identitiesin A. thalianaa)

Cs 1 800.41600.8

EGLELLK VNQIGSVTESIEAVR Enolase 47.72/5.31 42521309Glycine max

87%

Cs 2 800.41221.61600.82251.1

EGLELLKISGDALKDLYKVNQIGSVTESIEAVRSGETEDTFIADLAVGLSTGQIK

Enolase 47.72/5.31 42521309Glycine max

87%

Cs 3 905.5988.51089.6

EKVGSSSGRVVEGMDIVKAIEKVGSSSGR

Cyclophilin 18.15/8.95 18076088Ricinus communis

77%

Cs 4 1006.4 VIDAESFAR ABC transporter substrate-binding protein

38.39/8.38 27375524Bradyrhizobium

japonicum

28%

Cs 5 962.41566.7

GDFAIDIGRKIIGATNPAESPPGTI

Nucleoside diphosphatekinase B

16.22/6.31 2498076Helianthus annuus

74%

Cs 6 1062.6 VVDIVDTFR Translationally controlledtumor protein homolog (TCTP)

19.04/4.51 9979196Hevea brasiliensis

73%

Cs 7 1103.5 EDDNIETIR Uridylate kinase 22.48/5.73 2497486A. thaliana

100%

Cs 8 1072.51489.6

IINDRETGRDAIEGMNGQDLDGR

GRP 16.89/5.86 544424A. thaliana

100%

Cs 9 1448.21705.81716.4

DLSYYTLVLKGDQDPALDSEYAANLKQSDSALTTNSFTK

Putative peroxidase 34.92/8.74 21536908A. thaliana

100%

Cs 10 978.5 GNLDIFSGR Elicitor inducible protein EIG-J7 20.34/5.07 40287496Capsicum annuum

69%

Cs 11 n.d.

Cs 12 1197.71325.81367.71444.8

VPIEVTIGELKVPIEVTIGELKKYIGLSEASASTIRELGIGIVAYSPLGR

Aldo/keto reductase 37.47/5.7 2462763A. thaliana

100%

Cs 13 1592.01696.6

FFDTSDIYGEKYIDLYYIHR

Putative auxin induced protein 37.92/6.3 54290851Oryza sativa

60%

Cs 15 1318.7 VIDLFSSPDVVK 40S ribosomal protein S20 13.88/9.73 21542443A. thaliana

100%

Cs 16 1147.61176.51287.61445.71855.91953.1

EITALAPASMKGYMFTTTAERYPIEHGIVSNWDDMEKGEYDESGPSIVHRLAYVALDYEQELETAKVAPKEHPVLLTEAPLNPK

Actin 41.73/5.6 9082317Helianthus annuus

97%

Cs 17 794.4816.4824.51306.61380.71818.92344.1

AYDLEGKTVGTVGAGRIVGVFYKFEEDLDAMLPKGEDFPAENYIVKYFKGEDFPAENYIVKYMPNQAMTPHISGTTIDAQLR

Mitochondrial FDH 42.02/6.6 11991527Solanum tuberosum

80%

Cs 18 1631.02251.8

ALGLELDLSEKNVENGGEFSVSSA

Thioredoxin-dependent peroxidase 17.39/5.3 52851172Plantago major

85%



Table 2. Continued

Spot Precursorion massm/z

Sequence Protein Mr (kDa)/pITheoretical

AC number (gi NCBI)and referenceorganism

% Identitiesin A. thalianaa)

Cs 19 1253.81343.71621.0

VSVV PSAAALVIKVTGGEVGAASSLAPKISLDDVIEIAK

60S rpL12 17.71/8.8 40287508Capsicum annuum

88%

Cs 20 1006.51056.21104.21192.61333.6

GFDVVDDIKTITAGLI/LRNSANNASRGYEIIDEAKRADILTLATR

Peroxidase N 31.97/6.36 7245407A. thaliana

100%

Cs 21 959.51218.7

SAGLQDQIKVSTAIDTTLIGK

Endo-beta 1,3 glucanase 23.58/8.62 41584400Glycine tabacina

50%

Cs 22 n.d.

Cs 23 827.41740.7

GLVHFQKIDYAPGGLNPPHTHPR

Nectarin 1 precursor (Mn-SOD) 24.75/7.71 18203447Nicotiana plumbaginifolia

64%

a) Percentage of residue identities in the homolog protein of A. thaliana proteome.n.d., determined

Figure 3. 2-DE of C. sativa root proteins stained with CBB G-250.The IEF was performed with 13 cm linear IPG strip pH 3–10, fol-lowed by SDS-PAGE on 12% gel. The boxed areas are: (A) (40–80 kDa molecular weight proteins), (B) (low molecular weightproteins), and (C) (basic proteins).

analysis has been performed on five replicates and variationson spots’ area were confirmed by statistical analysis. Asshown in Figs. 5a and b, copper treatment induced the dis-appearance of two spots (Cs18 and Cs19) corresponding tothioredoxin-dependent peroxidase and 60S ribosomal pro-tein L12 (rpL12). While seven spots were down-regulated(Cs1, Cs3, Cs4, Cs8, Cs9, Cs10, Cs11, respectively enolase,cyclophilin, ABC transporter substrate-binding protein, gly-cine rich RNA binding protein (GRP), putative peroxidase,

Figure 4. Enlargements of the following boxed areas: (A) 40–80 kDa molecular weight proteins, (B) low molecular weight pro-teins, and (C) basic proteins. Differentially expressed spots areindicated for both control- and Cu-treated maps.



Figure 5. (a) and (b) Differential expression of down-regulatedproteins from control- and Cu-treated C. sativa roots. For eachprotein, the normalized od average of five replicates (6 SEM) isshown. Equal letters (a, a) indicate no significant statistical dif-ferences and different letters (a, b) indicate significant statisticaldifferences (ANOVA using StatView 4.5, p�0.05).

and elicitor inducible protein), in particular Cs1 was reducedto about 50%, Cs3, Cs4, and Cs8 to 65, Cs9 to 70, Cs10 to 34,and Cs11 to 40% with respect to the controls. Figure 6 showsthat five spots were up-regulated (Cs12, Cs13, Cs15, Cs16,Cs17), among them Cs12 (aldo/keto reductase) increased to5.5-fold, Cs13 (putative auxin induced protein), Cs15 (40Sribosomal protein S20), and Cs17 (mitochondrial formatedehydrogenase) to two-fold and Cs16 (actin) to 1.3-fold.

4 Discussion

C. sativa plants were grown for 6 wk in the presence of cop-per (150 ppm), a concentration higher than the mean worldsoil copper concentration (20 ppm) and above which plant

Figure 6. Differential expression of up-regulated proteins fromcontrol- and Cu-treated roots. For each protein, the normalizedod average of five replicates (6 SEM) is shown. Equal letters (a, a)indicate no significant statistical differences and different letters(a, b) indicate significant statistical differences (ANOVA usingStatView 4.5, p�0.05).

toxicity events can occur. The copper concentration in shootsresulted considerably lower compared to the root system,which proved that Cu movement along the plant vascularsystem was strongly limited. Our data were in agreementwith Angelova et al. [13], who showed the followingcopper gradient of accumulation in hemp organs: roots.

stems.leaves.seeds. Since morphological parameters wereindicative of metal accumulation in roots and with the aim ofdisclosing at a protein level the uptake and the destiny ofcopper, the proteomic analysis of the extracted hemp rootproteins has been evaluated.

4.1 Copper up-regulated proteins

We detected a marked increase of aldo/keto reductase (Cs12)which is a NAD(P)H-dependent enzyme, widely distributedfrom mammals to insects, fungi, including yeasts [32]; theincrease in aldo/keto reductase is linked to its involvement indetoxification process by improving scavenging capacity ofthe cell [33, 34]. In fact, it has been shown a potential role ofhuman aldehyde dehydrogenases in the biotransformationof drugs, dietary substances, and xenobiotics [35] and threeproteins of the aldo/keto reductase family were correlatedwith herbicide stress in Triticum tauschii [33]. Copper treat-ment also induced the up-regulation of a putative auxin-induced protein (Cs13). On analyzing the primary structureof this protein, its homology with the above-mentioned aldo/keto reductase was evident (with 54.8% of identity); con-sidering the cross-species identification, it is very reliablethat the two spots represent the same protein belonging tothe family of aldo/keto reductase, the pI shift can thereforebe ascribed to PTMs. It is interesting to note the massive



involvement (5.5-fold and 2-fold increase respectively forCs12 and Cs13 spots) of aldo/keto reductase in response tocopper stress.

Since both proteins Cs12 and Cs13 of the aldo-ketoreductase family are recorded in NCBI database as putativeauxin-induced proteins, we can underline the involvement ofauxin as plant growth regulator in mediating the responsesof root architecture to nutrients (Cu) [36]. Root architectureremodeling is clearly evident in Fig. 2, moreover, we havedetected a significant increase in actin (Cs16). Actin is animportant component of plant cytoskeleton and microfila-ments, they contribute to cell elongation, probably throughinteractions with cortical microtubules [37] and by main-taining polar auxin transport [38].

The increase in formate dehydrogenase (FDH) (Cs17), amitochondrial NAD-dependent enzyme which catalyzes theoxidation of formate into CO2 is linked to its function inmaintaining a reduced environment. Hourton-Cabassa etal.[39] showed that FDH mRNA expression was stronglyincreased by stress factors like dark, hypoxia, cold, anddrought.

Protein synthesis machinery was affected by coppertreatment since an increase in 40S ribosomal protein S20(Cs15) has been detected. This protein has been shown to beup-regulated at early time from exposure to cadmium andsubsequently repressed in Schizosaccharomyces pombe [40].

4.2 Copper down-regulated proteins

One of the two protein spots that disappeared upon Cutreatment was Cs 19, identified as 60S rpL12, indicatingagain the involvement of protein synthesis machinery. Evenif it was not possible to ascribe specific functions to thisribosomal protein [41], it is important to underline thatmouse rpL12 differs from the bulk of ribosomal proteins anduses a distinct nuclear import, so rpL12 could play a role insignaling the final maturation of ribosome [42].

As for thioredoxin peroxidase (Cs18), we have detectedthe suppression of the corresponding spot in copper-treatedroots. It has been reported, using RNA interference, that thisenzyme might have both beneficial and detrimental effect oncells challenged with different stresses [43]. Thioredoxinperoxidase underexpressing Drosophyla cells were moreresistant to stressors like elevated temperature and copper;while cells overexpressing thioredoxin peroxidase were morevulnerable to copper [43]. Then we can suggest a directinvolvement of the decrease in this enzyme in root protectionagainst copper stress, as it has been proposed that Cu21 maybe reduced by thiol groups in the active site of thioredoxinperoxidase, thus competing with the reduction of hydrogenperoxide [43–45].

The detected down-regulation of a putative peroxidase(Cs9) was in accordance with the decrease in peroxidase,reported in rice leaves during treatment with H2O2 and inmaize roots treated with aluminum, to maintain as a bufferthe root growth rate [46, 47].

One (Cs1) of the two spots identified as enolasedecreased in treated roots. Enolase is a multifunctional pro-tein and in a plant proteomic study it has been reported as aresponsive protein to many environmental stresses, such assalt stress, drought, and cold [48].

We detected the reduction of elicitor inducible protein(Cs10), a protein with unknown function, containing aPLAT/LH2 domain, which consists of a b-sandwich; its pro-posed function (PROSITE database) is to mediate interactionwith lipids or membrane bound proteins, many members ofthis protein family are stress-induced.

A decrease in cyclophilin (Cs3) has been detected, even ifnot statistically significant; this can be attributed to theinvolvement of the cyclophilin, a peptidyl-prolyl cis-transisomerase, in the reducing system of eukaryotic organisms,including thioredoxin, glutaredoxin, and cyclophilin as di-sulfide reducers and correlates to the observed decreasedlevel of the above-mentioned peroxidases [44].

GRP (Cs8) was reported by Nishiyama et al. [49] as a cold-inducible protein; its overexpression resulted in reducedgrowth rate in mouse fibroblasts, besides a class of GRP hasbeen found in meristematic tissues of plants, where probablyGRPs are involved in the regulation of growth rate and/orresponse to external stimuli [50]. We have detected a decreasein GRP as response to copper stress, probably related withthe need of maintaining root growth rate or to the strongreduction of the number of root apical meristems induced bycopper.

A decrease in ABC transporter substrate binding protein(Cs4) has been detected. The ABC transporter superfamily isa large protein family, widespread from bacteria to plants andman; mutant plants for the A. thaliana AtMRP5 showed astrongly reduced root elongation, a formation of lateral roots,and increased levels of free auxin [51]. At present it is notknown which kind of substrate the C. sativa ABC transportersubstrate binding protein can bind, mostly considering thelow percent of identities between the cross-species-identifiedprotein and the corresponding A. thaliana homolog.

Among the down-regulated proteins spot Cs11 has notbeen identified, neither with QSTAR instrument, nor afterN-acetylation derivatization, it is very probable that Cs11 is anon-conserved protein, so its cross-species identificationbecomes very difficult employing homology tools.

4.3 C. sativa response to copper stress

The processes of heavy metal uptake, accumulation, dis-tribution, and detoxification have been studied in a widerange of plant species; however the mechanisms involved arestill only partially understood. The proteomic approach cantherefore help in disclosing new aspects of plant metalstress.

Our results confirm that C. sativa has not evolved a spe-cific copper accumulation mechanism [11], as we did notdetect any new root protein after metal treatment. Never-theless, hemp succeeded as a phytostabilizer of copper ions



in its root system. The development of plant mycorrhizalsymbiosis [52] could improve its phytoremediation potential,ameliorating root system efficiency and increasing root toshoot copper translocation.

The aldo/keto reductase is the most up-regulated pro-tein by copper treatment, it could directly interact with cop-per ions inside the cell and we hypothesize it could act as“copper chaperone” for C. sativa root cells. A copper–proteininteraction mechanism has been described for aldosereductase involving an essential residue of Cys in position303 [5]; in this case copper induces enzyme inactivationthrough a thiol-mediated protein oxidation. The enzyme isable to bind two copper ions, each copper ion coordinatestwo residues of Cys (80 and 303), the distance is compatiblewith an electron transfer process and this allows the sub-sequent formation of the disulfide bond. The inactivatedenzyme can be converted into the native form and theinteraction with copper results in an efficient system ofmetal sequestration [5].

We propose a model for the effect of copper on C. sativaroots, where the aldo/keto reductase is the first proteininteracting with copper ions, it could reduce copper ions toCu (I), so they could be available for interaction with otherpartner proteins, like phytochelatins which usually bind Cu(I) and from this location copper can be transported to thevacuole.

Our proteomic analysis did not show the identification ofa copper chaperone, nevertheless we cannot exclude itspresence in C. sativa, we can only report that a copper chap-erone, like bacterial CopZ, Arabidopsis CCH, or yeast Atx1p[3], is not included among the differentially expressed pro-teins upon copper stress.

We did not detect any increase in superoxide dismutase(SOD) and catalase, typical scavenging H2O2 enzymes; this isprobably linked to the temporal phase of copper treatment,our root samples reflect a chronic adaptation to copper, inagreement with Vensel et al. [53] who found that differentproteins related to stress/defense metabolic processes wereinvolved during the different developmental stages of wheatendosperm. Moreover, two different proteomic studies onArabidopsis have shown no change in Mn-SOD upon oxida-tive stress [54], neither a SOD involvement as a copper inter-acting protein in roots [17].

Functional analysis of C. sativa root proteome revealedthat five proteins increased, two proteins disappeared, andseven proteins decreased in response to copper treatment.Since cellular damage can occur both from an excess (copperis able to promote the formation of ROS) or a deficit of cop-per (it can act as cofactor in enzymatic antioxidant systems),inside the cell there is a fine control of free copper level.Among the differentially expressed proteins it was possibleto detect a defense network with the involvement of thescavenging system (represented by aldo/keto reductase),stress proteins (such as enolase, elicitor-inducible protein,and FDH), proteins conferring more resistance to copperstress and an efficient reducing system (thioredoxin peroxi-

dase, peroxidase, cyclophilin), and finally proteins regulatingand maintaining a suitable growth rate of the root (actin,ribosomal proteins, and GRP).

On the whole, we can hypothesize that the responses ofthe C. sativa root proteome reveal an adaptation to a chroniccopper stress, in order to reestablish the cellular home-ostasis.

4.4 Concluding remarks

Combining cross species identification with the differentialanalysis of 2D maps, it has been possible to describe a pat-tern of proteins up- and down-regulated upon copper stress.These findings would be useful in phytoremediation activi-ties, such as the development of plants for biomonitoringand for remediation of heavy metal polluted soil. In the nearfuture, the protein markers identified by the proteomicglobal approach could be the starting point for bioengineer-ing new plants for phytoremediation purposes.

We thank Dr. O. Repetto for technical support. Dr. E. Bonawas supported by a grant from the Europa Metalli factory. Theresearch was performed within the framework of “Associazioneper l’Ambiente ed il Territorio” (ATF, Alessandria) project.

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Proteomic characterization of copper stress response inCannabis sativa roots

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