RESEARCH ARTICLE SPECIAL ISSUE: PLANT CELL BIOLOGY

Calmodulin antagonist affects peroxisomal functionality bydisrupting both peroxisomal Ca2+ and protein importFrancisco J. Corpas1,* and Juan B. Barroso2

ABSTRACTCa2+ is a second messenger in many physiological andphytopathological processes. Peroxisomes are subcellularcompartments with an active oxidative and nitrosative metabolism.Previous studies have demonstrated that peroxisomal nitric oxide(NO) generation is dependent on Ca2+ and calmodulin (CaM). Here,we used Arabidopsis thaliana transgenic seedlings expressing cyanfluorescent protein (CFP) containing a type 1 peroxisomal-targetingsignal motif (PTS1; CFP–PTS1), which enables peroxisomes to bevisualized in vivo, and also used a cell-permeable fluorescent probefor Ca2+. Analysis by confocal laser-scanning microscopy (CLSM)enabled us to visualize the presence of endogenous Ca2+ in theperoxisomes of both roots and guard cells. The presence of Ca2+ inperoxisomes and the import of CFP–PTS1 are drastically disruptedby both CaM antagonist and glutathione (GSH). Furthermore, theactivity of three peroxisomal enzymes (catalase, glycolate oxidaseand hydroxypyruvate reductase) containing PTS1 was clearlyaffected in these conditions, with a decrease of between 41 and51%. In summary, data show that Ca2+ and CaM are strictlynecessary for protein import and normal functionality ofperoxisomal enzymes, including antioxidant and photorespiratoryenzymes, as well as for NO production.

KEY WORDS: Ca2+, Calmodulin, Catalase, Glutathione, Nitric oxide,Peroxisome

INTRODUCTIONCa2+ is a cellular second messenger whose homeostasis is highlyregulated in cells through different components including Ca2+

pumps, channels, cation exchangers and Ca2+-binding proteins,since this cation regulates many cellular activities (Choi et al., 2012,2014; Charpentier and Oldroyd, 2013; Cheval et al., 2013;Steinhorst and Kudla, 2013; Simeunovic et al., 2016). Moreover,crosstalk among Ca2+ and other signaling molecules, including H2O2

and nitric oxide (NO), which affects gene expression and Ca2+-dependent protein kinase, has also been described (Rentel and Knight,2004;González et al., 2012;Han et al., 2014; Niu andLiao, 2016). Ca2+

signals can remain local or be propagated throughout an entire cellwithin milliseconds to seconds (Monshausen, 2012). Ca2+ has beendetected in almost all cellular compartments including the cytosol,

chloroplasts, mitochondria and nucleus. But, there can be a 10,000-folddifference between cytoplasmic and non-cytoplasmic Ca2+

concentrations, which allows the formation of Ca2+ signals by rapidalterations of cytoplasmic Ca2+ levels via membrane-localized Ca2+-permeable channels (Stael et al., 2012). However, informationregarding Ca2+ in peroxisomes is scarce; to our knowledge, only onestudy has claimed that it is present in plants (Costa et al., 2010).Nevertheless, its potential function in peroxisomes has been analysed,as Ca2+ is necessary for key antioxidant peroxisomal enzymes, such ascatalase (Yang and Poovaiah, 2002; Schmidt et al., 2006; Costa et al.,2013), and for peroxisomal NO generation (Corpas et al., 2009; Corpasand Barroso, 2014b).

Peroxisomes are single-membrane-bound subcellularcompartments present in almost all types of eukaryotic cells. Theycontain catalase andH2O2-producing flavin oxidases as indispensableenzymatic elements (Reumann et al., 2007; Palma et al., 2009;Corpas, 2015). These organelles are characterized by metabolicflexibility, as their enzymatic content can vary according to theorganism (i.e. animal, plant or yeast), cell or tissue type (i.e. liver, rootor leaf), development stage and external environmental conditions(Mullen et al., 2001; del Río et al., 2002; Hayashi and Nishimura,2006; Pracharoenwattana and Smith, 2008; Hu et al., 2012).

In plant cells, peroxisomes house a large number of antioxidativeenzymes, such as catalase, superoxide dismutase, components ofthe ascorbate–glutathione cycle and several NADP dehydrogenases,which are involved in different functions (del Río et al., 2002;Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016;Leterrier et al., 2016; Corpas et al., 2017). On the other hand,accumulating data have shown that plant peroxisomes have thecapacity to generate NO through a L-arginine-dependent nitricoxide synthase (NOS) activity, which strictly depends on NADPHand requires calmodulin (CaM) and Ca2+ (Barroso et al., 1999;Corpas et al., 2004). Furthermore, peroxisomal NO, together withother related reactive nitrogen species (RNS) such as peroxynitrite,has been shown to participate in the response to abiotic stresses suchas salinity (Corpas et al., 2009) and heavy metals such as cadmiumor lead (Corpas and Barroso, 2014a, 2016). In addition, it has beendemonstrated that the protein responsible for generating NO appearsto be imported by a type 2 peroxisomal-targeting signal (PTS2) in aprocess that depends on the cytosolic receptor PEX7, and CaM andCa2+ (Corpas and Barroso, 2014b).

With the aim of gaining a deeper understanding of cross-talkbetween Ca2+ and the peroxisomal NO metabolism, apharmacological study was carried out using different NO donors,glutathione (GSH) and a CaM antagonist in Arabidopsis thalianaseedlings. The data show that Ca2+ is present in the peroxisomes ofArabidopsis stomata and root cells, and its presence is drasticallydisrupted by a CaM antagonist, which also blocks protein importinto peroxisomes. Furthermore, the activity of typical peroxisomalenzymes that containing a type 1 PTS motif (PTS1), such ascatalase, glycolate oxidase and hydroxypyruvate reductase, wasReceived 10 January 2017; Accepted 3 February 2017

1Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food andAgriculture, Department of Biochemistry, Cell and Molecular Biology of Plants,Estacion Experimental del Zaidın, CSIC, C/Profesor Albareda 1, Granada E-18008,Spain. 2Group of Biochemistry and Cell Signaling in Nitric Oxide, Department ofBiochemistry and Molecular Biology, Campus ‘Las Lagunillas’, University of Jaen,Jaen E-23071, Spain.

*Author for correspondence ( javier.corpas@eez.csic.es)

F.J.C., 0000-0002-1814-9212

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negatively affected by the CaM antagonist, suggesting that CaMmust also be necessary for plant peroxisome functionality.

RESULTSLocation of Ca2+ in the peroxisomes of roots and guard cellsas analysed by CLSMUsing Arabidopsis seedlings expressing CFP containing a PTS1motif (CFP–PTS1), the potential endogenous presence of Ca2+ inperoxisomes was studied by using the fluorescence probe Fluo-3AM. Although, theoretically, there is no overlap between theexcitation and emission wavelengths of CFP with the specificfluorescent probe used to detect Ca2+ (Fluo-3 AM), potentialoverlap was evaluated at the experimental level. Fig. S1A,D showsin vivo confocal laser-scanning microscopy (CLSM) visualizationof peroxisomes in the guard cells and root tips of transgenicArabidopsis seedlings expressing CFP–PTS1 (excitation maximumat 458 nm and emission maximum at 475 nm). The peroxisomesappeared in the form of spherical spots in the cells. Fig. S1B,E,shows the same field observed by CLSM using an excitationwavelength of 514 nm and an emission wavelength of 610 nm but inthe absence of the fluorescent probe (Fluo-3 AM) used to detect Ca2+

in which it is not possible to detect any fluorescence signal.Fig. 1 shows in vivo CLSM visualization of peroxisomes, Ca2+

and chloroplasts in the guard and root cells of transgenicArabidopsis seedlings expressing CFP–PTS1. Peroxisomes

appeared in the form of spherical spots of a green color in guardand root cells (Fig. 1A,F, respectively). On the other hand, Fig. 1B,G show the same fields analyzed using Fluo-3 AM as fluorescenceprobe, which also enabled Ca2+ (red color) to be detected. The redfluorescence appeared in both types of spherical spots, with apattern similar to that of CFP–PTS1, and also throughout thecells although with less intensity. Fig. 1C shows chlorophyllautofluorescence (purple color), enabling us to detect thelocalization of chloroplasts as spherical spots in guard cells;however, these were larger and in a different position in the cellsfrom the spots corresponding to peroxisomes (Fig. 1A). Fig. 1D,Hcontains merged images of the overlap of the corresponding panels,showing a complete overlap of the punctate patterns of peroxisomeswith the Ca2+ signals; in addition, the presence of the red color waslocalized throughout the cytosol, as well as being observed in otherparts both inside and between the cells, for example, in the cell wallof roots cells (Fig. 1G) and colocalized with chloroplasts (seeFig. 2B,C). Fig. 1E,I shows the bright field of the guard and rootcells, respectively. Apparently, peroxisomes have a high Ca2+

content in relation to rest of the cells, and this seems to be logicalconsidering that the concentration of cytosolic Ca2+ usually is lower(around 100 nM) in comparison with the non-cytoplamisc Ca2+,which has a millimolar concentration (Monshausen et al., 2008;Stael et al., 2012). However, the Ca2+ signal has a wide distribution,such that it could be observed in images taken at a lower

Fig. 1. In vivo detection of Ca2+, peroxisomes and chloroplasts inArabidopsis guard and root cells.Representative CLSM images of (A–E) guard cells and(F–I) root cells of 5-day-old transgenic Arabidopsis seedlings expressing CFP–PTS1. Ca2+ is shown in red, peroxisomes in green and chloroplasts in purple.(A,F) Punctuate structures (green) visualized through CFP–PTS1 fluorescence, indicating the localization of peroxisomes. (B,G) Fluorescence (red) attributableto the detection of Ca2+ in the same cell area. (C) Chlorophyll autofluorescence (purple) demonstrating location of chloroplasts. (D,H) Merged images ofA–C and F–H, respectively. (E,I) Bright-field image of guard and root cells. Ca2+ (red color) was detected by using 5 µMFluo-3 AM. Arrows indicate representativepunctuate spots corresponding to the colocalization of Ca2+ with peroxisomes. Asterisks indicate localization of Ca2+ in the cytosol or cell wall.

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magnification. For example, Fig. S2 shows root cells taken at alower magnification, illustrating the general distribution of Ca2+

signal using Fluo-3 AM as fluorescence probe.

Exogenous application of NO, GSH and CaM antagonist –effect on peroxisomal Ca2+ and import of CFP–PTS1 intoperoxisomesPrevious data have demonstrated that peroxisomal NO generationdepends on Ca2+ (Corpas et al., 2009) and that the import of theperoxisomal NOS-like protein responsible for this NO generationalso depends on Ca2+ and CaM (Corpas and Barroso, 2014b). Withthe aim of gaining a greater insight into the relationship betweenperoxisomal NO and Ca2+ metabolism, a pharmacological study wascarried out using different NO donors (GSNO and DEA NONOate)and the CaM antagonist trifluoperazine (TFP). These analyses were

performed in both the guard and root cells of transgenic Arabidopsisseedlings expressing CFP–PTS1 (Figs 2 and 3, respectively).

Fig. 2A–E corresponds to the untreated Arabidopsis seedlings inwhich it can be observed that Ca2+ clearly colocalized withperoxisomes and chloroplasts (Fig. 2D). Fig. 2F–J shows the effectof using S-nitrosoglutathione (GSNO) as anNO donor. It can be seenthat thismolecule significantlyaffects the import ofCFP–PTS1, sincethe green signal appears to diffuse to all the guard cells,with onlyveryfew green punctuate spots being observed (Fig. 2F), and also leads toa sharp reduction in Ca2+ content (Fig. 2G). However, thechloroplasts do not appear to be affected (Fig. 2H). Figs 2I,J showsthe merged image of the corresponding panels and the bright field,respectively. To corroborate this potential effect, another NO donor,DEA NONOate, was used. In this case, a smaller number of greenspots corresponding to CFP–PTS1 content was observed (Fig. 2K) in

Fig. 2. In vivo detection of Ca2+, peroxisomes and chloroplasts inArabidopsis guard cells upon pre-incubationwith different chemicals.RepresentativeCLSM images of guard cells of 5-day-old transgenic Arabidopsis seedlings expressing CFP–PTS1 pre-incubated with different chemicals visualized as in Fig. 1.Ca2+ is shown in red, peroxisomes in green and chloroplasts in purple. Control plants (A–E) and plants treated with (F–J) 2 mM S-nitrosoglutathione (GSNO) asan NO donor, (K–O) 2 mM DEA NoNoate as an NO donor, (P–T) 2 mM reduced glutathione (GSH) or (U–Y) 300 µM of the CaM antagonist TFP, are shown.Arrows indicate punctuate spots corresponding to the colocalization of Ca2+ with peroxisomes and angles (^) indicate colocalization of Ca2+ with chloroplasts.

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comparison to the untreated sample (Fig. 2A); in addition, the redsignal corresponding to the Ca2+ was diffused to the guard cells butshowed an accumulation in the innerwalls of the guard cells (Fig. 2L).In this regard, itmust bementioned thatNOcan have awide spectrumof actions because it can directly or indirectly modulate post-translational modifications affecting the function of the targetmolecules (Frungillo et al., 2014; Yun et al., 2016; Begara-Moraleset al., 2014, 2015, 2016; Mata-Pérez et al., 2016).Taking in consideration that GSNO releases both NO and

glutathione (GSH), the putative effect of GSH was also assessed asan internal control. We found that the pre-treatment of seedlingswith 2 mM GSH prevents the import of CFP–PTS1 since the greenfluorescence signal appears to diffuse through the guard cells(Fig. 2P); the red color corresponding to the Ca2+ signal was alsodiffused to the guard cells but with a strong accumulation in theinner walls of the guard cells (Fig. 2Q). On the other hand, Ca2+ inthe chloroplasts seems to be less affected (Fig. 2R,S). Fig. 2T shows

the corresponding bright field image. CaM is a Ca2+-binding proteininvolved in the signaling process. The chemical TFP, a CaMantagonist (Vandonselaar et al., 1994; Sengupta et al., 2007), wastherefore used to evaluate its possible effect on both peroxisomalprotein import and Ca2+. Fig. 2U shows that pretreatment with TFPsignificantly affects the import of CFP–PTS1 since the greenfluorescence signal appears to diffuse to all the guard cells. On theother hand, Fig. 2V shows that it also lowers the Ca2+ signal (redcolor), with very few red punctuate signals being observed, whichdo not seem to correspond to peroxisomes. Ca2+ signal wasobserved in the chloroplasts (Fig. 2W). Fig. 2X,Y show the mergedimage of the corresponding panels and the bright field, respectively.Thus, these results collectively suggest that NO also affects bothperoxisomal protein import and endogenous Ca2+, although theeffect is less strong than that seen with GSH and TFP.

Fig. 3 illustrates an analysis of root cell similar to that carried outon guard cells. Fig. 3A,B shows peroxisomes and Ca2+ detected in

Fig. 3. In vivo detection of Ca2+ andperoxisomes in Arabidopsis root tipcells upon pre-incubation withdifferent chemicals. RepresentativeCLSM images of root tip cells of 5-day-old transgenic Arabidopsis seedlingsexpressing CFP–PTS1 pre-incubatedwith different chemicals, visualized asin Fig. 1. Ca2+ is shown in red andperoxisomes in green. Control plants(A–D) and plants treated with (E–H)2 mM S-nitrosoglutathione (GSNO) asan NO donor, (I–L) 2 mM DEANoNoate as an NO donor, (M–P) 2 mMreduced glutathione (GSH) or (Q–T)300 µM of the calmodulin antagonistTFP, are shown. Arrows indicaterepresentative punctuate spotscorresponding to the colocalization ofCa2+ with peroxisomes.

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untreated Arabidopsis roots, in which it can be observed that Ca2+

clearly colocalizes with peroxisomes (Fig. 3C,D). Fig. 3E–H showsthe impact of GSNO. Thus, it can be observed that this molecule didnot affect the import of CFP–PTS1, as peroxisomes appear in theform of green spherical spots (Fig. 3E); however, the red signal(corresponding to Ca2+) appears to diffuse through the cells and alsoappears in a smaller number of red spherical spots (Fig. 3F).Fig. 3G,H shows the merged image of the corresponding panels andthe bright field, respectively. To corroborate this potential effect,another NO donor, DEANONOate, was used. In this case, the greencolor was also observed to appear in green spherical spots and alsowith some diffuse green signals (Fig. 3I). However, the redfluorescence signal corresponding to the Ca2+ signal appears to beonly very weakly present in spherical spots (Fig. 3J). Fig. 3K,Lshows the merged image of the corresponding panels and the brightfield, respectively. These effects of NO donors (GSNO and DEANONOate) in roots are quite different to that observed in guard cells(Fig. 2F,K). This could be due to the fact that guard cells arespecialized cells in the epidermis of leaves involved in stomatamovement, and its regulation there is dependent on a complexinterplay between NO, Ca2+, H2O2, abscisic acid (ABA), salicylicacid and Ca2+-dependent protein kinases (Desikan et al., 2004; Liet al., 2009; Zhang et al., 2009; Hao et al., 2010; Wang et al., 2014;Murata et al., 2015), where peroxisomes seem also to be involved(Leterrier et al., 2016). Therefore, we hypothesize that theapplication of exogenous NO could alter this equilibrium, withthe guard cells more affected that the root cells.As performed above, GSH was used as an internal control for

GSNO. We found that the pre-treatment of seedlings with 2 mMGSH avoids the import of CFP–PTS1 since the green signal appearsto diffuse through the cells, especially in the cells of the root cap(Fig. 3M); the red color corresponding to the Ca2+ signal was alsodiffused through the cells in the elongation area of the primary root(Fig. 3N). Fig. 3O,P shows the merged image of the correspondingpanels and the bright field, respectively. Fig. 3Q shows thatpretreatment with TFP (CaM antagonist) drastically affects theimport of CFP–PTS1, with the green color appearing to diffuse inthe cells of the root cap. On the other hand, Fig. 3R shows a veryweak red signal, indicating that the CaM antagonist verysignificantly affects Ca2+ content in root cells. Fig. 3S,T showsthe merged image of the corresponding panels and the bright field,respectively. Fig. 4 shows the change in the fluorescence intensity ofCa2+ (red color) in guard and root cells of Arabidopsis seedlingspretreated with GSNO, DEA NONOate, GSH and TFP whichcorresponds to results shown in Figs 2 and 3. In this sense, it canbe seen that GSH is apparently the chemical that had a strongereffect in Ca2+ content and distribution in guard and root cells.Although the chemical TFP acts as a CaM antagonist

(Vandonselaar et al., 1994; Sengupta et al., 2007), it might alsoinduce non-specific effects that could not be directly related toCa2+/CaM metabolism, such as inhibition of electron transport andphosphorylation in plant mitochondria (Dunn et al., 1984), inhibitionof Ca2+ ATPase, proton excretion or membrane properties (Lichtmanet al., 1982; Barr et al., 1990; Wilson, 1994; Sengupta et al., 2007).Consequently, to affirm the effect of TFP as a CaM antagonist in ourexperimental Arabidopsis model, another CaM antagonist was used,in particular N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide(W7), which has been previously described to have a similar effect toTFP in animal and plant cells (Sengupta et al., 2007; Ma et al., 2008).Fig. S3 illustrates the CLSM in vivo detection of Ca2+ (red signal) andperoxisomes (green signal) in root tip cells of 5-day-old transgenicArabidopsis seedlings expressing CFP–PTS1 pre-incubated with

either of the CaM antagonists TFP (Fig. S3D–F) orW7 (Fig. S3G–I).In both cases and, in comparison with the untreated seedlings(Fig. S3A,B), the green signal appears to be diffuse in the cells of theroot cap with a very weak red signal, indicating that both CaMantagonists caused identical effects. On the other hand, pre-incubation with the chemical W5 (Fig. S3J– L), which is aninactive form of W7, shows that Ca2+ clearly colocalizes withperoxisomes, similar to the localization observed in untreatedseedlings (Fig. S3A–C).

Additionally, to evaluate whether the CaM antagonist TFP couldaffect the cell viability, Arabidopsis seedlings were stained withpropidium iodide (PI). This chemical is a cationic dye that does notreadily cross intact membranes but that can penetrate throughout themeristem and bind to cell walls, thereby showing an outline of livingcells; PI binds to nuclear structures, causing bright punctatefluorescence. Fig. S4 shows the PI staining of root cells of 5-day-old transgenic Arabidopsis seedling expressing CFP–PTS1 thatwere pre-incubated with TFP. It can be observed that the CaMantagonist (Fig. S4D–F) does not affect the cell viability whencompared to untreated seedlings (Fig. S4A–D).

Given that the CaM antagonist TFP is the chemical with the mostdrastic effect on both peroxisomal protein import and Ca2+ content,we also performed in vitro assays with this CaM antagonist tomeasure the activity of representative peroxisomal enzymes,including catalase, glycolate oxidase and hydroxypyruvatereductase, using 14-day-old Arabidopsis seedlings pre-incubatedwith 300 µM TFP for 2 h at 25°C in darkness. Fig. 5A–C shows thatthese three activities were considerably affected, with a reductionof 45% for catalase, 41% for glycolate oxidase and 51% forhydroxypyruvate reductase. For the purposes of characterization,we also determined whether the CaM antagonist is capable ofmodifying the gene expression of these peroxisomal enzymes. TheArabidopsis genome contains three catalase genes (CAT1,CAT2 andCAT3) (Du et al., 2008), whose gene expression increased ∼2.0-,1.4- and 1.5-fold, respectively, following pretreatment ofArabidopsis seedlings with TFP for 2 h (Fig. 5D). The CaMantagonist also causes an induction of HPR1 expression of ∼2.8-fold. Finally, glycolate oxidase is encoded by five genes, three ofwhich, GOX1, GOX2 and GOX3, were selected for analysis in thisstudy. While GOX1 and GOX3 expression seems to be unaffected,

Fig. 4. Quantification of the relative amount of Ca2+ in guard and root cellsof upon treatment with different chemicals. The fluorescence intensity ofthe red Fluo-3 AM signal (detecting Ca2+) in guard and root cells of 5-day-oldtransgenic Arabidopsis seedlings expressing CFP–PTS1 pre-incubatedwith different chemicals [2 mM GSNO or 2 mM DEA NoNoate (NO donors);2 mM reduced glutathione (GSH); 300 µM TFP] as shown in the images inFigs 2 and 3. The fluorescence is expressed as mean±s.e.m. [arbitrary units(A.U.)], as determined using Image J software. *P<0.05 compared with thecorresponding control for guard or root cells.

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GOX2 expression increased 1.6-fold after pretreatment with theCaM antagonist (Fig. 5E).

DISCUSSIONCa2+ is a highly important second messenger whose presence indifferent plant subcellular compartments, including the cytosol,chloroplasts, mitochondria, nucleus and vacuoles, has been wellstudied (Raychaudhury et al., 2006; Monshausen et al., 2008; Doddet al., 2010; Kudla et al., 2010; Behera et al., 2015; Xu et al., 2015).However, information concerning Ca2+ in peroxisomes, particularlyin plants, is scarce. In a study using human HeLa cells transientlyexpressing a peroxisome-targeted the GFP-based Ca2+ indicatorD3cpv-SKL, the presence of Ca2+ in animal peroxisomeswas shownfor the first time, with the peroxisomal membrane representing asignificant barrier to Ca2+ diffusion into peroxisomes (Drago et al.,2008). In the same year, Lasorsa et al. (2008) performed additionalexperiments showing that peroxisomes contain between 20- and 50-fold more Ca2+ than the cytosol (reaching a maximum of 100 µM).To our knowledge, only one study in the literature analyses Ca2+

homeostasis in plant peroxisomes; this study used the GFP-basedCameleon Ca2+ indicator D3cpv-KVK-SKL and demonstrated thatperoxisomal Ca2+ affected catalase activity (Costa et al., 2010). Thepresent study therefore aimed to gain a deeper understanding of thesignificance of Ca2+ in the peroxisomal metabolism of plants.

CaM is necessary for normal peroxisomal Ca2+ homeostasisand for peroxisomal protein importCaM is a protein of ∼17 kDa that is expressed in all eukaryotic cellsand has the capacity to bind Ca2+ (Bouché et al., 2005). This protein,

together with CaM-like (CML) proteins, is the primary Ca2+ sensorthat controls the activity of various target proteins and consequentlydiverse cellular functions, including development processes andresponses to adverse biotic and abiotic conditions (Yang andPoovaiah, 2003; Bouché et al., 2005; Reddy et al., 2011; Chevalet al., 2013; Zhao et al., 2015; Zeng et al., 2015). For instance, in theArabidopsis genome, seven distinct CaM and 50 CMLs genes havebeen predicted (McCormack et al., 2005). Recently, Arabidopsisthaliana (At)CML3 (also known as AGD11) has been demonstratedto be targeted to peroxisomes (Chigri et al., 2012), where itappears to mediate the dimerization of peroxisomal processingprotease AtDEG15 (Dolze et al., 2013). Moreover, there is anotherfamily of proteins which act as Ca2+ sensors called Ca2+-dependent protein kinases (CDPKs) (Boudsocq and Sheen, 2013)and in Arabidopsis the isoform AtCPK1 is targeted to peroxisomes(Dammann et al., 2003) and seems to mediate pathogen resistance(Coca and San Segundo, 2010).

Using an alternative fluorescent probe to detect Ca2+, the presentstudy corroborates the presence of Ca2+ in the peroxisomes of twodifferent tissues and cells – guard cells in green cotyledons and rootcells – suggesting that Ca2+ must be necessary for the normalphysiological functioning of plant peroxisomes, as has been shownfor other subcellular compartments. Moreover, pharmacologicaltechniques using different chemicals have enabled us to demonstratethat peroxisomal Ca2+ must be regulated by CaM and GSH and also,though less intensely, by NO. Thus, the CaM antagonist TFP blocksperoxisomal function due to its negative effect on peroxisomalprotein import, as shown by CFP–PTS1 remaining in the cytosol.Additionally, the presence of Ca2+ in peroxisomes suggests that a

Fig. 5. Activity and gene expression ofperoxisomal enzymes in response to a CaMantagonist. (A–C) Peroxisomal enzymatic activityassays for (A) catalase, (B) glycolate oxidase (GOX)activity and (C) hydroxypyruvate reductase (HPR)activity. (D,E) Semi-quantitative RT-PCR analysesfor (D) CAT gene expression, and (E) HPR and GOXgene expression. Semi-quantitative RT-PCR wasperformed on total RNA isolated using ACT2 asinternal control. Representative agaroseelectrophoresis gels of the amplification productswere visualized by GelRed™ staining under UV light.T/C indicates the relative level of the eachamplification product (T) over the ACT2 internalcontrol (C) after normalization to the control samples,and it expresses the fold change in with respect to theuntreated control. In all panels, 14-day-oldArabidopsis seedlings were exposed to 300 µM TFPfor 2 h at 25°C in darkness. Results are the mean±s.e.m. of three different experiments. *P<0.05 inrelation to control values.

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CaM or CML protein is strictly necessary for the peroxisomalhomeostasis of Ca2+, although, to our knowledge there is noexperimental information about the potential effect of a CaMantagonist on the peroxisomal AtCML3 function. In any case, this isa reasonable assumption, since the function of CaM is to transportand regulate Ca2+ levels. Thus, these data are closely in line withprevious findings that show that Ca2+ is required for peroxisomalNO production (Corpas et al., 2009) and that enzyme protein importresponsible for its generation is blocked by CaM antagonist (Corpasand Barroso, 2014b).Hitherto, there is clear evidence to support the cross-talk between

NO and Ca2+/CaM in plant cells (Sang et al., 2008; Ma et al., 2012).However, in the present study, we find that NO appears to be lessinvolved in controlling peroxisomal Ca2+ content; by contrast,peroxisomal Ca2+ is necessary and indispensable for the productionof NO in peroxisomes. It has been demonstrated that the activity ofperoxisomal protein L-arginine-dependent nitric oxide synthase(NOS) responsible for NO generation requires Ca2+ (Barroso et al.,1999; Corpas et al., 2004). More recently, it has been reported thatthe import of this protein into the peroxisome also depends on Ca2+

and CaM (Corpas et al., 2009; Corpas and Barroso, 2014b).Reduced glutathione (GSH) constitutes an important antioxidant,

and it is present in plant peroxisomes (Zechmann and Müller, 2010)where it is indispensable for the functioning of glutathionereductase (GR), a component of the ascorbate–glutathione cyclethat functions in these organelles (Romero-Puertas et al., 2006;Reumann and Corpas, 2010). Under our experimental conditions,GSH was used as control during the analysis of the GSNOtreatment; however, our findings were surprising as GSH, whichcaused a general delocalization of the Ca2+ signal and a markedintensification throughout the cells, drastically affected bothperoxisomal protein import and the peroxisomal localization ofCa2+. This suggests that a change in the redox state or a possibleS-glutathionylation process could be involved in the mechanism ofperoxisomal protein import; the latter is regulated by a large numberof cytosolic and peroxisomal proteins, known as peroxins (PEXs),which are encoded by at least 22 PEX genes in Arabidopsis (Nitoet al., 2007; Brown and Baker, 2008; Hu et al., 2012).

Ca2+ deficiency negatively affects the activity of catalaseand photorespiratory enzymes but increases their geneexpressionAs part of the potential physiological functionality of Ca2+, theCaM antagonist TFP was observed to negatively affect the activityof the antioxidant catalase enzyme, as well as the glycolate oxidaseand hydroxypyruvate reductase enzymes, which are both involvedin photorespiration (Rojas et al., 2012). The decrease in theiractivity could be due to the blocking of the peroxisomal import ofthe corresponding proteins containing a PTS1; this is closely in linewith the results obtained, since the CaM antagonist TFP blocks theimport of CFP–PTS1. However, a previous study has demonstratedthat the activity of plant catalase is stimulated by both Ca2+ andCaM (Yang and Poovaiah, 2002; Afiyanti and Chen, 2014).Moreover, with the aid of a recombinant AtCat3 in a CaM-bindingassay, the same authors demonstrated that CaM specifically bindsto AtCat3 in the presence of Ca2+; protein sequence analysis ofCat3 specifically enabled them to identify a CaM-binding region of37 amino acids between Gly415 and Val451 (Yang and Poovaiah,2002). More recently, an increase in intraperoxisomal Ca2+, whichoccurs in response to adverse conditions accompanied by oxidativestress, has been shown to stimulate catalase activity (Costa et al.,2010). However, to our knowledge, there is no information

available on any potential relationship between CaM/Ca2+ andglycolate oxidase and hydroxypyruvate reductase activity.Nevertheless, the results show that their activity was alsoadversely affected, with the blocking of their import into theperoxisomes being the most plausible cause. Thus, the geneexpression of these enzyme groups either increased (CAT1 toCAT3, HPR1 and GOX2) or appeared to be unaffected (GOX1 andGOX3), which could be a way of compensating for the loss of theseenzyme activities.

In summary, our findings indicate that the presence of Ca2+ isnecessary for the physiological functioning of peroxisomes in rootand guard cells, since its absence negatively affects the activity ofkey enzymes as well as the generation of NO, as has previously beendemonstrated (Corpas et al., 2009). Moreover, we suggest thatCa2+/CaM is also necessary for the import of peroxisomal proteinscontaining a PTS1. However, a drastic alteration in the redox stateand/or S-glutathionylation of peroxisomal protein import ofperoxins could be involved. In this latter case, further analyseswould be necessary to determine which peroxins are affected by thisredox regulation.

MATERIALS AND METHODSPlant material and growth conditionsArabidopsis thaliana transgenic seeds expressing cyan fluorescent protein(CFP) containing a type 1 peroxisomal-targeting signal (PTS1) (Nelsonet al., 2007) were grown on Petri plates for 5 days or 14 days under a cycle of16 h light (under a light intensity of 100 µE m−2 s−1) at 22°C and 8 h dark at18°C as previously described (Corpas and Barroso, 2014a). For microscopeanalyses, 5-day-old seedlings were grown on vertically positioned plates.For enzymatic and gene expression assays, 14-day-old seedlings were grownon horizontally positioned plates.

Detection of Ca2+ in transgenic plants expressing CFP–PTS1 byCLSMCa2+ was detected with 5 µM Fluo-3 AM (Biotium) prepared in 10 mMTris-HCl (pH 7.4) (Zhang et al., 1998; Kanchiswamy et al., 2010, 2014).The Arabidopsis seedlings were incubated in darkness with 5 µM Fluo-3AM at 25°C for 1 h. Each sample was then washed twice in the same bufferfor 15 min and mounted on a microscope slide for examination with aCLSM (Leica TCS SP5 II). For Fluo-3 AM, the excitation laser wavelengthwas 514 nm, with an emission of 610 nm and a 40-nm band pass width(590–630 nm). For CFP, the excitation laser wavelength was 458 nm, withan emission of 475 nm and a 40-nm band pass width (455–495 nm).Chlorophyll autofluorescence was studied as follows: chlorophyll a and b,excitation of 429 and 450 nm, respectively; emission of 650 and 670 nm,respectively.

Pharmacological assays of peroxisomal Ca2+

Arabidopsis seedlings (5 days old) expressing CFP–PTS1 were pre-incubated for 120 min at 25°C in darkness with different chemicalsprepared in 10 mM Tris-HCl (pH 7.4) including 2 mM diethylamineNONOate (DEA NoNoate) and 2 mM S-nitrosoglutathione (GSNO), whichare both NO donors (Broniowska et al., 2013; Begara-Morales et al., 2014),and 2 mM glutathione (GSH) and 300 µM trifluoperazine (TFP, acalmodulin antagonist) (Vandonselaar et al., 1994). Additionally,seedlings were also pre-incubated with 200 µM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), which is another CaM antagonist(Ma et al., 2008), and 200 µMN-(6-aminohexyl)-1-naphthalenesulfonamide(W5), which is the inactive W7 structural analog (Li et al., 2004). Theseedlings were then incubated with 5 µM Fluo-3 AM for 1 h at 25°C indarkness to detect and visualize Ca2+ using CLSM. In all cases, the imagesobtained by CLSM from control and treatedArabidopsis seedlings were keptconstant during the course of the experiment in order to produce comparabledata. For each treatment, at least three independent experiments were madeusing, at least, four or five seedlings in each experiment.

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Cell wall integrityPlant cell integrity was evaluated with propidium iodide (PI) staining (Truernitand Haseloff, 2008). Arabidopsis seedlings were incubated for 5 min at roomtemperature in a 10 μg/ml PI solution (P4170 Sigma) in water. For PI, theexcitation laser wavelength was 536 nm, with an emission of 617 nm and a 40-nm band pass width (597–637 nm).

Crude extracts of Arabidopsis seedlingsCrude extracts of 14-day-old Arabidopsis seedlings were frozen in liquid N2

and ground in a mortar with a pestle. The powder was suspended in ahomogenizing medium containing 50 mM Tris-HCl (pH 7.8, ratio 1:3; w/v)containing 0.1 mMEDTA, 0.2% (v/v) Triton X-100 and 10% (v/v) glycerol.Homogenates were centrifuged at 27,000 g for 20 min, and the supernatantswere used for the enzymatic assays.

Enzyme activity assayCatalase activity (EC 1.11.1.6) was assayed by measuring thedisappearance of H2O2 as described by Aebi (1984). NADH-dependenthydroxypyruvate reductase (HPR; EC 1.1.1.29) was determined accordingto the method used by Schwitzguébel and Siegenthaler (1984). Glycolateoxidase (GOX; EC 1.1.3.1) was assayed as previously described (Kerr andGroves, 1975), by measuring the formation of glyoxylate-phenylhydrazone.

RNA isolation and semiquantitative RT-PCRTotal RNAwas extracted with Trizol according to instructions provided byGibco BRL (Life Technologies); 5 μg of total RNA were used to producecDNA for the reverse transcriptase (RT) reaction by adding 0.5 mM dNTPs,poly-dT23, 5x RT buffer, 40 U RNase inhibitor (Invitrogen) and 200 UReverse Transcriptase (Thermo Fisher) in a final volume of 20 μl. Thereaction was carried out at 50°C for 30 min.

Semiquantitative RT-PCR amplification of actin 2 (ACT2) cDNA fromArabidopsis was chosen as control. CAT1, CAT2, CAT3, HPR1, GOX1,GOX2 and GOX3 cDNAs were amplified by a PCR as previouslydescribed (Corpas and Barroso, 2016). PCR products were then detectedafter electrophoresis in 2.8% (w/v) agarose gels and by staining withGelRed™. Quantification of the bands was performed using a Gel Docsystem (Bio-Rad Laboratories) coupled with a high-sensitivity charge-coupled device (CCD) camera. The T/C ratio indicates the relative levelof the each amplification product (T) over the ACT2 used as internalcontrol (C) after normalization to the control samples, and it expressesthe fold change with respect to the untreated control (Marone et al.,2001).

Other assaysProtein concentration was determined with the aid of the Bio-Rad ProteinAssay (Hercules, CA) using bovine serum albumin as a standard. Formicroscope analyses, around five to ten 5-day-old Arabidopsis seedlingswere used for each experiment and treatment. For enzymatic activity andgene expression assays, ∼150 14-day-old Arabidopsis seedlings were usedfor each experiment. Data are means of at least three sets of independentexperiments with three repetitions each. To estimate the statisticalsignificance between means, the data was analyzed by the Student’s test.Relative fluorescence was quantified by using ImageJ software.

AcknowledgementsAuthors are grateful to Mr. Carmelo Ruiz-Torres for his excellent technical support.The authors thank Dr Juan D Alche (CSIC) for his advice on the analysis of images.Technical and human support provided by CICT of Universidad de Jaen (UJA,MINECO, Junta de Andalucıa, FEDER) is also acknowledged. We also thankMichael O’Shea for proofreading the text.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: F.J.C; Methodology: F.J.C., J.B.B.; Formal analysis andinvestigation: F.J.C., J.B.B.; Writing - original draft preparation: F.J.C.; Writing -review and editing: F.J.C., J.B.B.; Funding acquisition: F.J.C., J.B.B.

FundingResearch in F.J.C. and J.B.B. laboratory is supported by a European RegionalDevelopment Fund (ERDF) grant co-financed by the Ministerio de Economıa yCompetitividad (projects AGL2015-65104-P and BIO2015-66390-P, respectively),and Consejerıa de Economıa, Innovacion, Ciencia y Empleo, Junta de Andalucıa(groups BIO192 and BIO286) in Spain.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.201467.supplemental

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RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs201467. doi:10.1242/jcs.201467

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