© 2017. Published by The Company of Biologists Ltd.

Calmodulin (CaM) antagonist affects peroxisomal functionality by

disrupting both peroxisomal Ca2+

and protein import

Francisco J Corpasa,1

, Juan B. Barrosob

aGroup of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and

Agriculture, Department of Biochemistry, Cell and Molecular Biology of Plants, Estación

Experimental del Zaidín, CSIC, C/ Profesor Albareda 1, E-18008 Granada, Spain bGroup of Biochemistry and Cell Signaling in Nitric Oxide. Department of Biochemistry and

Molecular Biology, Campus "Las Lagunillas", E-23071, University of Jaén, Jaén, Spain.

1Corresponding author: javier.corpas@eez.csic.es

Dr. Francisco J Corpas

Department of Biochemistry, Cell and Molecular Biology of Plants

Estación Experimental del Zaidín (CSIC)

C/ Profesor Albareda, 1,

E-18008 Granada, SPAIN

SPAIN

[Fax: 34 958 129600; E-mail: javier.corpas@eez.csic.es]

Keywords Calcium; calmodulin; catalase; glutathione; nitric oxide; peroxisomes

Summary statement

Ca2+

is indispensable for the physiological functioning of peroxisomes in roots and leaves,

since its absence negatively affects the activity of key enzymes as well as the nitric oxide

generation.

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JCS Advance Online Article. Posted on 9 February 2017

Abstract

Calcium (Ca2+

) is a second messenger in many physiological and phyto-pathological

processes. Peroxisomes are subcellular compartments with an active oxidative and nitrosative

metabolism. Previous studies have demonstrated that peroxisomal nitric oxide (NO)

generation is dependent on calcium and calmodulin (CaM). We used Arabidopsis thaliana

transgenic seedlings expressing cyan fluorescent protein (CFP) through the addition of

peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo,

and also used a cell-permeable fluorescent probe for Ca2+

. Analysis by confocal laser

scanning microscopy (CLSM) enabled us to visualize the presence of endogenous Ca2+

in the

peroxisomes of both roots and guard cells. The presence of calcium in peroxisomes and the

import of CFP-PTS1 are drastically disrupted by both CaM antagonist and glutathione

(GSH). Furthermore, the activity of three peroxisomal enzymes (catalase, glycolate oxidase

and hydroxypyruvate reductase) containing PTS1 was clearly affected, with a decrease of

between 41 and 51 %. In summary, data show that CaM/Ca2+

is strictly necessary for protein

import, normal functionality of peroxisomal enzymes, including antioxidant and

photorespiratory enzymes, as well as for NO production.

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Introduction

Calcium (Ca2+

) is a cellular second messenger whose homeostasis is highly regulated in cells

by different components including calcium-pumps, channels, cation exchangers and calcium-

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, cross-talk among calcium and other signalling molecules, including

H2O2 and NO, which affects gene expression and Ca-dependent protein kinase, has also been

described (Rentel and Knight, 2004; Gonzalez et al., 2012; Niu and Liao, 2016). Ca2+

signals

can remain local or be propagated throughout an entire cell within milliseconds to seconds

(Monshausen, 2012). Calcium has been detected in almost all cellular compartments

including the cytosol, chloroplasts, mitochondria and nucleus. But, it can be 10,000-fold

difference between cytoplasmic and non-cytoplasmic Ca2+

concentrations which allows the

formation of Ca2+

signals by rapid alterations of cytoplasmic Ca2+

levels via membrane-

localized Ca2+

permeable channels (Stael et al., 2012). However, as information regarding

calcium in peroxisomes is scarce, to our knowledge, only one study has claimed that it is

present in plants (Costa et al., 2010). Nevertheless, its potential function in peroxisomes has

been analysed, as calcium is necessary for key antioxidant peroxisomal enzymes, such as

catalase (Yang and Pooviah, 2002; Schmidt et al., 2006; Costa et al., 2013), and for

peroxisomal NO-generation (Corpas et al., 2009; Corpas and Barroso, 2014b).

Peroxisomes are single membrane-bound subcellular compartments present in almost

all types of eukaryotic cells. They contain catalase and H2O2-producing flavin oxidases as

indispensable enzymatic elements (Reumann et al., 2007; Palma et al., 2009; Corpas, 2015).

These organelles are characterized by metabolic flexibility, as their enzymatic content can

vary according to the organism (i.e. animal, plant or yeast), cell/tissue type (i.e. liver, root or

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 antioxidative enzymes, such as

catalase, superoxide dismutase, components of the ascorbate-glutathione cycle and several

NADP-dehydrogenases, 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 the

capacity to generate NO through a L-arginine-dependent nitric oxide synthase (NOS) activity

which strictly depends on NADPH and requires calmodulin (CaM) and Ca2+

(Barroso et al.,

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1999; Corpas et al., 2004). Furthermore, peroxisomal NO, together with other related reactive

nitrogen species (RNS) such as peroxynitrite, has been shown to participate in the response to

abiotic stresses such as salinity (Corpas et al., 2009) and heavy metals such as cadmium or

lead (Corpas and Barroso, 2014a, 2016). In addition, it has been demonstrated that the

protein responsible for generating NO appears to be imported by a peroxisomal targeting

signal type 2 (PTS-2) in a process that depends on the cytosolic receptor PEX7, CaM and

Ca2+

(Corpas and Barroso, 2014b).

With the aim of gaining a deeper understanding of cross-talk between Ca2 +

and the

peroxisomal NO metabolism, a pharmacological study was carried out using different NO

donors, glutathione (GSH) and a CaM antagonist in Arabidopsis seedlings. The data show

that Ca2+

is present in the peroxisomes of Arabidopsis stomata and root cells, and its presence

is drastically disrupted by a CaM antagonist which also blocks protein import into

peroxisomes. Furthermore, the activity of typical peroxisomal enzymes, containing a PTS1

such as catalase, glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), was

negatively affected by the CaM antagonist, suggesting that CaM must also be necessary for

plant peroxisome functionality.

Results

Location of Ca2+

in the peroxisomes of roots and guard cells analysed by CLSM

Using Arabidopsis seedlings expressing CFP-PTS1, the potential endogenous presence of

Ca2+

in peroxisomes was studied using the fluorescence probe, Fluo-3 AM. Although

theoretically, there is no overlap between the excitation/emission wavelengths of CFP with

the specific fluorescent probe used to detect calcium (Fluo-3 AM), potential overlap was

evaluated at the experimental level. Fig. S1, panels A and D, shows in vivo CLSM

visualization of peroxisomes in the guard cells and root tips of transgenic Arabidopsis

seedlings expressing CFP-PTS1 (excitation maximum at 458 nm and emission maximum at

475 nm). The peroxisomes appeared in the form of spherical spots in the cells. Fig. S1, panels

B and E, shows the same field observed by CLSM using an excitation wavelength of 514 nm

and an emission wavelength of 610 nm but in the 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 transgenic Arabidopsis seedlings expressing CFP-PTS1. Peroxisomes

appeared in the form of spherical spots of a green colour in guard and root cells (Fig. 1A and

1F, respectively). On the other hand, Fig. 1B and 1G show the same fields analyzed using

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Fluo-3 AM as fluorescence probe, which also enabled Ca2+

(red color) to be detected. The red

fluorescence appeared in both types of spherical spots, with a pattern similar to that of CFP-

PTS1, and also throughout the cells although with less intensity. Fig. 1C shows chlorophyll

autofluorescence (purple colour), enabling us to detect the localization of chloroplasts as

spherical spots in guard cells; however, these were larger and in a different position in the

cells from the spots corresponding to peroxisomes (Fig. 1A). Fig. 1D and 1H contain merged

images of the overlap of the corresponding panels, showing a complete overlap of the

punctate patterns of peroxisomes with the Ca2+

signals; in addition, the presence of the color

red was localized throughout the cytosol as well it was also observed in other parts both

inside and between the cells, for example in the cell wall of roots cells (Fig. 1G) and

colocalized with chloroplasts (see Fig. 2B and Fig. 2C). . Fig. 1E and 1I show the bright field

of the guard and root cells, respectively. Apparently, peroxisomes appear with a high content

of Ca2+

in relation to rest of the cells, and this seems to be logical considering that the

concentration of cytosolic calcium usually is lower (around 100 nM) in comparison with the

non-cytoplamisc Ca2+

which is in the range of mM (Monshausen et al., 2008; Stael et al et al.,

2012). However, Ca2+

signal has a wide distribution that it could be observed under pictures

with lower magnification. For example, Fig. S2 shows root cells with a lower 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 into peroxisomes.

Previous data have demonstrated that peroxisomal NO generation depends on Ca2+

(Corpas et

al., 2009) and that the import of the peroxisomal NOS-like protein responsible for this NO

generation also depends on Ca2+

and calmodulin (Corpas and Barroso, 2014b). With the aim

of gaining a greater insight into the relationship between peroxisomal NO and Ca2+

metabolism, a pharmacological study was carried out using different NO donors (GSNO and

DEA NONOate) and a CaM antagonist (TFP, trifluoperazine). These analyses were

performed in both the guard and root cells of transgenic Arabidopsis seedlings expressing

CFP-PTS1 (Figs. 2 and 3, respectively).

Fig. 2, panels A to E, corresponds to the untreated Arabidopsis seedlings in which it

can be observed that Ca2+

clearly co-localized with peroxisomes and chloroplasts (Fig. 2D).

Fig. 2, panels F to J, shows the effect of S-nitrosoglutathione (GSNO) used as NO donor.

Thus, this molecule can be observed to significantly affect the import of CFP-PTS1 since

the green signal appears to diffuse to all the guard cells, with only very few green

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punctuates being observed (Fig. 2F), and also leads to a sharp reduction in Ca2+

content

(Fig. 2G). However, the chloroplasts do not appear to be affected (Fig. 2H). Figures 2I and 2J

show the 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 green punctuates corresponding to CFP-PTS1 content was observed (Fig.

2K); also, the red signal corresponding to the Ca2+

was diffused to the guard cells but with an

accumulation in the inner walls of the guard cells (Fig. 2L). In this regard, it must be

mentioned that NO can have a wide spectrum of actions because it can mediate directly or

indirectly post-translational modifications affecting the function of the target molecules

(Frungillo et al. 2014; Yun et al 2016; Begara-Morales et 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 as an internal control. Thus, the pre-treatment of

seedlings with 2 mM GSH avoids the import of CFP-PTS1 since the green fluorescence

signal appears to diffuse through the guard cells (Fig. 2P); the red colour corresponding to

the Ca2+

signal was also diffused to the guard cells but with a strong accumulation in the

inner walls of the guard cells (Fig. 2Q). On the other hand, calcium in the chloroplasts seems

to be less affected (Fig. 2R and 2S). Fig. 2T shows the corresponding bright field.

Calmodulin is a calcium-binding protein involved in the signaling process. The chemical

trifluoperazine (TFP), a CaM antagonist (Vandonselaar et al., 1994; Sengupta et al. 2007),

was therefore used to evaluate its possible effect on both peroxisomal protein import and

calcium. Figure 2U shows that pretreatment with TFP significantly affects the import of CFP-

PTS1 since the green fluorescence signal appears to diffuse to all the guard cells. On the

other hand, Fig. 2V shows lower Ca2+

signal (red colour), with very few red punctuates being

observed which apparently do not look like peroxisomes. Calcium signal was observed in the

chloroplasts (Fig. 2W). Figures 2X and 2Y show the merged image of the corresponding

panels and the bright field, respectively. Thus, these results collectively suggest that NO also

affects both peroxisomal protein import and endogenous Ca2+

, though with less intensity than

GSH and TFP.

Fig. 3 illustrates an analysis of root cell, which is similar to that carried out on guard

cells. Figures 3A and 3B show peroxisomes and Ca2+

detected in untreated Arabidopsis roots,

in which it can be observed that Ca2+

clearly co-localizes with peroxisomes (Figs 3C and 3D).

Figs. 3E to 3H show the impact of GSNO. Thus, it can be observed that this molecule did not

affect the import of CFP-PTS1, as peroxisomes appear in the form of green spherical spots

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(Fig. 3E); however, the red signal (corresponding to calcium) appears to diffuse through the

cells and also in a smaller number of red spherical spots (Fig. 3F). Figs. 3G and 3H show the

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, the green

color was also observed to appear in green spherical spots and also with some diffuse green

signals (Fig. 3I). However, the red fluorescence signal corresponding to the Ca2+

signal

appears to be very weak in spherical spots (Fig. 3J). Figs 3K and 3L show the merged image

of the corresponding panels and the bright field, respectively. These effects of NO donors

(GSNO and DEA NONOate) in roots are quite different with that observed in guard cells

(Fig. 2F and 2K). This could be due to the fact that guard cells are specialized cells in the

epidermis of leaves involved in stomata movement and its regulation is under the control of a

complex interplay amongst NO, Ca2+

, H2O2, ABA, salicylic acid and calcium-dependent

protein kinases (Desikan et al. 2004; Li et 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, it could be hypothesized that the application of exogenous NO could alter

this equilibrium being the guard cells more affected that the root cells.

As done previously, GSH was used as an internal control of GSNO. Thus, the pre-

treatment of seedlings with 2 mM GSH avoids the import of CFP-PTS1 since the green

signal appears to diffuse through the cells, especially in the cells of the root cap (Fig. 3M);

the red colour corresponding to the Ca2+

signal was also diffused through the cells in the

primary root’s elongation area (Fig. 3N). Figures 3O and 3P show the merged image of the

corresponding panels and the bright field, respectively. Figure 3Q shows that pretreatment

with TFP (CaM antagonist) drastically affects the import of CFP-PTS1, with the green

colour appearing to diffuse in the cells of the root cap. On the other hand, Fig. 3R shows a

very weak red signal, indicating that the CaM antagonist very significantly affects Ca2+

content in root cells. Figs. 3S and 3T show the merged image of the corresponding panels and

the bright field, respectively. Fig. 4 shows the change in the fluorescence intensity of Ca2+

(red colour) in guard and root cells of Arabidopsis seedling pretreated with GSNO, DEA

NONOate, GSH and TFP which corresponds to the Fig. 2 and 3. In this sense, it can be

mentioned that GSH is apparently the chemical which had a stronger effect in Ca2+

content/distribution in guard and root cells.

Although the chemical trifluoperazine (TFP) has CaM antagonist effects

(Vandonselaar et al., 1994; Sengupta et al. 2007), there are also additional reactions which

can be or not directly related to calcium/calmodulin metabolism such as inhibition of electron

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transport and phosphorylation in plant mitochondria (Dunn et al, 1984), inhibition of calcium

ATPase, proton excretion or membrane properties (Barr et al. 1990; Wilson et al., 1994;

Sengupta et al. 2007). Consequently, to corroborate the effect of TFP as CaM antagonist in

our experimental Arabidopsis model, another CaM antagonist was used, in particular the N-

(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) which it has been previously

described to have similar effect to TFP 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) and

peroxisomes (green signal) in root tip cells of 5-day-old transgenic Arabidopsis seedlings

expressing CFP-PTS1 pre-incubated with both CaM antagonists, TFP (supplementary Fig. 3,

panels D to F) and W7 (Fig. S3, panels G to I). In both cases and in comparison with the

untreated seedlings (Fig. S3, panels A to B), the green signal appears diffuse in the cells of

the root cap with a very weak red signal, indicating that both CaM antagonists caused

identical effects. On the other, the pre-incubation with the chemical W5 (Fig. 3S, panels J to

L) which is an inactive form of W7 shows that Ca2+

clearly co-localizes with peroxisomes,

similar to the observed in untreated seedlings (supplemental Fig. 3, panels A to C).

Additionally, to evaluate if the CaM antagonist (TFP) could affect the cell viability,

Arabidopsis seedlings were stained with propidium iodide (PI). This chemical is a cationic

dye that does not readily cross intact membranes but it penetrates throughout the meristem

and binds to cell walls, forming an outline of living cells but also PI binds in the nucleus,

causing bright punctate fluorescence. Fig. S4 shows the PI staining of root cells of 5-day-old

transgenic Arabidopsis seedling expressing CFP-PTS1 that were preincubated with TFP. It

can be observed that the CaM antagonist (Fig. S4, panels D to F) does not affect the cell

viability if it is compared to untreated seedlings (Fig. S4, panels A to D).

Given that the CaM antagonist is the chemical with the most drastic effect on both

peroxisomal protein import and Ca2+

content, it was carry out also an in vitro assays of this

CaM antagonist in representative peroxisomal enzymes including CAT, GOX and HPR,

using 14-day-old Arabidopsis seedlings pre-incubated with 300 µM TFP for 2 h at 25ºC in

darkness. Fig. 5 (panels A to C) shows that these three activities were considerably affected,

with a reduction of 45% for catalase, 41% for GOX and 51% for HPR. For the purposes of

characterization, we also determined whether the CaM antagonist is capable of modifying the

gene expression of these peroxisomal enzymes. The Arabidopsis genome contains three

catalase genes (CAT1, CAT2 and CAT3), whose gene expression increased around 2.0, 1.4

and 1.5-fold respectively following pretreatment of Arabidopsis seedlings with TFP for 2 h

(Fig. 5D). The CaM antagonist also causes an induction of HPR1 expression, around 2.8-fold.

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Finally, glycolate oxidase is encoded by five genes, three of which, GOX1, GOX2 and

GOX3, were selected for this study. While GOX1 and GOX3 expression seems to be

unaffected, GOX2 expression increased 1.6-fold after pretreatment with the CaM antagonist

((Fig. 5E).

Discussion

Calcium is a highly important second messenger whose presence in different plant

subcellular compartments including the cytosol, chloroplasts, mitochondria, nucleus and

vacuoles has been abundantly studied (Monshausen et al., 2008; Dodd et al., 2010; Kudla et

al., 2010; Xu et al., 2015). However, information concerning calcium in peroxisomes,

particularly in plants, is scarce. In a study using human HeLa cells transiently expressing a

peroxisome targeting the GFP-based Ca2+

indicator D3cpv-SKL, the presence of Ca2+

in

animal peroxisomes was shown for the first time, with the peroxisomal membrane

representing a significant barrier to calcium diffusion into peroxisomes (Drago et al., 2008).

In the same year, Lasorsa et al. (2008) performed additional experiments showing that

peroxisomes contain between 20 and 50-fold more calcium than 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-based Cameleon Ca2+

indicator

D3cpv-KVK-SKL and demonstrated that peroxisomal calcium affected catalase activity

(Costa et al., 2010). The present study has therefore aimed to gain a deeper understanding of

the significance of Ca2+

in the peroxisomal metabolism of plants.

CaM is necessary for normal peroxisomal Ca2+

homeostasis and for peroxisomal

protein import

Calmodulin (CaM) is a protein of approximately 17 kDa expressed in all eukaryotic

cells with the capacity of binding Ca2+

(Bouché et al. 2005). This protein together with

calmodulin-like (CML) proteins are primary Ca2+

sensors that control the activity of various

target proteins and consequently diverse cellular functions including development processes

and responses to adverse biotic and abiotic conditions (Bouché et al., 2005; Reddy et al.,

2011; Cheval et al., 2013; Zeng et al., 2015). For instance, in the Arabidopsis genome, seven

distinct CaM and 50 CMLs genes have been predicted (McCormack et al., 2005). Recently,

AtCML3 has been demonstrated to be targeted to peroxisomes (Chigri et al., 2012) which

appears to mediate the dimerization of peroxisomal processing protease AtDEG15 (Dolze et

al., 2013). Moreover, there is another family of proteins which act as Ca2+

sensors called

Ca2+

-dependent protein kinases (CDPKs) (Boudsocq and Sheen, 2013) and in Arabidopssis

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the isoform AtCPK1 is targeted to peroxisomes (Dammann et al 2003) which seems to

mediate pathogen resistance (Coca and San Segundo, 2010).

Using an alternative fluorescent probe to detect Ca2+

, the present study corroborates

the presence of Ca2+

in the peroxisomes of two different tissues/cells – guar cells in green

cotyledons and root cells - suggesting that Ca2+

must be necessary for the normal

physiological functioning of plant peroxisomes, as has been shown with respect to other

subcellular compartments. Moreover, pharmacological techniques using different chemicals

have enabled us to demonstrate that peroxisomal Ca2+

must be regulated by CaM and GSH

and also, though less intensely, by nitric oxide. Thus, the CaM antagonist blocks peroxisomal

function due to its negative effect on peroxisomal protein import as CFP-PTS1 remains in the

cytosol. Additionally, the presence of Ca2+

in peroxisomes suggests that a CaM or CML

protein is strictly necessary for the peroxisomal homeostasis of Ca2+

; although, to our

knowledge there is not experimental information about the potential effect of a CaM

antagonist on the peroxisomal AtCML3 function. In any case, this is a reasonable assumption

since the function of CaM is to transport and regulate Ca2+

levels. Thus, these data are closely

in line with previous findings which show that Ca2+

is required for peroxisomal NO

production (Corpas et al., 2009) and that enzyme protein import responsible for its

generation is blocked by the CaM antagonist (Corpas and Barroso, 2014b).

Hitherto, there is clear evidence to support the cross-talk between NO and calcium-

calmodulin in plant cells (Sang et al. 2008; Ma et al., 2012). However, in the present study,

NO appears to be less involved in controlling peroxisomal Ca2+

content, by contrast,

peroxisomal Ca2+

is necessary and indispensable for the production of NO in peroxisomes. It

has been demonstrated that the activity of peroxisomal 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 that the import of this protein

responsible for NO generation into the peroxisome also depends on calcium and CaM

(Corpas et al., 2009; Corpas and Barroso, 2014b).

Reduced glutathione (GSH) constitutes an important antioxidant which has been

demonstrated to be present in plant peroxisomes (Zechmann and Müller, 2010), as it is

indispensable for the functioning of glutathione reductase (GR), a component of the

ascorbate-glutathione cycle present 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 GSNO treatment; however, our findings were surprising as GSH,

which caused a general delocalization of the Ca2+

signal and a marked intensification

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throughout the cells, drastically affected both peroxisomal protein import and the

peroxisomal localization of Ca2+

. This suggests that a change in the redox state or a possible

S-glutathionylation process could be involved in the mechanism of peroxisomal protein

import; the latter is regulated by a complex of cytosolic and peroxisomal proteins, known as

peroxins (PEXs), encoded by at least 22 PEX genes in Arabidopsis (Nito et al., 2007; Brown

and Baker A. 2008; Hu et al., 2012).

Ca2+

deficient negatively affects the activity of catalase and photorespiratory enzymes

but increases their gene expression

As part of the potential physiological functionality of Ca2+

, the CaM antagonist was observed

to negatively affect the activity of the antioxidant catalase enzyme as well as the glycolate

oxidase (GOX) and hydroxypyruvate reductase (HPR) enzymes, which are both involved in

photorespiration. The decrease in their activity could be due to the blocking of the

peroxisomal import of the corresponding proteins containing a PTS1; this is closely in line

with the results obtained, since the CaM antagonist blocks the import of CFP-PTS1.

However, using a purified tobacco catalase, a previous study has demonstrated that its

activity was stimulated by both Ca2+

and CaM (Yang and Poovaiah, 2002). Moreover, with

the aid of a recombinant Arabidopsis Cat3 in a CaM-binding assay, the same authors

demonstrated that CaM specifically binds to AtCat3 in the presence of calcium; protein

sequence analysis of Cat3 specifically enabled them to identify a CAM-binding region of 37

amino acids between Gly415 and Val451 (Yang and Poovaiah, 2002). More recently, an

increase in intraperoxisomal Ca2+

, in response to adverse conditions accompanied by

oxidative stress, 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+

and GOX or HPR activity. Nevertheless, the results show that its activity was also

adversely affected, with the blocking of its import into the peroxisomes being the most

plausible cause. Thus, the gene expression of these enzyme groups either increased (CAT1 to

CAT3, HPR1 and GOX2) or appeared to be unaffected (GOX1 and GOX3), which could be a

way of compensating for the loss of these enzyme activities.

In summary, our findings indicate that the presence of Ca2+

is necessary for the

physiological functioning of peroxisomes in root and guard cells, since its absence negatively

affects the activity of key enzymes as well as the generation of NO, as has previously been

demonstrated (Corpas et al 2009). Moreover, it is suggested that this could be due to the

possibility that Ca2+

/CaM is also necessary for the peroxisomal protein import containing a

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PTS1. However, a drastic alteration in the redox state and/or S-glutathionylation of

peroxisomal protein import peroxins could be involved. Nevertheless, further analyses will

be necessary to determine which peroxins are affected by this redox regulation.

Material and methods

Plant material and growth conditions

Arabidopsis thaliana transgenic seeds expressing cyan fluorescent protein (CFP) through the

addition of peroxisomal targeting signal 1 (PTS1) (Nelson et al., 2007) were grown on Petri

plates for 5 d or 14 d at 16 h light, 22°C/8 h dark, at 18 ºC under a light intensity of 100 µE

m-2

s-1

such as it has been previously described (Corpas and Barroso, 2014a). For microscope

analyses, 5-d-old seedlings were grown on vertical-positioned plates. For enzymatic and gene

expression assays, 14-d-old seedlings were grown on horizontal-positioned plates.

Detection of Ca2+

in transgenic plants expressing CFP-PTS1 using confocal laser

scanning microscopy (CLSM)

Calcium was detected with 5 µM Fluo-3 AM (Biotium) prepared in 10 mM Tris-HCl (pH

7.4) (Zhang et al., 1998; Kanchiswamy et al. 2010, 2014). The Arabidopsis seedlings were

incubated in darkness with 5 µM Fluo-3 AM at 25°C for 1 h. Each sample was then washed

twice in the same buffer for 15 min and mounted on a microscope slide for examination with

a CLSM (Leica TCS SP5 II). For Fluo-3 AM, the excitation laser wavelength was 514 nm,

with an emission of 610 and a 40-nm band pass width (590-630 nm). For cyan fluorescent

protein (CFP), the excitation laser wavelength was 458 nm, with an 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 calcium

Five-day-old Arabidopsis seedlings expressing CFP-PTS1 were preincubated for 120 min at

25ºC in darkness with different chemicals prepared in 10 mM Tris-HCl (pH 7.4) including 2

mM diethylamine NONOate (DEA NoNoate, nitric oxide donor), 2 mM S-nitrosoglutathione

(GSNO, nitric oxide donor) (Broniowska et al., 2013; Begara-Morales et al., 2014), 2 mM

glutathione (GSH) and 300 µM trifluoperazine (TFP, a calmodulin antagonist)

(Vandonselaar et al., 1994). Additionally, seedlings were also preincubated with 200 µM N-

(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) which is another CaM antagonist

(Ma et al., 2008); and 200 µM N-(6-aminohexyl)-1-naphthalenesulfonamide (W5) which is

the inactive W7 structural analog (Li et al., 2004). The seedlings were then incubated with 5

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µM Fluo-3 AM for 1h at 25 ºC in darkness to detect and visualize calcium using CLSM. In

all cases, the images obtained by CLSM from control and treated Arabidopsis seedlings were

kept constant during the course of the experiment in order to produce comparable data. For

each treatment, at least three independent experiments were made using, at least, four or five

seedlings in each one.

Cell wall integrity

Plant cell integrity was evaluated with propidium iodide (PI) staining (Truernit and Haseloff,

2008). Arabidopsis seedlings were incubated for 5 min at room temperature in a solution

propidium iodide (P4170 Sigma) of 10 μg/ml in water. For PI, the excitation 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 seedlings

Crude 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 a homogenizing medium containing 50

mM Tris-HCl, pH 7.8, 0.1mM EDTA, 0.2% (v/v) Triton X-100 and 10% (v/v) glycerol.

Homogenates were centrifuged at 27,000 g for 20 min, and the supernatants were used for the

enzymatic assays.

Enzyme activity assay

Catalase activity (EC 1.11.1.6) was assayed by measuring the disappearance of H2O2 as

described by Aebi (1984). NADH-dependent hydroxypyruvate reductase (HPR; EC 1.1.1.29)

was determined according to the method used by Schwitzguébel and Siegenthaler (1984).

Glycolate oxidase (GOX; EC 1.1.3.1) was assayed, as previously described (Kerr and

Groves, 1975), by measuring the formation of glyoxylate-phenylhydrazone.

RNA isolation and semiquantitative RT-PCR.

Total RNA was extracted with Trizol according to instructions provided by Gibco BRL (Life

Technologies). Five μg of total RNA were used to produce cDNA for the reverse

transcriptase (RT) reaction by adding 0.5 mM dNTPs, poly-dT23, 5x RT buffer, 40 U RNase

inhibitor (Invitrogen) and 200 U Reverse Transcriptase (Termofisher) in a final volume of 20

μl. The reaction was carried out at 50ºC for 30 min.

Semiquantitative reverse transcription-PCR amplification of ACTIN2 cDNA from

Arabidopsis was chosen as control. CAT1, CAT2, CAT3, HPR1, GOX1, GOX2 and GOX3

cDNAs were applied by the PCR as previously described (Corpas and Barroso, 2016). PCR

products were then detected by electrophoresis in 2.8% (w/v) agarose gels and by staining

with GelRed™. Quantification of the bands was performed using a Gel Doc system (Bio-Rad

Laboratories) coupled with a high-sensitivity charge-coupled device (CCD) camera. T/C ratio

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indicates the relative level of the each amplification product (T) over the ACTIN 2 used as

internal control (C) after normalization to the control samples, and it expresses the change in

folds with respect to the untreated control (Marone et al., 2001).

Other Assays

Protein concentration was determined with the aid of the Bio-Rad Protein Assay (Hercules,

CA) using bovine serum albumin as standard. For microscope analyses around five to ten 5-

d-old Arabidopsis seedlings were used for each experiment and treatment. For enzymatic

activity and gene expression assays around one hundred and fifty 14-d-old Arabidopsis

seedlings were used for each experiment. Data are means of at least three sets of independent

experiments with three repetitions each. To estimate the statistical significance between

means, the data was analyzed by the Student’s test. Relative fluorescence was quantified

using ImageJ software.

Acknowledgements

Authors are grateful to Mr. Carmelo Ruiz-Torres for his excellent technical support. The

authors thank Dr Juan D Alché (CSIC) for his advice on the analysis of images. Technical

and human support provided by CICT of Universidad de Jaén (UJA, MINECO, Junta de

Andalucía, FEDER) is also acknowledged.

Competing interests

The authors declare no competing or financial interests.

Author contributions

J.B.B. performed experiments, interpreted and discussed data. F.J.C. conceived, designed the

study, performed experiments, interpreted data, and wrote the manuscript.

Funding

Research in FJC and JBB laboratory are supported by an ERDF grant co-financed by the

Ministry of Economy and Competitiveness (projects AGL2015-65104-P and BIO2015-

66390-P, respectively) and the Junta de Andalucía (groups BIO192 and BIO286) in Spain.

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References

Aebi, H. (1984) Catalase in vitro. Methods in Enzymol. 105, 121-126.

Afiyanti, M. and Chen, H.J. (2014). Catalase activity is modulated by calcium and

calmodulin in detached mature leaves of sweet potato. Journal Plant Physiology 171, 35-

47.

Barr, R., Böttger M., Frederick., L. and Crane, F.L. (1990). The effect of selected

inhibitors on plasma membrane redox reactions and proton excretion by carrot cells.

Plant Sci. 69, 33–38

Barroso, J.B., Corpas, F.J., Carreras, A., Sandalio, L.M., Valderrama, R., Palma, J.M.,

Lupiáñez, J.A. and del Río, L.A. (1999). Localization of nitric-oxide synthase in plant

peroxisomes. J. Biol. Chem. 274, 36729-33.

Begara-Morales, J.C., Sánchez-Calvo, B., Luque, F., Leyva-Pérez, M.O., Leterrier, M.,

Corpas, F.J. and Barroso, J.B. (2014). Differential transcriptomic analysis by RNA-Seq

of GSNO-responsive genes between Arabidopsis roots and leaves. Plant Cell Physiology

55, 1080-1095.

Begara-Morales, J.C., Sánchez-Calvo, B., Chaki, M., Mata-Pérez C, Valderrama R, Padilla

MN, López-Jaramillo J, Luque F, Corpas FJ, Barroso JB. (2015) Differential molecular

response of monodehydroascorbate reductase and glutathione reductase by nitration and

S-nitrosylation. J Exp Bot. 66, 5983-5996.

Begara-Morales, J.C., Sánchez-Calvo, B., Chaki, M., Valderrama, R., Mata-Pérez, C.,

Padilla, M.N., Corpas, F.J. and Barroso, J.B. (2016). Antioxidant systems are

regulated by nitric oxide-mediated post-translational Modifications (NO-PTMs). Front

Plant Sci. 7, 152.

Behera S, Wang N, Zhang C, Schmitz-Thom I, Strohkamp S, Schültke S, Hashimoto K,

Xiong L. and Kudla J. (2015). Analyses of Ca2+

dynamics using a ubiquitin-10

promoter-driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and

differences in cytoplasmic Ca2+

responses in Arabidopsis and rice (Oryza sativa) roots.

New Phytol. 206, 751-60.

Boudsocq M, Sheen J. (2013). CDPKs in immune and stress signaling. Trends in Plant

Science 18, 30-40.

Bouché N, Yellin A, Snedden WA. and Fromm, H. (2005). Plant-specific calmodulin-

binding proteins. Annual Review Plant Biology 56,435-466.

Broniowska KA, Diers AR, Hogg N. (2013). S-nitrosoglutathione. Biochimica et

Biophysica Acta 1830,3173-3181.

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Brown LA, Baker A. (2008). Shuttles and cycles: transport of proteins into the peroxisome

matrix (review). Molecular Membrane Biology 25, 363-375.

Cheval C, Aldon D, Galaud JP, Ranty B. (2013). Calcium/calmodulin-mediated regulation

of plant immunity. Biochim Biophys Acta 1833, 1766-17671.

Charpentier M, Oldroyd GE. (2013) Nuclear calcium signaling in plants. Plant Physiology

163, 496-503.

Chigri F, Flosdorff S, Pilz S, Kölle E, Dolze E, Gietl C, Vothknecht UC. (2012). The

Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to

mitochondria and peroxisomes, respectively. Plant Molecular Biology 78, 211-222.

Choi WG, Swanson SJ, Gilroy S. (2012). High-resolution imaging of Ca2+

, redox status,

ROS and pH using GFP biosensors. Plant Journal 70, 118-28.

Choi, W.G., Toyota, M., Kim, S.H., Hilleary, R. and Gilroy, S. (2014) Salt stress-induced

Ca2+

waves are associated with rapid, long-distance root-to-shoot signaling in plants. Proc

Natl Acad Sci U S A. 111, 6497-6502.

Coca M, San Segundo B. (2010). AtCPK1 calcium-dependent protein kinase mediates

pathogen resistance in Arabidopsis. Plant Journal 63, 526-540.

Corpas FJ. (2015). What is the role of hydrogen peroxide in plant peroxisomes? Plant

Biology (Stuttg). 17, 1099-1103.

Corpas, F.J. and Barroso, J.B. (2014a). Peroxynitrite (ONOO-) is endogenously produced

in arabidopsis peroxisomes and is overproduced under cadmium stress. Annals of Botany

113, 87-96.

Corpas, F.J. and Barroso J.B. (2014b). Peroxisomal plant nitric oxide synthase (NOS)

protein is imported by peroxisomal targeting signal type 2 (PTS2) in a process that

depends on the cytosolic receptor PEX7 and calmodulin. FEBS Letters 588, 2049-2054.

Corpas, F.J, and Barroso J.B. (2016) Lead-induced stress, which triggers the production of

nitric oxide (NO) and superoxide anion (O2·-) in Arabidopsis peroxisomes, affects catalase

activity. Nitric Oxide Biology and Chemistry doi 10.1016/j.niox.2016.12.010

Corpas, FJ, Barroso, J.B., Palma JM. and Rodriguez-Ruiz, M. (2017) Plant Peroxisomes:

a nitro-oxidative cocktail . Redox Biol. 11, 535-542.

Corpas, FJ, Barroso, J.B., Carreras A, Quirós M, León AM, Romero-Puertas MC,

Esteban FJ, Valderrama R, Palma JM, Sandalio LM, Gómez M, del Río LA. (2004).

Cellular and subcellular localization of endogenous nitric oxide in young and senescent

pea plants. Plant Physiology 136, 2722-2733.

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Corpas, F.J., Hayashi, M., Mano, S., Nishimura, M. and Barroso, J.B. (2009).

Peroxisomes are required for in vivo nitric oxide accumulation in the cytosol following

salinity stress of Arabidopsis plants. Plant Physiology 151, 2083-2094.

Costa, A., Drago, I., Behera, S., Zottini, M., Pizzo, P., Schroeder, J.I., Pozzan, T. and

Lo Schiavo, F. (2010). H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+

)-

dependent scavenging system. Plant Journal 62, 760-772.

Costa A, Drago I, Zottini M, Pizzo P, and Pozzan, T. (2013). Peroxisome Ca2+

homeostasis in animal and plant cells. Subcellular Biochemistry 69, 111-133.

Dammann C, Ichida A, Hong B, Romanowsky SM, Hrabak EM, Harmon AC, Pickard

BG. and Harper JF. (2003). Subcellular targeting of nine calcium-dependent protein

kinase isoforms from Arabidopsis. Plant Physiology 13, 1840-1848.

del Río LA, Corpas FJ, Sandalio LM, Palma JM, Gómez M, Barroso JB. (2002).

Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. Journal

Experimental Botany 53,1255-72.

Desikan, R., Cheung, M.K., Bright, J., Henson, D., Hancock, J.T. and Neill, S.J. (2004)

ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J Exp Bot 55,

205-212

Dodd AN, Kudla J, Sanders D. (2010). The language of calcium signaling

Annual Review of Plant Biology 61, 593-620.

Dolze E, Chigri F, Höwing T, Hierl G, Isono E, Vothknecht UC, Gietl C. (2013).

Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing

protease AtDEG15 and contributes to normal peroxisome metabolism. Plant Molecular

Biology 83, 607-624.

Drago I, Giacomello M, Pizzo P, Pozzan T. (2008). Calcium dynamics in the peroxisomal

lumen of living cells. Journal Biological Chemistry 283, 14384-14390.

Du YY, Wang PC, Chen J, Song CP. (2008). Comprehensive functional analysis of the

catalase gene family in Arabidopsis thaliana. J Integr Plant Biol. 50(10),1318-26.

Dunn PP, Slabas AR, Cottingham IR, Moore AL. (1984) Trifluoperazine inhibition of

electron transport and adenosine triphosphatase in plant mitochondria. Arch Biochem

Biophys. 229, 287-294.

Fernández-Fernández ÁD, Corpas FJ. (2016). In silico analysis of Arabidopsis thaliana

peroxisomal 6-Phosphogluconate dehydrogenase. Scientifica (Cairo) 2016,3482760

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Frungillo L, Skelly MJ, Loake GJ, Spoel SH, Salgado I. (2014) S-nitrosothiols regulate

nitric oxide production and storage in plants through the nitrogen assimilation pathway.

Nat Commun. 5, 5401.

González A, Cabrera Mde L, Henríquez MJ, Contreras RA, Morales B, Moenne A.

(2012). Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of

gene expression involving calmodulins and calcium-dependent protein kinases in Ulva

compressa exposed to copper excess. Plant Physiology 158, 1451-1462.

Hao F, Zhao S, Dong H, Zhang H, Sun L, Miao C (2010) Nia1 and Nia2 are involved in

exogenous salicylic acid-induced nitric oxide generation and stomatal closure in

Arabidopsis. J Integr Plant Biol 52: 298–307.

Han, S., Wang, C.W., Wang, W.L. and Jiang, J. (2014) Mitogen-activated protein kinase

6 controls root growth in Arabidopsis by modulating Ca2+

-based Na+ flux in root cell

under salt stress. Journal of Plant Physiology 171, 26-34.

Hu, J., Baker, A., Bartel, B., Linka, N., Mullen, R.T., Reumann, S. and Zolman, B.K.

(2012). Plant peroxisomes: biogenesis and function. The Plant Cell 24, 2279-2303.

Kanchiswamy, C.N., Takahashi, H., Quadro, S., Maffei, M.E., Bossi, S., Bertea, C.,

Zebelo, S.A., Muroi, A., Ishihama, N., Yoshioka, H., Boland, W., Takabayashi, J.,

Endo, Y., Sawasaki, T. and Arimura, G. (2010). Regulation of Arabidopsis defense

responses against Spodoptera littoralis by CPK-mediated calcium signaling. BMC Plant

Biology 10,97.

Hölscher, C., Lutterbey, M.C., Lansing, H., Meyer, T., Fischer, K. and von Schaewen,

A. (2016) Defects in peroxisomal 6-phosphogluconate dehydrogenase isoform PGD2

prevent gametophytic interaction in Arabidopsis thaliana. Plant Physiol. 171,192-205.

Kanchiswamy CN, Malnoy M, Occhipinti A, Maffei ME. (2014). Calcium imaging

perspectives in plants. International J. Mol. Sci. 15, 3842-3859.

Kerr MW, Groves D. (1975). Purification and properties of glycollate oxidase from Pisum

sativum leaves. Phytochemistry 14, 359–362.

Kudla J, Batistic O, Hashimoto K. (2010). Calcium signals: the lead currency of plant

information processing. Plant Cell. 22, 541-63.

Lasorsa FM, Pinton P, Palmieri L, Scarcia P, Rottensteiner H, Rizzuto R, Palmieri F.

(2008). Peroxisomes as novel players in cell calcium homeostasis. J. Biol. Chem. 283,

15300-15308.

Leterrier M, Barroso JB, Valderrama R, Begara-Morales JC, Sánchez-Calvo B, Chaki

M, Luque F, Viñegla B, Palma JM, Corpas FJ. (2016). Peroxisomal NADP-isocitrate

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

dehydrogenase is required for Arabidopsis stomatal movement. Protoplasma 253, 403-

415.

Li B, Liu HT, Sun DY, Zhou RG. (2004). Ca(2+

) and calmodulin modulate DNA-binding

activity of maize heat shock transcription factor in vitro. Plant Cell Physiol. 45, 627-34.

Lichtman, A.H, Segel, G.B. and Lichtman, M.A. (1982) Effects of trifluoperazine and

mitogenic lectins on calcium ATPase activity and calcium transport by human

lymphocyte plasma membrane vesicles. J Cell Physiol. 111, 213-217.

Ma F, Lu R, Liu H, Shi B, Zhang J, Tan M, Zhang A, Jiang M. (2012) Nitric oxide-

activated calcium/calmodulin-dependent protein kinase regulates the abscisic acid-

induced antioxidant defence in maize. J Exp Bot. 63, 4835-4847.

Ma W, Smigel A, Tsai YC, Braam J, Berkowitz GA. (2008). Innate immunity signaling:

cytosolic Ca2+

elevation is linked to downstream nitric oxide generation through the

action of calmodulin or a calmodulin-like protein. Plant Physiol. 148, 818-828.

Mata-Pérez, C., Sánchez-Calvo, B., Padilla, M.N., Begara-Morales, J.C., Luque F,

Melguizo, M., Jiménez-Ruiz, J., Fierro-Risco, J., Peñas-Sanjuán, A., Valderrama,

R., Corpas, F.J. and Barroso JB. (2016). Nitro-fatty acids in plant signaling: nitro-

linolenic acid induces the molecular chaperone network in Arabidopsis. Plant Physiol.

170, 686-701.

Marone M, Mozzetti S, De Ritis D, Pierelli L, Scambia G. (2001). Semiquantitative RT-

PCR analysis to assess the expression levels of multiple transcripts from the same sample.

Biological Procedures Online 3,19-25.

McCormack E., Tsai YC. and Braam J. (2005) Handling calcium signaling: Arabidopsis

CaMs and CMLs. Trends Plant Sci. 10, 383-389.

Monshausen GB. (2012). Visualizing Ca2+

signatures in plants. Curr. Op.Plant Biol. 15,

677–682

Monshausen GB, Messerli MA, Gilroy S. (2008) Imaging of the Yellow Cameleon 3.6

indicator reveals that elevations in cytosolic Ca2+

follow oscillating increases in growth in

root hairs of Arabidopsis. Plant Physiol. 147, 1690-1698.

Murata Y, Mori IC. and Munemasa, S. (2015) Diverse stomatal signaling and the signal

integration mechanism. Annu Rev Plant Biol. 66, 369-392.

Nelson BK, Cai X, Nebenführ A. (2007). A multicolored set of in vivo organelle markers

for co-localization studies in arabidopsis and other plants. Plant J. 51, 1126–1136.

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Nito K, Kamigaki A, Kondo M, Hayashi M, Nishimura M. (2007). Functional

classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of

knockdown mutants. Plant Cell Physiol. 48, 763–774

Niu, L. and Liao, W. (2016). Hydrogen peroxide signaling in plant development and abiotic

responses: crosstalk with nitric oxide and calcium. Front. Plant Sci. 7, 230.

Pinfield-Wells H, Rylott EL, Gilday AD, Graham S, Job K, Larson TR, Graham IA.

(2005). Sucrose rescues seedling establishment but not germination of Arabidopsis

mutants disrupted in peroxisomal fatty acid catabolism. Plant J.l 43,861–72

Raychaudhury B, Gupta S, Banerjee S, Datta SC. (2006). Peroxisome is a reservoir of

intracellular calcium. Biochim Biophys Acta. 1760, 989-92.

Reddy AS, Ali GS, Celesnik H, Day IS. (2011). Coping with stresses: roles of calcium- and

calcium/calmodulin-regulated gene expression. Plant Cell 23, 2010-32.

Rentel MC, Knight MR. (2004). Oxidative stress-induced calcium signaling in Arabidopsis.

Plant Physiol. 135, 1471-1479.

Reumann S, Corpas FJ. (2010). The Peroxisomal Ascorbate–Glutathione Pathway:

Molecular Identification and Insights into Its Essential Role Under Environmental Stress

Conditions. In Ascorbate-Glutathione Pathway and Stress Tolerance in Plants, 387-404

(Anjum et al. eds.). Springe (doi 10.1007/978-90-481-9404-9_14).

Rojas CM, Senthil-Kumar M, Wang K, Ryu CM, Kaundal A, Mysore KS. (2012).

Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during

nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 24, 336-352.

Romero-Puertas MC, Corpas FJ, Sandalio LM, Leterrier M, Rodríguez-Serrano M, del

Río LA, Palma JM. (2006). Glutathione reductase from pea leaves: response to abiotic

stress and characterization of the peroxisomal isozyme. New Phytol.t 170, 43-52.

Sang J, Zhang A, Lin F, Tan M, Jiang M. (2008) Cross-talk between calcium-calmodulin

and nitric oxide in abscisic acid signaling in leaves of maize plants. Cell Res. 18, 577-

588.

Schmidt M, Grief J, Feierabend J. (2006). Mode of translational activation of the catalase

(cat1) mRNA of rye leaves (Secale cereale L.) and its control through blue light and

reactive oxygen. Planta 223, 835-846.

Schwitzguébel JP, Siegenthaler PA. (1984). Purification of peroxisomes and mitochondria

from spinach leaf by percoll gradient centrifugation. Plant Physiol. 75, 670–674,

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Schlicht M, Ludwig-Müller J, Burbach C, Volkmann D, Baluska F. (2013). Indole-3-

butyric acid induces lateral root formation via peroxisome-derived indole-3-acetic acid

and nitric oxide. New Phytol. 200, 473–482.

Sengupta P, Ruano MJ, Tebar F, Golebiewska U, Zaitseva I, Enrich C, McLaughlin S,

Villalobo A. (2007). Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind

to membranes, changing the electrostatic surface potential: dual effect of W-13 on

epidermal growth factor receptor activation. J Biol Chem. 282, 8474-8486.

Simeunovic A, Mair A, Wurzinger B, Teige M. (2016) Know where your clients are:

subcellular localization and targets of calcium-dependent protein kinases. J. Exp. Bot. 67,

3855-3872.

Stael S, Wurzinger B, Mair A, Mehlmer N, Vothknecht UC, Teige M. (2012). Plant

organellar calcium signalling: an emerging field. J. Exp. Bot. 63, 1525-42.

Steinhorst L, Kudla J. (2013). Calcium and reactive oxygen species rule the waves of

signaling. Plant Physiol. 163, 471-485.

Truernit E, Haseloff J. (2008). A simple way to identify non-viable cells within living plant

tissue using confocal microscopy. Plant Methods. 4,15.

Vandonselaar M, Hickie RA, Quail JW, Delbaere LT. (1994). Trifluoperazine-induced

conformational change in Ca(2+)

-calmodulin. Nat. Struct. Biol. 1, 795-801.

Wang P, Du Y, Hou Y, Zhao Y, Hsu C, Yuan F, Zhu X, TaoWA, Song C, Zhu J (2014)

Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation

of OST1. Proc Natl Acad Sci USA 112, 613-618.

Wilson, S.B. (1994) Complex V of plant mitochondria - Another target for agrochemicals?

Biochemical Society Transactions 22, 73S.

Xu H, Martinoia E, Szabo I. (2015) Organellar channels and transporters. Cell Calcium. 58,

1-10.

Yang T, Poovaiah BW. (2002). Hydrogen peroxide homeostasis: activation of plant catalase

by calcium/calmodulin. Proc Natl Acad Sci U S A. 99, 4097-4102.

Yang T, Poovaiah BW. (2003). Calcium/calmodulin-mediated signal network in plants.

Trends Plant Sci. 8, 505-512.

Yun BW, Skelly MJ, Yin M, Yu M, Mun BG, Lee SU, Hussain A, Spoel SH, Loake GJ.

(2016). Nitric oxide and S-nitrosoglutathione function additively during plant immunity. New

Phytol. 211, 516-526

Jour

nal o

f Cel

l Sci

ence

• A

dvan

ce a

rtic

le

Zhang W-H, Rengel Z, Kuo J. (1998). Determination of intracellular Ca2+

in cells of intact

wheat roots: loading of acetoxymethyl ester of Fluo-3 under low temperature. Plant J. 15,

147-151

Zhao Q, Zhang C, Jia Z, Huang Y, Li H, Song S. (2015). Involvement of calmodulin in

regulation of primary root elongation by N-3-oxo-hexanoyl homoserine lactone in

Arabidopsis thaliana. Frontiers Plant Science 5, 807.

Zechmann B, Müller M. (2010). Subcellular compartmentation of glutathione in

dicotyledonous plants. Protoplasma 246, 15-24.

Zeng H, Xu L, Singh A, Wang H, Du L, Poovaiah BW. (2015) Involvement of calmodulin

and calmodulin-like proteins in plant responses to abiotic stresses. Front Plant Sci. 6,

600.

Jour

nal o

f Cel

l Sci

ence

• A

dvan

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Figures

Figure 1. Representative images illustrating the CLSM in vivo detection of Ca2+

(red

colour), peroxisomes (green colour), and chloroplasts in guard (panels A to E) and root

(panels F tp I) cells of 5-day-old transgenic Arabidopsis seedlings expressing CFP-PTS1.

A and F, show fluorescence punctuates (green) attributable to CFP-PTS1 indicating the

localization of peroxisomes. B and G, show the fluorescence (red) attributable to the

detection in the same cell area of calcium. C, shows the chlorophyll autofluorescence

(purple) attributable to the detection of chloroplasts. D and H, show merged images of the

correspoding panels. E and I, show the bright-field image of guard and root cells. Calcium

(red colour) was detected using 5µM Fluo-3 AM. Arrows indicate representative punctuate

spots corresponding to the co-localization of calcium with peroxisomes. Asterics indicate

localization of Ca2+

in cytosol or cell wall. Jo

urna

l of C

ell S

cien

ce •

Adv

ance

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Figure 2. Representative images illustrating the CLSM in vivo detection of Ca2+

(red),

peroxisomes (green), and chloroplasts (purple) in guard cells of 5-day-old transgenic

Arabidopsis seedlings expressing CFP-PTS1 preincubated with different chemicals. A to

E, control. F to J, 2 mM S-nitrosoglutathione (GSNO) as NO donor. K to O, 2 mM DEA

NoNoate as NO donor. P to T, 2 mM reduced glutathione (GSH). U to Y, 300 µM

trifluoperazine (TFP) calmodulin antagonist. Arrows indicate punctuate spots corresponding

to the co-localization of calcium with peroxisomes and angles (^) indicate co-localization of

Ca2+

with chloroplasts.

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Figure 3. Representative images illustrating the CLSM in vivo detection of Ca2+

(red)

and peroxisomes (green) in root tip cells of 5-day-old transgenic Arabidopsis seedlings

expressing CFP-PTS1 preincubated with different chemicals. A to D, control. E to H, 2

mM S-nitrosoglutathione (GSNO) as NO donor. I to L, 2 mM DEA NoNoate as NO donor.

M to P, 2 mM reduced glutathione (GSH). Q to T, 300 µM trifluoperazine (TFP)

calmodulin antagonist. Arrows indicate representative punctuate spots corresponding to the

co-localization of calcium with peroxisomes.

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Figure 4. Fluorescence intensity of Ca2+

(red) detection in guard and root cells of 5-day-old

transgenic Arabidopsis seedlings expressing CFP-PTS1 preincubated with different

chemicals (2 mM GSNO; 2 mM DEA NoNoate; as NO donor; 2 mM reduced glutathione

(GSH); and 300 µM trifluroperazine (TFP) calmodulin antagonist) according to Figures 2

and 3. The fluorescence produced was expressed as arbitrary units (A.U.) using Image J

software. *P < 0.05 compared with the corresponding control for guard and root cells,

respectively.

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Figure 5. Activity and gene expression of peroxisomal enzymes (CAT, HPR and GOX)

in response to a calmodulin (CaM) antagonist. (A) Catalase activity. (B) Glycolate

oxidase (GOX) activity. (C) Hydroxypyruvate reductase (HPR) activity. (D) CAT gene

expression. (E) HPR and GOX gene expression. For peroxisomal enzymatic activity assay

and Semiquantitative RT-PCR analyses, 14-day-old Arabidopsis seedlings were exposed to

300 µM TFP (CaM antagonist) for 2 h at 25ºC in darkness. Results are the mean of three

different experiments ± SEM. ∗Differences in relation to control values were significant at P

< 0.05. Semiquantitative RT-PCR was performed on total RNA isolated using ACT2 as

internal control. Representative agarose electrophoresis gels of the amplification products

visualized by GelRed™ staining under UV light. T/C indicates the relative level of the each

amplification product (T) over the ACT2 internal control (C) after normalization to the

control samples, and it expresses the change in folds with respect to the untreated control.

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CFP-PTS1 Without Fluo-3 AM

A B C

10 µm

D E F

Supplemental Figure 1 Representative images illustrating the CLSM

30 µm

Supplemental Figure 1. Representative images illustrating the CLSMin vivo detection of peroxisomes (green) in guard cells (A to C) androot tips (D to F) of transgenic Arabidopsis 5-days-old seedlingsexpressing CFP-PTS1 without the presence of Fluo-3 AM, fluorescentprobe used to detect Ca2+, and observed under two conditions. PanelsA and D show fluorescence punctuates (green) attributable to CFP-PTS1 (excitation 458 nm; emission 475 nm) indicating the localizationof peroxisomes Panels B and E show the absence of anyof peroxisomes. Panels B and E show the absence of anyfluorescence in the same area observed in panels A and D,respectively, without any fluorescence probe Fluo-3 AM (negativecontrol) using the wavelength conditions to detect Ca2+ with Fluo-3 AM(excitation 514 nm and emission 610 nm). Panels C and F show brightfield plus merged images of the corresponding samples. Arrowsindicate representative punctuate spots corresponding to thelocalization of peroxisomesp

J. Cell Sci. 130: doi:10.1242/jcs.201467: Supplementary information

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A B C D

Supplemental Figure 2. Low magnification images illustrating the CLSM in

40 µm

vivo detection of Ca2+ (red) and peroxisomes (green) in root cells of 5-day-oldtransgenic Arabidopsis seedlings expressing CFP-PTS1. Panel A showsfluorescence punctuates (green) attributable to CFP-PTS1 indicating thelocalization of peroxisomes. Panel B shows the fluorescence (red) attributable tothe detection in the same cell area of Ca2+. Panel C shows merged images of thecorresponding panels. Panel D shows the bright-field image of root cells. Calcium(red colour) was detected using 5µM Fluo-3 AM. Arrows indicate representative

t t t di t th l li ti f l i ith ipunctuate spots corresponding to the co-localization of calcium with peroxisomes.

J. Cell Sci. 130: doi:10.1242/jcs.201467: Supplementary information

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A Control B C

CPF-PTS1 Calcium Bright field

20 µm

TFPD E F

20 µm

30 µm

W7G H I

30 µm

W5J K L

Supplementary Figure 3. Representative images illustring theCLSM in vivo detection of Ca2+ (red colour) and peroxisomes (greencolour) in root tipc cells of 5-day-old transgenic Arabidopsisseedlings expressing CFP-PTS1 preincubated with different

µ

g p g pchemicals. A to C, control. D to F, 300 µM TFP (CaM antagonist). Gto I, 200 µM W7 (CaM antagonist). J to L, 200 µM W5, inactiveform of W7.

J. Cell Sci. 130: doi:10.1242/jcs.201467: Supplementary information

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CFP-PTS1 Propidium iodide Mergedp g

A B CControl

30 µm

D E FTFP

30 µm

Supplementary Figure 4. Representative images illustrating theCLSM in vivo detection of peroxisomes (green) in root cells of 5-day-old transgenic Arabidopsis seedling expressing CFP-PTS1which were preincubated with TFP (calmodulin antagonist) and

30 µm

which were preincubated with TFP (calmodulin antagonist) andstaining with propidium iodide (red). A to D, control. D to F, 300 µMtrifluroperazine (TFP). Arrow, nucleus.

J. Cell Sci. 130: doi:10.1242/jcs.201467: Supplementary information

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