- Email: [email protected]
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 xxx (2014) xxx–xxx
journal homepage: www.FEBSLetters.org
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 Francisco J. Corpas a,⇑, Juan B. Barroso b a Group 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, Apartado 419, E-18080 Granada, Spain b Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus ‘‘Las Lagunillas’’, E-23071 Jaén, Spain
a r t i c l e
i n f o
Article history: Received 22 February 2014 Revised 18 April 2014 Accepted 23 April 2014 Available online xxxx Edited by Ulf-Ingo Flügge Keywords: Nitric oxide Nitric oxide synthase Peroxin Peroxisome Peroxisomal targeting signal Arabidopsis thaliana
a b s t r a c t Nitric oxide (NO) production in plant peroxisomes by L-arginine-dependent NO synthase activity has been proven. The PEX5 and PEX7 PTS receptors, which recognize PTS1- and PTS2-containing proteins, are localized in the cytosol. Using AtPex5p and AtPex7p knockdown in Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i, we found that the L-arginine-dependent protein responsible for NO generation in peroxisomes appears to be imported through an N-terminal PTS2. Pharmacological analyzes using a calcium channel blocker and calmodulin (CaM) antagonist show that the import of the peroxisomal NOS protein also depends on calcium and calmodulin. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Nitric oxide (NO) is a free radical messenger with a broad spectrum of regulatory functions in many physiological and pathological processes in higher plants [1–5]. At subcellular level, NO has been reported to be generated in different cell types and compartments including peroxisomes, chloroplasts, mitochondria and cytosol [6–11]. However, identification of the protein responsible for L-arginine-dependent NO production is still an open question even though supporting evidence has been found [12] which includes the identification of a gene which codified for a putative NOS protein in a green alga that showed a 40% identity with animal NOSs [13]. Peroxisomes are single membrane-bounded subcellular organelles with an essentially oxidative type of metabolism and a simple morphology that does not mirror the complexity of their enzymatic composition. They harbour variable metabolic pathways that differ depending on cell type, developmental stage and environmental conditions [6,14]. In higher plants, previous data have demonstrated that peroxisomes have an active NO metabolism given ⇑ Corresponding author. Fax: +34 958 129600. E-mail address: [email protected] (F.J. Corpas).
the presence of L-arginine-dependent NOS activity in pea peroxisomes [15], NO modulation during senescence [16], salinity [17] and cadmium stress [18] as well as the peroxisomal localization of other NO-derived molecules such as S-nitrosoglutathione [19] and peroxynitrite [18]. As peroxisomes do not have DNA, all its proteins are nuclear-encoded. There are two principal signals that direct proteins into peroxisomes found at the C-terminus (peroxisomal targeting signal type 1 or PTS1) and N-terminus (peroxisomal targeting signal type 2 or PTS2) of the protein [20–22]. Peroxins (PEX) are peroxisomal biogenesis factors encoded by PEX genes, some of which are involved in matrix protein transport [23]. Previously, it has been shown that peroxins PEX12 and PEX13 appear to be involved in transporting the putative NOS protein to peroxisomes, as pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content [17]. PEX5p and PEX7p are cytosolic receptors for PTS1 and PTS2, respectively, and form a receptor– cargo complex in the cytosol during transport to peroxisomes [24–27]. Using Arabidopsis knockdown mutants obtained by RNA interference (RNAi) AtPex5p (pex5i) and AtPex7p (pex7i) [24], the aim of the present study is to analyze the potential involvement of PEX5 and/or PEX7 in the import of the peroxisomal protein involved in NO generation in plants.
http://dx.doi.org/10.1016/j.febslet.2014.04.034 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
2
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
2. Materials and methods 2.1. Plant material and growth conditions Arabidopsis thaliana Columbia wild-type and mutants expressing GFP-PTS1 [28] were used as control plants. Constructs of transgenic plants and characterization of mutant AtPex5p expressing GFP-PTS1 and mutant AtPex7p expressing PTS2-GFP knockdown Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i were characterized previously [24]. Seeds were surface-sterilized for 5 min in 70% ethanol containing 0.1% SDS, then placed for 20 min in sterile water containing 20% bleach and 0.1% SDS and washed four times in sterile water. The seeds were sown for 2 days at 4 °C in the dark for vernalization on the basal growth medium composed of 4.32 g/l commercial Murashige and Skoog medium (Sigma) with a pH of 5.5, containing 1% sucrose and 0.8% phytoagar. The Arabidopsis seeds were then grown on Petri dishes in the dark at 22 °C for 5 days for the pex5i and pex7i mutants and at 16 h light/8 h dark (long-day conditions) under a light intensity of 100 lE m 2 s 1 for the GFP-PTS1 mutants. 2.2. Detection of NO in mutants expressing GFP-PTS1 or PTS2-GFP using confocal laser scanning microscopy (CLSM) Nitric oxide was detected using fluorescent reagent diaminorhodamine-4M acetoxymethyl ester (DAR-AM AM, Calbiochem). Arabidopsis seedlings were incubated at 25 °C for 1 h in darkness with 5 lM DAR-AM AM prepared in 100 mM potassium phosphate buffer (pH 7.4) [17]. Each sample was then washed twice in the same buffer for 15 min and mounted on a slide for examination with a confocal laser scanning microscope (Leica TCS SL or Leica TCS SP5 II). For green fluorescent protein (GFP), the excitation laser wavelength was 496 nm and emission of 515 with a 10-nm band pass width (500–525 nm). For DAR-AM T, the excitation laser wavelength was 543 nm and emission of 575 nm with a 40-nm band pass width (570–600 nm). As control for DAR-AM AM as fluorescent probe to detect NO in peroxisomes, Supplemental Fig. 1 shows a highly magnified image of root cells from Arabidopsis wild-type plants, with red spherical spots resembling peroxisomes inside the cells. 2.3. Pharmacological analyzes using calcium channel blocker and calmodulin antagonist Five-day-old Arabidopsis seedlings expressing GFP-PTS1 were preincubated for 120 min at 25 °C with either 1.5 mM CaCl2, 5 mM lanthanum chloride (LaCl3, a calcium channel blocker) [29] or 300 lM trifluroperazine (TFP, a calmodulin antagonist) [30] and then incubated with 5 lM DAR-AM AM for 1 h at 25 °C to detect and visualize NO 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. The images were processed and analyzed using statistical Leica confocal software. 3. Results 3.1. Analysis of calcium and calmodulin in peroxisomal NO localization Fig. 1A showed in vivo CLSM visualization of peroxisomes in the root cells of transgenic arabidopsis plants expressing GFP-PTS1. The peroxisomes appeared in the form of spherical spots (green color). Fig. 1B illustrated the same field analyzed using the DAR4M AM fluorescence probe, which also enabled NO (red in color) to be detected. Spherical spots (red) were found to have a pattern
identical to that of GFP-PTS1 (Fig. 1C). Fig. 1D and E provided images of an arabidopsis seedling root pre-incubated with 1.5 mM calcium. Fig. 1D displayed spherical spots (green color) which correspond to peroxisomes and Fig. 1E showed the same field analyzed using DAR-4M AM (red color). Spherical spots (red) were found to have a pattern similar to that of GFP-PTS1 (Fig. 1F). When these panels were compared to Fig. 1A and B, calcium supplementation is seen to enable peroxisomes to be visualized on an apparently larger scale but does not affect visualization of NO generated for a putative NOS activity. Arabidopsis seedlings were also pre-incubated with different chemicals that have previously been reported to affect the calcium metabolism which included 5 mM lanthanum chloride (LaCl3, a calcium channel blocker) (Fig. 1G–I) and trifluroperazine (TFP, a calmodulin antagonist) (Fig. 1J–L). When the effect of LaCl3 was compared to the control samples (Fig. 1A–C), the calcium channel blocker appeared to affect the visualization of GFP-PTS1 (Fig. 1H), indicating that calcium possibly regulates peroxisomal protein import and almost eliminates red fluorescence corresponding to the detection of NO. This suggests that the peroxisomal protein responsible for NO generation was specifically affected by this chemical which blocks calcium channels (Fig. 1G). On the other hand, when roots treated with a calmodulin antagonist (Fig. 1J– L) were compared with the control root (Fig. 1A to C), both green (corresponding to peroxisomes) and red (corresponding to NO) fluorescence did not appear as spherical spots and the fluorescence can be seen on the periphery of the cells, indicating that the calmodulin antagonist affected GFP-PTS1 import including the peroxisomal protein responsible for NO generation. To quantify the effect of 5 mM LaCl3 on the import of GFP-PTS1, we determined the fluorescence intensity of GFP (green spots) for control Arabidopsis seedlings and those treated with 5 mM LaCl3. Fig. 2 illustrated that this calcium channel blocker significantly reduced (73%) the import of GFP-PTS1. 3.2. Analysis of pex5i/GFP-PTS1 and pex7i/PTS2-GFP Arabidopsis mutants in peroxisomal NO localization Previously, Hayashi et al. [24] have provided a full characterization of each gene construct for pex5i and pex7i which were inserted under the control of a constitutive 35S promoter from a cauliflower mosaic virus in a Ti vector containing a hygromycin-resistant gene as a selectable marker in plants. They were then integrated into the genomes of two kanamycin-resistant transgenic plants, AtGFPPTS1 and AtPTS2-GFP, through Agrobacterium-mediated transformation [31]. The same authors showed that the absence of sucrose in the growth medium significantly affected the size of both pex5i/ GFP-PTS1 and pex7i/PTS2-GFP mutants, which were smaller in comparison to the parental Arabidopsis plants expressing GFPPTS1 [24]. Both mutants were also affected by peroxisomal fatty acid b-oxidation and were resistant to 2,4-diclrophenoxybutiric acid [32]. On the other hand, under normal atmospheric conditions (36 Pa CO2), both mutants were characterized by dwarf phenotype with yellow/green leaves, indicating that the photorespiration pathway was also affected [24]. To evaluate the type of peroxisomal targeting signal (PTS) present in the protein responsible for NO generation in the peroxisomal matrix, these two Arabidopsis mutants, pex5i and pex7i, were used. The Arabidopsis pex5i mutant was defective in the import of both PTS1- and PTS2-containing proteins, and the pex7i mutant was defective in the import of PTS2-containing proteins. Fig. 3 show in vivo CLSM visualization of NO and peroxisomes in the root of transgenic Arabidopsis seedlings expressing GFP through the addition of PTS1 or PTS2. Fig. 3A illustrated in vivo CLSM visualization of peroxisomes in the root cells of transgenic Arabidopsis plants expressing GFP through the addition of PTS1 used as a posi-
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
3
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
GFP-PTS1 A
NO B
C
→
→
→ →
→
→
→ D
Merged
→
→
10 µm
F
E
+ Ca
G
I
H
+ LaCl3
J
L
K
+ TFP Fig. 1. Representative images illustrating the CLSM in vivo detection of NO (red color) and peroxisomes (green color) in root of 5-d-old Arabidopsis mutant seedlings expressing GFP-PTS1. A, D, G and J show peroxisome detection with GFP (excitation 495 nm; emission 515 nm). B, E, H, and K show NO detection (red color) with DAR-4M AM (excitation 543 nm; emission 575 nm). C, F, I and L show the merged images of the corresponding panels. A–C, control seedlings without any treatment. D–F, seedlings preincubated with 1.5 mM CaCl2. G–I, seedlings preincubated with 5 mM lanthanum chloride (LaCl3 a calcium channel blocker).J–L, seedling preincubated with trifluroperazine (TFP, a calmodulin antagonist). Arrows indicate representative punctate spots corresponding to NO and peroxisome localization. Bar = 10 lm.
tive control to observe both peroxisomes and NO. The peroxisomes appeared in the form of spherical green spots in the cells. Fig. 3B represented the same field, analyzed using DAR-4M AM as a fluorescence probe, which also enabled NO to be detected. An intense red fluorescence was found in the spherical spots with a pattern identical to that of GFP-PTS1. Fig. 3C illustrated the bright-field image of the corresponding sample. Fig. 3D displayed the CLSM visualization of pex5i/GFP-PTS1 in root cells, with, in this case, the green fluorescence being located at the periphery of the cells that coincided with the cytosol surrounding the central vacuoles. Fig. 3E showed NO detection in this mutant, with the distribution of the fluorescence being identical to that observed in Fig. 3D, indicating that the protein responsible for NO generation was not
imported into the peroxisomes. Fig. 3F represented the bright-field image of the corresponding panels. Fig. 3G exhibited the CLSM visualization of pex7i/PTS2-GFP in root cells. The green fluorescence was located at the periphery of the cells, which also coincided with the cytosol surrounding the central vacuoles. Fig. 3H established NO detection in this mutant, and the distribution of the red fluorescence was also identical to that observed in Fig. 3G, indicating that the protein responsible for NO generation was not imported into the peroxisomes. Fig. 3I represented the bright-field image of the corresponding sample. Although, in theory, there is no overlap between the excitation/ emission wavelengths of GFP (excitation 495 nm; emission 515 nm) and the fluorescent probe used to detect nitric oxide
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
4
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
rescence in the root tips of transgenic Arabidopsis plants expressing GFP-PTS1 (panels A to C), pex5i expressing GFP-PTS1 (panels D– F) and pex7i expressing PTS2-GFP (panels G–I). We consequently observed green fluorescence attributable to GFP (panels A, D and G) under conditions of excitation and emission of 495 nm and 515 nm, respectively. However, no fluorescence was observed under conditions of excitation and emission of 543 nm and 575 nm, respectively (panels B, E and H). Therefore, it could be concluded that there is no experimental overlap between the two fluorescence probes used in these experiments.
Pixel Sum · µm-2
150
100
50
*
0
0 0 mM
5 5 mM
4. Discussion
LaCl3 (mM)
Nitric oxide is widely recognized as an outstanding signal molecule under physiological and stress conditions [1–3,5]. How and where NO is produced in plant cells are important questions to be studied in order to advance our knowledge of this radical molecule’s metabolism. In higher plants, previous studies have shown that peroxisomes are compartments with the capacity to generate both reactive oxygen species (ROS) and NO [6,33]. Thus, an initial study using isolated pea leaf peroxisomes showed that these organelles contain a L-arginine-dependent nitric oxide synthase (NOS) activity immunorelated to animal NOS which require all the co-factors needed for animal NOS activity including NADPH, FAD, FMN, BH4, calmodulin and calcium [15]. Later, an inducible nitric oxide synthase was also reported to be located in the peroxisomes of rat hepatocytes [34,35]. This supports the claim that NOS activity could be an enzymatic component common to both plant and animal peroxisomes. Additionally, it was demonstrated, using
Fig. 2. Effect of 5 mM lanthanum chloride (LaCl3, a calcium channel blocker) in the fluorescence intensity of GFP (green spots) in root tips of 5-d-old Arabidopsis seedlings expressing GFP-PTS1. The fluorescence produced is expressed as Pixel Sum lm-2 using Leica confocal software. Results are the means ± SEM obtained in various root areas from ten different seedlings preincubated or not with 5 mM LaCl3. Asterisks indicate that variations in relation to control values were significant at P < 0.05.
(DAR-AM AM; excitation 543 nm; emission 575 nm), several controls were carried out to evaluate potential overlap at the experimental level. Thus, all the transgenic Arabidopsis plants expressing GFP-PTS1, pex5i expressing GFP-PTS1 and pex7i expressing PTS2-GFP were observed using CLSM under both types of conditions but in the absence of the fluorescence probe for NO. Supplementary Fig. 2 illustrated in vivo CLSM visualization of fluo-
→
C
NO
→
B
GFP-PTS1
→
A
→
→
→
→
10 μm
D
pex5i/GFP-PTS1
E
NO
F
15 μm
G
pex7i/PTS2-GFP
H
NO
I
14 μm
Fig. 3. Representative images illustrating the CLSM in vivo detection of NO (red color) and peroxisomes (green color) in root of 5-d-old Arabidopsis seedlings of parent plants (panels A–C), mutant pex5i (panels D–F) expressing GFP-PTS1 and pex7i (panels G–I) expressing PTS2-GFP. A, D, and G show peroxisome detection with GFP (excitation 495 nm; emission 515 nm). B, E, and H show NO detection (red color) with DAR-4M AM (excitation 543 nm; emission 575 nm). C, F, and I show the bright-field image of the corresponding samples. Arrows indicate representative punctate spots corresponding to NO and peroxisome localization.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
5
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
spin trapping electron paramagnetic resonance (EPR) spectroscopy, that pea peroxisomes produce NO which is down regulated during the natural senescence of leaves [16]. Furthermore, peroxisomal NO has also been shown to be important in other plant species and processes such as pollen tube growth [36], auxin-induced root organogenesis [37] as well participation in the mechanism of response to abiotic stress conditions such as salinity [17] or cadmium [18]. More recently, in Arabidopsis peroxisomes, it has been demonstrated that peroxins PEX12 and PEX13, which are peroxisomal membrane proteins that participate in the import of the peroxisomal matrix [14,28], were involved in transporting the putative NOS protein to peroxisomes, as pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, showed lower NO content. Consequently, the aim of this study is to gain a deeper understanding of the peroxisomal protein responsible for NO generation and thus to determine whether this peroxisomal protein is imported into peroxisomes throughout PTS1 or PTS2. The Arabidopsis pex5i mutant exhibits defects in relation to the import of both PTS-1 and PTS2-containing proteins, whereas the pex7i mutant appeared to be defective in terms of importing PTS2-containing proteins [24]. However, it has recently been established that the import of the PTS2 cargo into plant peroxisomes requires not only PTS2 receptor PEX7 but also PTS1 receptor PEX5 [24,38–42]. Our findings suggest that the plant peroxisomal protein responsible for NO generation must contain PTS2. This is based in two factors: (i) in pex5i mutants defective in terms of the import of GFP–PTS1, the red fluorescence resulting from NO generation for NOS protein activity does not appear in peroxisomes; (ii) also, in pex7i mutants defective in terms of the import of PTS2-GFP but with the capacity to import protein with PTS1 as PEX5 is unaffected in this mutant, red fluorescence resulting from NO generation for NOS protein activity is not observed into peroxisomes. These findings are in line with a recent study which proves that the peroxisomal isoform of iNOS present in rat hepatocytes is also imported by PTS2 [43]. These data suggest that both plants and animals contain a similar mechanism for importing the protein responsible for NO generation into peroxisomes. Calcium (Ca2+) is a secondary messenger in many signal transduction pathways in plants although very little information exists on calcium signalling in peroxisomes [44]. Previous data have demonstrated that peroxisomal NOS activity assayed by L-[3H] citrulline production depends on calcium, as pre-incubation with a calcium chelator (EGTA) inhibited activity by 73% [15]. This calcium-dependent NO production was corroborated by an alternative technique which assayed the NO production of peroxisomal NOS activity using ozone chemiluminescence [16] and by the reduction in NO detected by the fluorescent probe DAR-4M AM in Arabidopsis mutants expressing GFP-PTS1 pre-incubated with EGTA [17]. However, our findings provide additional information as the calcium channel blocker (LaCl3) appears to affect the protein import of peroxisomal proteins and consequently the protein responsible for peroxisomal NO production. Calmodulin (CaM) is a ubiquitous transducer of Ca2+ signals in eukaryotic cells. Although plant CaM has been regarded as a cytosolic protein, it is now reported to be present in other cell compartments including plant peroxisomes [42]. The peroxisomal CaM is necessary for the activation of catalase [45] and has also been shown to participate in the proteolytic processing of peroxisomal proteins by PTS2 after these proteins are in the peroxisomal matrix [46,47]. Based on a previous model of peroxisomal import systems [26], Fig. 4 shows a straightforward hypothetical model which summarizes the components known to be specifically involved in importing the peroxisomal protein responsible for NO generation. Thus, the putative peroxisomal plant NOS contains PTS2. This PTS2-NOS can interact with the PEX5–PEX7 complex, two cytosolic receptors which can
PEX5 PEX7 PTS2 Putative-NOS CaM Ca2+
Cytosol
PEX12 Protease
Matrix
PTS2 Putative-NOS
L-arginine
NADPH CaM Ca2+
NO
Fig. 4. Simplify hypothetical model of the known elements involved in the mechanism of import of the peroxisomal protein responsible of NO generation into the peroxisome. CaM, calmodulin. NOS, nitric oxide synthase. PEX5, pexosin 5. PEX7, pexosin 7. PEX12, pexosin 12.
also interact with PTS1-containing proteins. The PEX5–PEX7– PTS2-NOS complex is transferred to PEX12, a peroxisomal membrane protein which enables PTS2-NOS to be imported to the peroxisomal matrix that requires CaM and calcium. The PTS2-NOS will then be processed by peroxisomal proteases present in the matrix [48–50]. Subsequently, the L-arginine-dependent NOS can generate NO which requires NAPDH, calcium and CaM. In summary, our findings provide new insights into the mechanism involved in importing the peroxisomal protein responsible for NO generation in plants which appears to have a type 2 (PTS2) peroxisomal targeting signal that requires both calcium and CaM. Further studies will be necessary to determine the identity of this peroxisomal NOS in higher plants. Acknowledgements This work was supported by grants from the Ministry of Science and Innovation (projects BIO2009-12003-C02-01, BIO2009-12003C02-02 and BIO2012-33904) and Junta de Andalucía (groups BIO286 and BIO192). Drs M. Nishimura, M. Hayashi and S. Mano from National Institute for Basic Biology, Okazaki (Japan) are deeply acknowledged for providing Arabidopsis pex5i and pex7i mutants. Technical and human support provided by CICT of Universidad de Jaén (UJA, MINECO, Junta de Andalucía, FEDER) is also acknowledged. Authors are grateful to Mr. Carmelo Ruíz-Torres for his excellent technical support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014. 04.034. References [1] Lamattina, L., García-Mata, C., Graziano, M. and Pagnussat, G. (2003) Nitric oxide: the versatility of an extensive signal molecule. Annu. Rev. Plant Biol. 54, 109–136. [2] Shapiro, A.D. (2005) Nitric oxide signaling in plants. Vitam. Horm. 72, 339– 398. [3] Besson-Bard, A., Pugin, A. and Wendehenne, D. (2008) New insights into nitric oxide signaling in plants. Annu. Rev. Plant Biol. 59, 21–39. [4] Baudhouin, E. (2011) The language of nitric oxide signaling. Plant Biol. (Stuttg) 13, 233–242.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
6
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
[5] Corpas, F.J., Leterrier, M., Valderrama, R., Airaki, M., Chaki, M., Palma, J.M. and Barroso, J.B. (2011) Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress. Plant Sci. 181 (5), 604–611. [6] Corpas, F.J., Barroso, J.B. and del Río, L.A. (2001) Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci. 6, 145–150. [7] Takahashi, S. and Yamasaki, H. (2002) Reversible inhibition of photophosphorylation in chloroplasts by nitric oxide. FEBS Lett. 512, 145–148. [8] Gabaldón, C., Gómez-Ros, L.V., Pedreño, M.A. and Ros-Barceló, A. (2005) Nitric oxide production by the differentiating xylem of Zinnia elegans. New Phytol. 165, 121–130. [9] Jasid, S., Simontacchi, M., Bartoli, C.G. and Puntarulo, S. (2006) Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins. Plant Physiol. 142, 1246–1255. [10] Gupta, K.J., Igamberdiev, A.U., Manjunatha, G., Segu, S., Moran, J.F., Neelawarne, B., Bauwe, H. and Kaiser, W.M. (2011) The emerging roles of nitric oxide (NO) in plant mitochondria. Plant Sci. 181, 520–526. [11] Tewari, R.K., Prommer, J. and Watanabe, M. (2013) Endogenous nitric oxide generation in protoplast chloroplasts. Plant Cell Rep. 32, 31–44. [12] Corpas, F.J., Palma, J.M., del Río, L.A. and Barroso, J.B. (2009) Evidence supporting the existence of L-arginine-dependent nitric oxide synthase (NOS) activity in plants. New Phytol. 184, 9–14. [13] Foresi, N., Correa-Aragunde, N., Parisi, G., Caló, G., Salerno, G. and Lamattina, L. (2010) Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent. Plant Cell 22, 3816–3830. [14] Mullen, R.T., Flynn, C.R. and Trelease, R.N. (2001) How are peroxisomes formed? The role of the endoplasmic reticulum and peroxins. Trends Plant Sci. 6, 256–261. [15] 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, 36729–36733. [16] Corpas, F.J., Barroso, J.B., Carreras, A., Quirós, M., León, A.M., Romero-Puertas, M.C., Esteban, F.J., Valderrama, R., Palma, J.M., Sandalio, L.M., Gómez, M. and del Río, L.A. (2004) Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiol. 136, 2722–2733. [17] 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 Physiol. 151, 2083–2094. [18] Corpas, F.J. and Barroso, J.B. (2014) Peroxynitrite (ONOO-) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress. Ann. Bot. 113, 87–96. [19] Barroso, J.B., Valderrama, R. and Corpas, F.J. (2013) Immunolocalization of Snitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles. Acta Physiol. Plant 35, 2635–2640. [20] Reumann, S. (2004) Specification of the peroxisome targeting signals type 1 and type 2 of plant peroxisomes by bioinformatics analyses. Plant Physiol. 135, 783–800. [21] Reumann, S., Babujee, L., Ma, C., Wienkoop, S., Siemsen, T., Antonicelli, G.E., Rasche, N., Lüder, F., Weckwerth, W. and Jahn, O. (2007) Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19, 3170–3193. [22] Hu, J., Baker, A., Bartel, B., Linka, N., Mullen, R.T., Reumann, S. and Zolman, B.K. (2012) Plant peroxisomes: biogenesis and function. Plant Cell 24, 2279–2303. [23] Hayashi, M. and Nishimura, M. (2006) Arabidopsis thaliana – a model organism to study plant peroxisomes. Biochim. Biophys. Acta 1763, 1382–1391. [24] Hayashi, M., Yagi, M., Nito, K., Kamada, T. and Nishimura, M. (2005) Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis. J. Biol. Chem. 280, 14829–14835. [25] Nito, K., Kamigaki, A., Kondo, M., Hayashi, M. and Nishimura, M. (2007) Functional classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of knockdown mutants. Plant Cell Physiol. 48, 763– 774. [26] Singh, T., Hayashi, M., Mano, S., Arai, Y., Goto, S. and Nishimura, M. (2009) Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana. Plant J. 60, 488–498. [27] Ramón, N.M. and Bartel, B. (2010) Interdependence of the peroxisometargeting receptors in Arabidopsis thaliana: PEX7 facilitates PEX5 accumulation and import of pts1 cargo into peroxisomes. Mol. Biol. Cell 21, 1263–1271. [28] Mano, S., Nakamori, C., Nito, K., Kondo, M. and Nishimura, M. (2006) The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2dependent protein transport to peroxisomes. Plant J. 47, 604–618.
[29] Friedman, H., Meir, S., Rosenberger, I., Halevy, A.H., Kaufman, P.B. and Philosoph-Hadas, S. (1998) Inhibition of the gravitropic response of snapdragon spikes by the calcium-channel blocker lanthanum chloride. Plant Physiol. 118, 483–492. [30] Chen, T., Wu, X., Chen, Y., Li, X., Huang, M., Zheng, M., Baluska, F., Samaj, J. and Lin, J. (2009) Combined proteomic and cytological analysis of Ca2+-calmodulin regulation in Picea meyeri pollen tube growth. Plant Physiol. 149, 1111–1126. [31] Mano, S., Nakamori, C., Hayashi, M., Kato, A., Kondo, M. and Nishimura, M. (2002) Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement. Plant Cell Physiol. 43, 331–341. [32] Hayashi, M., Toriyama, K., Kondo, M. and Nishimura, M. (1998) 2,4Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid beta-oxidation. Plant Cell 10, 183–195. [33] del Río, L.A., Corpas, F.J., Sandalio, L.M., Palma, J.M., Gómez, M. and Barroso, J.B. (2002) Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. J. Exp. Bot. 53, 1255–1272. [34] Stolz, D.B., Zamora, R., Vodovotz, Y., Loughran, P.A., Billiar, T.R., Kim, Y.M., Simmons, R.L. and Watkins, S.C. (2002) Peroxisomal localization of inducible nitric oxide synthase in hepatocytes. Hepatology 36, 81–93. [35] Loughran, P.A., Stolz, D.B., Vodovotz, Y., Watkins, S.C., Simmons, R.L. and Billiar, T.R. (2005) Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 102, 13837–13842. [36] Prado, A.M., Porterfield, D.M. and Feijo, J.A. (2004) Nitric oxide is involved in growth regulation and re-orientation of pollen tubes. Development 131, 2707–2714. [37] Schlicht, M., Ludwig-Müller, J., Burbach, C., Volkmann, D. and Baluska, F. (2013) Indole-3-butyric acid induces lateral root formation via peroxisomederived indole-3-acetic acid and nitric oxide. New Phytol. 200, 473–482. [38] Khan, B.R. and Zolman, B.K. (2010) Pex5 mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis. Plant Physiol. 154, 1602–1615. [39] Braverman, N., Dodt, G., Gould, S.J. and Valle, D. (1998) An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum. Mol. Genet. 7, 1195–1205. [40] Otera, H., Okumoto, K., Tateishi, K., Ikoma, Y., Matsuda, E., Nishimura, M., Tsukamoto, T., Osumi, T., Ohashi, K., Higuchi, O. and Fujiki, Y. (1998) Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants. Mol. Cell. Biol. 18, 388–399. [41] Lee, J.R., Jang, H.H., Park, J.H., Jung, J.H., Lee, S.S., Park, S.K., Chi, Y.H., Moon, J.C., Lee, Y.M., Kim, S.Y., Kim, J.Y., Yun, D.J., Cho, M.J., Lee, K.O. and Lee, S.Y. (2006) Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis. Plant J. 47, 457–466. [42] Woodward, A.W. and Bartel, B. (2005) The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5. Mol. Biol. Cell 16, 573–583. [43] Loughran, P.A., Stolz, D.B., Barrick, S.R., Wheeler, D.S., Friedman, P.A., Rachubinski, R.A., Watkins, S.C. and Billiar, T.R. (2013) PEX7 and EBP50 target iNOS to the peroxisome in hepatocytes. Nitric Oxide 31, 9–19. [44] Stael, S., Wurzinger, B., Mair, A., Mehlmer, N., Vothknecht, U.C. and Teige, M. (2012) Plant organellar calcium signalling: an emerging field. J. Exp. Bot. 63, 1525–1542. [45] Al-Quraan, N.A., Locy, R.D. and Singh, N.K. (2010) Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress. Plant Physiol. Biochem. 48, 697–702. [46] Yang, T. and Poovaiah, B.W. (2002) Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin. Proc. Natl. Acad. Sci. U.S.A. 99, 4097–4102. [47] Chigri, F., Flosdorff, S., Pilz, S., Kölle, E., Dolze, E., Gietl, C. and Vothknecht, U.C. (2012) The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively. Plant Mol. Biol. 78, 211–222. [48] Dolze, E., Chigri, F., Höwing, T., Hierl, G., Isono, E., Vothknecht, U.C. and Gietl, C. (2013) Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism. Plant Mol. Biol. 83, 607–624. [49] Corpas, F.J., Palma, J.M. and del Río, L.A. (1993) Evidence for the presence of proteolytic activity in peroxisomes. Eur. J. Cell Biol. 61, 81–85. [50] Distefano, S., Palma, J.M., Gómez, M. and Río, L.A. (1997) Characterization of endoproteases from plant peroxisomes. Biochem. J. 327 (Pt 2), 399–405.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
journal homepage: www.FEBSLetters.org
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 Francisco J. Corpas a,⇑, Juan B. Barroso b a Group 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, Apartado 419, E-18080 Granada, Spain b Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus ‘‘Las Lagunillas’’, E-23071 Jaén, Spain
a r t i c l e
i n f o
Article history: Received 22 February 2014 Revised 18 April 2014 Accepted 23 April 2014 Available online xxxx Edited by Ulf-Ingo Flügge Keywords: Nitric oxide Nitric oxide synthase Peroxin Peroxisome Peroxisomal targeting signal Arabidopsis thaliana
a b s t r a c t Nitric oxide (NO) production in plant peroxisomes by L-arginine-dependent NO synthase activity has been proven. The PEX5 and PEX7 PTS receptors, which recognize PTS1- and PTS2-containing proteins, are localized in the cytosol. Using AtPex5p and AtPex7p knockdown in Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i, we found that the L-arginine-dependent protein responsible for NO generation in peroxisomes appears to be imported through an N-terminal PTS2. Pharmacological analyzes using a calcium channel blocker and calmodulin (CaM) antagonist show that the import of the peroxisomal NOS protein also depends on calcium and calmodulin. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Nitric oxide (NO) is a free radical messenger with a broad spectrum of regulatory functions in many physiological and pathological processes in higher plants [1–5]. At subcellular level, NO has been reported to be generated in different cell types and compartments including peroxisomes, chloroplasts, mitochondria and cytosol [6–11]. However, identification of the protein responsible for L-arginine-dependent NO production is still an open question even though supporting evidence has been found [12] which includes the identification of a gene which codified for a putative NOS protein in a green alga that showed a 40% identity with animal NOSs [13]. Peroxisomes are single membrane-bounded subcellular organelles with an essentially oxidative type of metabolism and a simple morphology that does not mirror the complexity of their enzymatic composition. They harbour variable metabolic pathways that differ depending on cell type, developmental stage and environmental conditions [6,14]. In higher plants, previous data have demonstrated that peroxisomes have an active NO metabolism given ⇑ Corresponding author. Fax: +34 958 129600. E-mail address: [email protected] (F.J. Corpas).
the presence of L-arginine-dependent NOS activity in pea peroxisomes [15], NO modulation during senescence [16], salinity [17] and cadmium stress [18] as well as the peroxisomal localization of other NO-derived molecules such as S-nitrosoglutathione [19] and peroxynitrite [18]. As peroxisomes do not have DNA, all its proteins are nuclear-encoded. There are two principal signals that direct proteins into peroxisomes found at the C-terminus (peroxisomal targeting signal type 1 or PTS1) and N-terminus (peroxisomal targeting signal type 2 or PTS2) of the protein [20–22]. Peroxins (PEX) are peroxisomal biogenesis factors encoded by PEX genes, some of which are involved in matrix protein transport [23]. Previously, it has been shown that peroxins PEX12 and PEX13 appear to be involved in transporting the putative NOS protein to peroxisomes, as pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content [17]. PEX5p and PEX7p are cytosolic receptors for PTS1 and PTS2, respectively, and form a receptor– cargo complex in the cytosol during transport to peroxisomes [24–27]. Using Arabidopsis knockdown mutants obtained by RNA interference (RNAi) AtPex5p (pex5i) and AtPex7p (pex7i) [24], the aim of the present study is to analyze the potential involvement of PEX5 and/or PEX7 in the import of the peroxisomal protein involved in NO generation in plants.
http://dx.doi.org/10.1016/j.febslet.2014.04.034 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
2
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
2. Materials and methods 2.1. Plant material and growth conditions Arabidopsis thaliana Columbia wild-type and mutants expressing GFP-PTS1 [28] were used as control plants. Constructs of transgenic plants and characterization of mutant AtPex5p expressing GFP-PTS1 and mutant AtPex7p expressing PTS2-GFP knockdown Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i were characterized previously [24]. Seeds were surface-sterilized for 5 min in 70% ethanol containing 0.1% SDS, then placed for 20 min in sterile water containing 20% bleach and 0.1% SDS and washed four times in sterile water. The seeds were sown for 2 days at 4 °C in the dark for vernalization on the basal growth medium composed of 4.32 g/l commercial Murashige and Skoog medium (Sigma) with a pH of 5.5, containing 1% sucrose and 0.8% phytoagar. The Arabidopsis seeds were then grown on Petri dishes in the dark at 22 °C for 5 days for the pex5i and pex7i mutants and at 16 h light/8 h dark (long-day conditions) under a light intensity of 100 lE m 2 s 1 for the GFP-PTS1 mutants. 2.2. Detection of NO in mutants expressing GFP-PTS1 or PTS2-GFP using confocal laser scanning microscopy (CLSM) Nitric oxide was detected using fluorescent reagent diaminorhodamine-4M acetoxymethyl ester (DAR-AM AM, Calbiochem). Arabidopsis seedlings were incubated at 25 °C for 1 h in darkness with 5 lM DAR-AM AM prepared in 100 mM potassium phosphate buffer (pH 7.4) [17]. Each sample was then washed twice in the same buffer for 15 min and mounted on a slide for examination with a confocal laser scanning microscope (Leica TCS SL or Leica TCS SP5 II). For green fluorescent protein (GFP), the excitation laser wavelength was 496 nm and emission of 515 with a 10-nm band pass width (500–525 nm). For DAR-AM T, the excitation laser wavelength was 543 nm and emission of 575 nm with a 40-nm band pass width (570–600 nm). As control for DAR-AM AM as fluorescent probe to detect NO in peroxisomes, Supplemental Fig. 1 shows a highly magnified image of root cells from Arabidopsis wild-type plants, with red spherical spots resembling peroxisomes inside the cells. 2.3. Pharmacological analyzes using calcium channel blocker and calmodulin antagonist Five-day-old Arabidopsis seedlings expressing GFP-PTS1 were preincubated for 120 min at 25 °C with either 1.5 mM CaCl2, 5 mM lanthanum chloride (LaCl3, a calcium channel blocker) [29] or 300 lM trifluroperazine (TFP, a calmodulin antagonist) [30] and then incubated with 5 lM DAR-AM AM for 1 h at 25 °C to detect and visualize NO 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. The images were processed and analyzed using statistical Leica confocal software. 3. Results 3.1. Analysis of calcium and calmodulin in peroxisomal NO localization Fig. 1A showed in vivo CLSM visualization of peroxisomes in the root cells of transgenic arabidopsis plants expressing GFP-PTS1. The peroxisomes appeared in the form of spherical spots (green color). Fig. 1B illustrated the same field analyzed using the DAR4M AM fluorescence probe, which also enabled NO (red in color) to be detected. Spherical spots (red) were found to have a pattern
identical to that of GFP-PTS1 (Fig. 1C). Fig. 1D and E provided images of an arabidopsis seedling root pre-incubated with 1.5 mM calcium. Fig. 1D displayed spherical spots (green color) which correspond to peroxisomes and Fig. 1E showed the same field analyzed using DAR-4M AM (red color). Spherical spots (red) were found to have a pattern similar to that of GFP-PTS1 (Fig. 1F). When these panels were compared to Fig. 1A and B, calcium supplementation is seen to enable peroxisomes to be visualized on an apparently larger scale but does not affect visualization of NO generated for a putative NOS activity. Arabidopsis seedlings were also pre-incubated with different chemicals that have previously been reported to affect the calcium metabolism which included 5 mM lanthanum chloride (LaCl3, a calcium channel blocker) (Fig. 1G–I) and trifluroperazine (TFP, a calmodulin antagonist) (Fig. 1J–L). When the effect of LaCl3 was compared to the control samples (Fig. 1A–C), the calcium channel blocker appeared to affect the visualization of GFP-PTS1 (Fig. 1H), indicating that calcium possibly regulates peroxisomal protein import and almost eliminates red fluorescence corresponding to the detection of NO. This suggests that the peroxisomal protein responsible for NO generation was specifically affected by this chemical which blocks calcium channels (Fig. 1G). On the other hand, when roots treated with a calmodulin antagonist (Fig. 1J– L) were compared with the control root (Fig. 1A to C), both green (corresponding to peroxisomes) and red (corresponding to NO) fluorescence did not appear as spherical spots and the fluorescence can be seen on the periphery of the cells, indicating that the calmodulin antagonist affected GFP-PTS1 import including the peroxisomal protein responsible for NO generation. To quantify the effect of 5 mM LaCl3 on the import of GFP-PTS1, we determined the fluorescence intensity of GFP (green spots) for control Arabidopsis seedlings and those treated with 5 mM LaCl3. Fig. 2 illustrated that this calcium channel blocker significantly reduced (73%) the import of GFP-PTS1. 3.2. Analysis of pex5i/GFP-PTS1 and pex7i/PTS2-GFP Arabidopsis mutants in peroxisomal NO localization Previously, Hayashi et al. [24] have provided a full characterization of each gene construct for pex5i and pex7i which were inserted under the control of a constitutive 35S promoter from a cauliflower mosaic virus in a Ti vector containing a hygromycin-resistant gene as a selectable marker in plants. They were then integrated into the genomes of two kanamycin-resistant transgenic plants, AtGFPPTS1 and AtPTS2-GFP, through Agrobacterium-mediated transformation [31]. The same authors showed that the absence of sucrose in the growth medium significantly affected the size of both pex5i/ GFP-PTS1 and pex7i/PTS2-GFP mutants, which were smaller in comparison to the parental Arabidopsis plants expressing GFPPTS1 [24]. Both mutants were also affected by peroxisomal fatty acid b-oxidation and were resistant to 2,4-diclrophenoxybutiric acid [32]. On the other hand, under normal atmospheric conditions (36 Pa CO2), both mutants were characterized by dwarf phenotype with yellow/green leaves, indicating that the photorespiration pathway was also affected [24]. To evaluate the type of peroxisomal targeting signal (PTS) present in the protein responsible for NO generation in the peroxisomal matrix, these two Arabidopsis mutants, pex5i and pex7i, were used. The Arabidopsis pex5i mutant was defective in the import of both PTS1- and PTS2-containing proteins, and the pex7i mutant was defective in the import of PTS2-containing proteins. Fig. 3 show in vivo CLSM visualization of NO and peroxisomes in the root of transgenic Arabidopsis seedlings expressing GFP through the addition of PTS1 or PTS2. Fig. 3A illustrated in vivo CLSM visualization of peroxisomes in the root cells of transgenic Arabidopsis plants expressing GFP through the addition of PTS1 used as a posi-
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
3
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
GFP-PTS1 A
NO B
C
→
→
→ →
→
→
→ D
Merged
→
→
10 µm
F
E
+ Ca
G
I
H
+ LaCl3
J
L
K
+ TFP Fig. 1. Representative images illustrating the CLSM in vivo detection of NO (red color) and peroxisomes (green color) in root of 5-d-old Arabidopsis mutant seedlings expressing GFP-PTS1. A, D, G and J show peroxisome detection with GFP (excitation 495 nm; emission 515 nm). B, E, H, and K show NO detection (red color) with DAR-4M AM (excitation 543 nm; emission 575 nm). C, F, I and L show the merged images of the corresponding panels. A–C, control seedlings without any treatment. D–F, seedlings preincubated with 1.5 mM CaCl2. G–I, seedlings preincubated with 5 mM lanthanum chloride (LaCl3 a calcium channel blocker).J–L, seedling preincubated with trifluroperazine (TFP, a calmodulin antagonist). Arrows indicate representative punctate spots corresponding to NO and peroxisome localization. Bar = 10 lm.
tive control to observe both peroxisomes and NO. The peroxisomes appeared in the form of spherical green spots in the cells. Fig. 3B represented the same field, analyzed using DAR-4M AM as a fluorescence probe, which also enabled NO to be detected. An intense red fluorescence was found in the spherical spots with a pattern identical to that of GFP-PTS1. Fig. 3C illustrated the bright-field image of the corresponding sample. Fig. 3D displayed the CLSM visualization of pex5i/GFP-PTS1 in root cells, with, in this case, the green fluorescence being located at the periphery of the cells that coincided with the cytosol surrounding the central vacuoles. Fig. 3E showed NO detection in this mutant, with the distribution of the fluorescence being identical to that observed in Fig. 3D, indicating that the protein responsible for NO generation was not
imported into the peroxisomes. Fig. 3F represented the bright-field image of the corresponding panels. Fig. 3G exhibited the CLSM visualization of pex7i/PTS2-GFP in root cells. The green fluorescence was located at the periphery of the cells, which also coincided with the cytosol surrounding the central vacuoles. Fig. 3H established NO detection in this mutant, and the distribution of the red fluorescence was also identical to that observed in Fig. 3G, indicating that the protein responsible for NO generation was not imported into the peroxisomes. Fig. 3I represented the bright-field image of the corresponding sample. Although, in theory, there is no overlap between the excitation/ emission wavelengths of GFP (excitation 495 nm; emission 515 nm) and the fluorescent probe used to detect nitric oxide
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
4
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
rescence in the root tips of transgenic Arabidopsis plants expressing GFP-PTS1 (panels A to C), pex5i expressing GFP-PTS1 (panels D– F) and pex7i expressing PTS2-GFP (panels G–I). We consequently observed green fluorescence attributable to GFP (panels A, D and G) under conditions of excitation and emission of 495 nm and 515 nm, respectively. However, no fluorescence was observed under conditions of excitation and emission of 543 nm and 575 nm, respectively (panels B, E and H). Therefore, it could be concluded that there is no experimental overlap between the two fluorescence probes used in these experiments.
Pixel Sum · µm-2
150
100
50
*
0
0 0 mM
5 5 mM
4. Discussion
LaCl3 (mM)
Nitric oxide is widely recognized as an outstanding signal molecule under physiological and stress conditions [1–3,5]. How and where NO is produced in plant cells are important questions to be studied in order to advance our knowledge of this radical molecule’s metabolism. In higher plants, previous studies have shown that peroxisomes are compartments with the capacity to generate both reactive oxygen species (ROS) and NO [6,33]. Thus, an initial study using isolated pea leaf peroxisomes showed that these organelles contain a L-arginine-dependent nitric oxide synthase (NOS) activity immunorelated to animal NOS which require all the co-factors needed for animal NOS activity including NADPH, FAD, FMN, BH4, calmodulin and calcium [15]. Later, an inducible nitric oxide synthase was also reported to be located in the peroxisomes of rat hepatocytes [34,35]. This supports the claim that NOS activity could be an enzymatic component common to both plant and animal peroxisomes. Additionally, it was demonstrated, using
Fig. 2. Effect of 5 mM lanthanum chloride (LaCl3, a calcium channel blocker) in the fluorescence intensity of GFP (green spots) in root tips of 5-d-old Arabidopsis seedlings expressing GFP-PTS1. The fluorescence produced is expressed as Pixel Sum lm-2 using Leica confocal software. Results are the means ± SEM obtained in various root areas from ten different seedlings preincubated or not with 5 mM LaCl3. Asterisks indicate that variations in relation to control values were significant at P < 0.05.
(DAR-AM AM; excitation 543 nm; emission 575 nm), several controls were carried out to evaluate potential overlap at the experimental level. Thus, all the transgenic Arabidopsis plants expressing GFP-PTS1, pex5i expressing GFP-PTS1 and pex7i expressing PTS2-GFP were observed using CLSM under both types of conditions but in the absence of the fluorescence probe for NO. Supplementary Fig. 2 illustrated in vivo CLSM visualization of fluo-
→
C
NO
→
B
GFP-PTS1
→
A
→
→
→
→
10 μm
D
pex5i/GFP-PTS1
E
NO
F
15 μm
G
pex7i/PTS2-GFP
H
NO
I
14 μm
Fig. 3. Representative images illustrating the CLSM in vivo detection of NO (red color) and peroxisomes (green color) in root of 5-d-old Arabidopsis seedlings of parent plants (panels A–C), mutant pex5i (panels D–F) expressing GFP-PTS1 and pex7i (panels G–I) expressing PTS2-GFP. A, D, and G show peroxisome detection with GFP (excitation 495 nm; emission 515 nm). B, E, and H show NO detection (red color) with DAR-4M AM (excitation 543 nm; emission 575 nm). C, F, and I show the bright-field image of the corresponding samples. Arrows indicate representative punctate spots corresponding to NO and peroxisome localization.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
5
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
spin trapping electron paramagnetic resonance (EPR) spectroscopy, that pea peroxisomes produce NO which is down regulated during the natural senescence of leaves [16]. Furthermore, peroxisomal NO has also been shown to be important in other plant species and processes such as pollen tube growth [36], auxin-induced root organogenesis [37] as well participation in the mechanism of response to abiotic stress conditions such as salinity [17] or cadmium [18]. More recently, in Arabidopsis peroxisomes, it has been demonstrated that peroxins PEX12 and PEX13, which are peroxisomal membrane proteins that participate in the import of the peroxisomal matrix [14,28], were involved in transporting the putative NOS protein to peroxisomes, as pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, showed lower NO content. Consequently, the aim of this study is to gain a deeper understanding of the peroxisomal protein responsible for NO generation and thus to determine whether this peroxisomal protein is imported into peroxisomes throughout PTS1 or PTS2. The Arabidopsis pex5i mutant exhibits defects in relation to the import of both PTS-1 and PTS2-containing proteins, whereas the pex7i mutant appeared to be defective in terms of importing PTS2-containing proteins [24]. However, it has recently been established that the import of the PTS2 cargo into plant peroxisomes requires not only PTS2 receptor PEX7 but also PTS1 receptor PEX5 [24,38–42]. Our findings suggest that the plant peroxisomal protein responsible for NO generation must contain PTS2. This is based in two factors: (i) in pex5i mutants defective in terms of the import of GFP–PTS1, the red fluorescence resulting from NO generation for NOS protein activity does not appear in peroxisomes; (ii) also, in pex7i mutants defective in terms of the import of PTS2-GFP but with the capacity to import protein with PTS1 as PEX5 is unaffected in this mutant, red fluorescence resulting from NO generation for NOS protein activity is not observed into peroxisomes. These findings are in line with a recent study which proves that the peroxisomal isoform of iNOS present in rat hepatocytes is also imported by PTS2 [43]. These data suggest that both plants and animals contain a similar mechanism for importing the protein responsible for NO generation into peroxisomes. Calcium (Ca2+) is a secondary messenger in many signal transduction pathways in plants although very little information exists on calcium signalling in peroxisomes [44]. Previous data have demonstrated that peroxisomal NOS activity assayed by L-[3H] citrulline production depends on calcium, as pre-incubation with a calcium chelator (EGTA) inhibited activity by 73% [15]. This calcium-dependent NO production was corroborated by an alternative technique which assayed the NO production of peroxisomal NOS activity using ozone chemiluminescence [16] and by the reduction in NO detected by the fluorescent probe DAR-4M AM in Arabidopsis mutants expressing GFP-PTS1 pre-incubated with EGTA [17]. However, our findings provide additional information as the calcium channel blocker (LaCl3) appears to affect the protein import of peroxisomal proteins and consequently the protein responsible for peroxisomal NO production. Calmodulin (CaM) is a ubiquitous transducer of Ca2+ signals in eukaryotic cells. Although plant CaM has been regarded as a cytosolic protein, it is now reported to be present in other cell compartments including plant peroxisomes [42]. The peroxisomal CaM is necessary for the activation of catalase [45] and has also been shown to participate in the proteolytic processing of peroxisomal proteins by PTS2 after these proteins are in the peroxisomal matrix [46,47]. Based on a previous model of peroxisomal import systems [26], Fig. 4 shows a straightforward hypothetical model which summarizes the components known to be specifically involved in importing the peroxisomal protein responsible for NO generation. Thus, the putative peroxisomal plant NOS contains PTS2. This PTS2-NOS can interact with the PEX5–PEX7 complex, two cytosolic receptors which can
PEX5 PEX7 PTS2 Putative-NOS CaM Ca2+
Cytosol
PEX12 Protease
Matrix
PTS2 Putative-NOS
L-arginine
NADPH CaM Ca2+
NO
Fig. 4. Simplify hypothetical model of the known elements involved in the mechanism of import of the peroxisomal protein responsible of NO generation into the peroxisome. CaM, calmodulin. NOS, nitric oxide synthase. PEX5, pexosin 5. PEX7, pexosin 7. PEX12, pexosin 12.
also interact with PTS1-containing proteins. The PEX5–PEX7– PTS2-NOS complex is transferred to PEX12, a peroxisomal membrane protein which enables PTS2-NOS to be imported to the peroxisomal matrix that requires CaM and calcium. The PTS2-NOS will then be processed by peroxisomal proteases present in the matrix [48–50]. Subsequently, the L-arginine-dependent NOS can generate NO which requires NAPDH, calcium and CaM. In summary, our findings provide new insights into the mechanism involved in importing the peroxisomal protein responsible for NO generation in plants which appears to have a type 2 (PTS2) peroxisomal targeting signal that requires both calcium and CaM. Further studies will be necessary to determine the identity of this peroxisomal NOS in higher plants. Acknowledgements This work was supported by grants from the Ministry of Science and Innovation (projects BIO2009-12003-C02-01, BIO2009-12003C02-02 and BIO2012-33904) and Junta de Andalucía (groups BIO286 and BIO192). Drs M. Nishimura, M. Hayashi and S. Mano from National Institute for Basic Biology, Okazaki (Japan) are deeply acknowledged for providing Arabidopsis pex5i and pex7i mutants. Technical and human support provided by CICT of Universidad de Jaén (UJA, MINECO, Junta de Andalucía, FEDER) is also acknowledged. Authors are grateful to Mr. Carmelo Ruíz-Torres for his excellent technical support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014. 04.034. References [1] Lamattina, L., García-Mata, C., Graziano, M. and Pagnussat, G. (2003) Nitric oxide: the versatility of an extensive signal molecule. Annu. Rev. Plant Biol. 54, 109–136. [2] Shapiro, A.D. (2005) Nitric oxide signaling in plants. Vitam. Horm. 72, 339– 398. [3] Besson-Bard, A., Pugin, A. and Wendehenne, D. (2008) New insights into nitric oxide signaling in plants. Annu. Rev. Plant Biol. 59, 21–39. [4] Baudhouin, E. (2011) The language of nitric oxide signaling. Plant Biol. (Stuttg) 13, 233–242.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034
6
F.J. Corpas, J.B. Barroso / FEBS Letters xxx (2014) xxx–xxx
[5] Corpas, F.J., Leterrier, M., Valderrama, R., Airaki, M., Chaki, M., Palma, J.M. and Barroso, J.B. (2011) Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress. Plant Sci. 181 (5), 604–611. [6] Corpas, F.J., Barroso, J.B. and del Río, L.A. (2001) Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci. 6, 145–150. [7] Takahashi, S. and Yamasaki, H. (2002) Reversible inhibition of photophosphorylation in chloroplasts by nitric oxide. FEBS Lett. 512, 145–148. [8] Gabaldón, C., Gómez-Ros, L.V., Pedreño, M.A. and Ros-Barceló, A. (2005) Nitric oxide production by the differentiating xylem of Zinnia elegans. New Phytol. 165, 121–130. [9] Jasid, S., Simontacchi, M., Bartoli, C.G. and Puntarulo, S. (2006) Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins. Plant Physiol. 142, 1246–1255. [10] Gupta, K.J., Igamberdiev, A.U., Manjunatha, G., Segu, S., Moran, J.F., Neelawarne, B., Bauwe, H. and Kaiser, W.M. (2011) The emerging roles of nitric oxide (NO) in plant mitochondria. Plant Sci. 181, 520–526. [11] Tewari, R.K., Prommer, J. and Watanabe, M. (2013) Endogenous nitric oxide generation in protoplast chloroplasts. Plant Cell Rep. 32, 31–44. [12] Corpas, F.J., Palma, J.M., del Río, L.A. and Barroso, J.B. (2009) Evidence supporting the existence of L-arginine-dependent nitric oxide synthase (NOS) activity in plants. New Phytol. 184, 9–14. [13] Foresi, N., Correa-Aragunde, N., Parisi, G., Caló, G., Salerno, G. and Lamattina, L. (2010) Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent. Plant Cell 22, 3816–3830. [14] Mullen, R.T., Flynn, C.R. and Trelease, R.N. (2001) How are peroxisomes formed? The role of the endoplasmic reticulum and peroxins. Trends Plant Sci. 6, 256–261. [15] 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, 36729–36733. [16] Corpas, F.J., Barroso, J.B., Carreras, A., Quirós, M., León, A.M., Romero-Puertas, M.C., Esteban, F.J., Valderrama, R., Palma, J.M., Sandalio, L.M., Gómez, M. and del Río, L.A. (2004) Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiol. 136, 2722–2733. [17] 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 Physiol. 151, 2083–2094. [18] Corpas, F.J. and Barroso, J.B. (2014) Peroxynitrite (ONOO-) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress. Ann. Bot. 113, 87–96. [19] Barroso, J.B., Valderrama, R. and Corpas, F.J. (2013) Immunolocalization of Snitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles. Acta Physiol. Plant 35, 2635–2640. [20] Reumann, S. (2004) Specification of the peroxisome targeting signals type 1 and type 2 of plant peroxisomes by bioinformatics analyses. Plant Physiol. 135, 783–800. [21] Reumann, S., Babujee, L., Ma, C., Wienkoop, S., Siemsen, T., Antonicelli, G.E., Rasche, N., Lüder, F., Weckwerth, W. and Jahn, O. (2007) Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19, 3170–3193. [22] Hu, J., Baker, A., Bartel, B., Linka, N., Mullen, R.T., Reumann, S. and Zolman, B.K. (2012) Plant peroxisomes: biogenesis and function. Plant Cell 24, 2279–2303. [23] Hayashi, M. and Nishimura, M. (2006) Arabidopsis thaliana – a model organism to study plant peroxisomes. Biochim. Biophys. Acta 1763, 1382–1391. [24] Hayashi, M., Yagi, M., Nito, K., Kamada, T. and Nishimura, M. (2005) Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis. J. Biol. Chem. 280, 14829–14835. [25] Nito, K., Kamigaki, A., Kondo, M., Hayashi, M. and Nishimura, M. (2007) Functional classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of knockdown mutants. Plant Cell Physiol. 48, 763– 774. [26] Singh, T., Hayashi, M., Mano, S., Arai, Y., Goto, S. and Nishimura, M. (2009) Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana. Plant J. 60, 488–498. [27] Ramón, N.M. and Bartel, B. (2010) Interdependence of the peroxisometargeting receptors in Arabidopsis thaliana: PEX7 facilitates PEX5 accumulation and import of pts1 cargo into peroxisomes. Mol. Biol. Cell 21, 1263–1271. [28] Mano, S., Nakamori, C., Nito, K., Kondo, M. and Nishimura, M. (2006) The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2dependent protein transport to peroxisomes. Plant J. 47, 604–618.
[29] Friedman, H., Meir, S., Rosenberger, I., Halevy, A.H., Kaufman, P.B. and Philosoph-Hadas, S. (1998) Inhibition of the gravitropic response of snapdragon spikes by the calcium-channel blocker lanthanum chloride. Plant Physiol. 118, 483–492. [30] Chen, T., Wu, X., Chen, Y., Li, X., Huang, M., Zheng, M., Baluska, F., Samaj, J. and Lin, J. (2009) Combined proteomic and cytological analysis of Ca2+-calmodulin regulation in Picea meyeri pollen tube growth. Plant Physiol. 149, 1111–1126. [31] Mano, S., Nakamori, C., Hayashi, M., Kato, A., Kondo, M. and Nishimura, M. (2002) Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement. Plant Cell Physiol. 43, 331–341. [32] Hayashi, M., Toriyama, K., Kondo, M. and Nishimura, M. (1998) 2,4Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid beta-oxidation. Plant Cell 10, 183–195. [33] del Río, L.A., Corpas, F.J., Sandalio, L.M., Palma, J.M., Gómez, M. and Barroso, J.B. (2002) Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. J. Exp. Bot. 53, 1255–1272. [34] Stolz, D.B., Zamora, R., Vodovotz, Y., Loughran, P.A., Billiar, T.R., Kim, Y.M., Simmons, R.L. and Watkins, S.C. (2002) Peroxisomal localization of inducible nitric oxide synthase in hepatocytes. Hepatology 36, 81–93. [35] Loughran, P.A., Stolz, D.B., Vodovotz, Y., Watkins, S.C., Simmons, R.L. and Billiar, T.R. (2005) Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 102, 13837–13842. [36] Prado, A.M., Porterfield, D.M. and Feijo, J.A. (2004) Nitric oxide is involved in growth regulation and re-orientation of pollen tubes. Development 131, 2707–2714. [37] Schlicht, M., Ludwig-Müller, J., Burbach, C., Volkmann, D. and Baluska, F. (2013) Indole-3-butyric acid induces lateral root formation via peroxisomederived indole-3-acetic acid and nitric oxide. New Phytol. 200, 473–482. [38] Khan, B.R. and Zolman, B.K. (2010) Pex5 mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis. Plant Physiol. 154, 1602–1615. [39] Braverman, N., Dodt, G., Gould, S.J. and Valle, D. (1998) An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum. Mol. Genet. 7, 1195–1205. [40] Otera, H., Okumoto, K., Tateishi, K., Ikoma, Y., Matsuda, E., Nishimura, M., Tsukamoto, T., Osumi, T., Ohashi, K., Higuchi, O. and Fujiki, Y. (1998) Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants. Mol. Cell. Biol. 18, 388–399. [41] Lee, J.R., Jang, H.H., Park, J.H., Jung, J.H., Lee, S.S., Park, S.K., Chi, Y.H., Moon, J.C., Lee, Y.M., Kim, S.Y., Kim, J.Y., Yun, D.J., Cho, M.J., Lee, K.O. and Lee, S.Y. (2006) Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis. Plant J. 47, 457–466. [42] Woodward, A.W. and Bartel, B. (2005) The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5. Mol. Biol. Cell 16, 573–583. [43] Loughran, P.A., Stolz, D.B., Barrick, S.R., Wheeler, D.S., Friedman, P.A., Rachubinski, R.A., Watkins, S.C. and Billiar, T.R. (2013) PEX7 and EBP50 target iNOS to the peroxisome in hepatocytes. Nitric Oxide 31, 9–19. [44] Stael, S., Wurzinger, B., Mair, A., Mehlmer, N., Vothknecht, U.C. and Teige, M. (2012) Plant organellar calcium signalling: an emerging field. J. Exp. Bot. 63, 1525–1542. [45] Al-Quraan, N.A., Locy, R.D. and Singh, N.K. (2010) Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress. Plant Physiol. Biochem. 48, 697–702. [46] Yang, T. and Poovaiah, B.W. (2002) Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin. Proc. Natl. Acad. Sci. U.S.A. 99, 4097–4102. [47] Chigri, F., Flosdorff, S., Pilz, S., Kölle, E., Dolze, E., Gietl, C. and Vothknecht, U.C. (2012) The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively. Plant Mol. Biol. 78, 211–222. [48] Dolze, E., Chigri, F., Höwing, T., Hierl, G., Isono, E., Vothknecht, U.C. and Gietl, C. (2013) Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism. Plant Mol. Biol. 83, 607–624. [49] Corpas, F.J., Palma, J.M. and del Río, L.A. (1993) Evidence for the presence of proteolytic activity in peroxisomes. Eur. J. Cell Biol. 61, 81–85. [50] Distefano, S., Palma, J.M., Gómez, M. and Río, L.A. (1997) Characterization of endoproteases from plant peroxisomes. Biochem. J. 327 (Pt 2), 399–405.
Please cite this article in press as: Corpas, F.J. and Barroso, J.B. 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 Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.034