Protoplasma DOI 10.1007/s00709-015-0819-0
ORIGINAL ARTICLE
Peroxisomal NADP-isocitrate dehydrogenase is required for Arabidopsis stomatal movement Marina Leterrier 1 & Juan B. Barroso 2 & Raquel Valderrama 2 & Juan C. Begara-Morales 2 & Beatriz Sánchez-Calvo 2 & Mounira Chaki 1 & Francisco Luque 2 & Benjamin Viñegla 3 & José M. Palma 1 & Francisco J. Corpas 1
Received: 26 February 2015 / Accepted: 8 April 2015 # Springer-Verlag Wien 2015
Abstract Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is
Handling Editor: Liwen Jiang Electronic supplementary material The online version of this article (doi:10.1007/s00709-015-0819-0) contains supplementary material, which is available to authorized users. * Francisco J. Corpas [email protected] 1
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, 18080 Granada, Spain
2
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus BLas Lagunillas^, 23071 Jaén, Spain
3
Departamento de Biología Animal, Biología Vegetal y Ecología (Ecología), Facultad de Ciencias Experimentales, Universidad de Jaén, Jaén, Spain
involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells. Keywords Nitric oxide . Hydrogen peroxide . NADP-isocitrate dehydrogenase . Peroxisomes . Stomata . Guard cells
Introduction Peroxisomes are subcellular organelles characterized by a simple morphological structure and a single boundary membrane but have a complex biochemical machinery involved in nitrooxidative-stress management and signaling (Palma et al. 2009; del Río 2011). In plants, peroxisomes participate in various biochemical pathways such as photorespiration, βoxidation of fatty acids, nitrogen metabolism (Beevers 1979; Hu et al. 2002, 2012), urate catabolism (Corpas et al. 1997), detoxification reactions, synthesis of plant hormones, backconversion of polyamines (Moschou et al. 2008), and the metabolism of reactive oxygen (ROS) and nitrogen species (RNS) (del Río et al. 2006; Corpas et al. 2009; Corpas and Barroso 2014a). Being an essential electron donor in many enzymatic reactions, biosynthetic pathways, and detoxification processes, the reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a key element in cellular redox homeostasis. As a result, NAPDH is a necessary reducing equivalent for the regeneration of ascorbate and reduced glutathione (GSH) by monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) in the ascorbate-glutathione cycle, an important cell antioxidant system against oxidative damage. NADP H is also a cofactor of important enzymes in the RNS and ROS metabolism (Corpas and Barroso 2014b). The main enzymes with the capacity to generate reducing power in the
M. Leterrier et al.
form of NADPH are ferredoxin-NADP reductase and a group of four NADP-dehydrogenases including glucose-6phosphate dehydrogenase (G6PDH) and 6phosphogluconate dehydrogenase (6PGDH), both belonging to the pentose phosphate pathway, the NADP-malic enzyme, and NADP-isocitrate dehydrogenase (NADP-ICDH). NADP-ICDH catalyzes the oxidative decarboxylation of Lisocitrate to 2-oxoglutarate with the production of the reduced coenzyme NADPH (Gálvez and Gadal 1995). In higher plant cells, NADP-ICDH activity has been detected and characterized in the cytosol (Curry and Ting 1976; Chen et al. 1988, 1989; Fieuw et al. 1995; Canino et al. 1996), mitochondria (Rasmusson and Moller 1990; Attucci et al. 1994; Møller and Rasmusson 1998; Gray et al. 2004), chloroplasts (Gálvez et al. 1994; Popova et al. 2002), and peroxisomes (Donaldson 1982; Corpas et al. 1999; Reumann et al. 2007). Significant progress is being made in our understanding of the way in which NADP-ICDH is involved in the nitrogen metabolism, redox regulation, senescence, and responses to oxidative stress (Scheible et al. 2000; Hodges et al. 2003; Sulpice et al. 2010; Mhamdi et al. 2010; Leterrier et al. 2007, 2012; Begara-Morales et al. 2013); however, the specific function of this enzyme in each subcellular compartment under physiological and stress conditions remains unclear. In the present study, Arabidopsis (Arabidopsis thaliana) is used as a model to analyze the potential function of the peroxisomal NADP-ICDH about which very little is known. The results suggest that this peroxisomal enzyme is necessary for stomatal movement.
Materials and methods Plant material A. thaliana wild-type and T-DNA insertion-line seeds SALK_072422 and SALK_039193C (ecotype Columbia) were obtained from the NASC (Nottingham, UK). Plants were grown on soil plus vermiculite (1:3) in a growth chamber at 22 °C under a 16-h photoperiod and a light intensity of 100– 120 μE m−2 s−1. Homozygous seeds from the SALK_072422 line were selected, and the homozygous status of both t-DNA mutants was checked by polymerase chain reaction (PCR) on the genomic DNA. Preparation of crude extracts All operations were carried out at 0–4 °C. Leaves and roots were frozen in liquid N2 and ground to a powder in a mortar with a pestle. The powder was suspended in homogenizing medium containing 50 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, 1 mM MgCl2, 2 mM DTT, 0.2 % (v/v) Triton X-100, and 10 % (v/v) glycerol (1:4, w/v). Homogenates were centrifuged at 13,
000×g for 20 min. Aliquots of supernatants were immediately used for the assays. Electron microscopy and immunocytochemistry Arabidopsis leaf segments of approximately 1 mm2 were fixed, dehydrated and embedded in LR White resin following the technique described by Corpas et al. (1994). Immunolabeling was performed as indicated by Corpas et al. (1999). Ultrathin sections were incubated for 3 h with IgG against pea NADP-ICDH (Chen et al. 1989) diluted 1/500 in TBS plus Tween-20 (TBST) buffer containing 2 % (w/v) BSA and 1 % (v/v) goat normal serum. The sections were then incubated for 1 h with goat anti-rabbit IgG conjugated to 15nm gold particles (Bio Cell, Cardiff, UK) diluted 1:40 in TBST plus 2 % (w/v) bovine serum albumin. Sections were post-stained in 2 % (v/v) uranyl acetate for 3 min and examined under a Zeiss EM 10C transmission electron microscope (Jena, Germany). NADP-ICDH labeling density is given as the number of gold particles per peroxisome. Two separately embedded blocks were used to cut sections of each class of leaves from Wt and both mutant lines. An average of 10 to 15 photographs was used for quantitative analysis for each type of leaves. Sequence analysis, database searches, and subcellular localization predictions NADP-ICDH sequences from completed genomes were retrieved from Plaza 2.0 (http://bioinformatics.psb.ugent.be/ plaza/) (Proost et al. 2009). Blast searches were carried out on the National Center for Biotechnology Information web site (http://www.ncbi.nlm.nih.gov/). Alignments were performed using OMIGA (2.0) and CLUSTAL W 2 (http:// www.ebi.ac.uk/Tools/msa/clustalw2/). Localization predictions were made using WoLF (http://wolfpsort.org/) (Horton et al. 2007) and peroxisomal targeting signal type 1 (PTS1) Predictor (http://mendel.imp.ac.at/mendeljsp/sat/pts1/ PTS1predictor.jsp) (Neuberger et al. 2003a, b). Molecular properties were estimated using the http://www.expasy.org/ cgibin/pi_tool. Phylogenetic analyses were conducted using MEGA software version 3.0 (http://www.megasoftware.net/). The molecular properties of Arabidopsis NADP-ICDH isozymes based on their predicted amino-acid sequences were used for the in silico predictions with the aid of http://www. expasy.org/tools/protparam.html. Microarray data were retrieved from the Arabidopsis eFP Browser (http://bar. utoronto.ca/efp/cgi-bin/efpWeb.cgi). RNA isolation and reverse transcription-PCR Total RNA was isolated from Arabidopsis leaves using the Trizol reagent kit followed by DNnase treatment (New
Peroxisomal NADP-isocitrate dehydrogenase
England Biolab), both according to manufacturer’s instructions. Approximately 2 μg of total RNA from leaves was used as a template for the reverse transcriptase (RT) reaction. This was added to a mixture containing 5 mM MgCl2, 1 mM dNTPs, 0.5 μg oligo (dT23) primers, 1× RT-buffer, 20 U Rnasin ribonuclease inhibitor, and 15 U AMV reverse transcriptase (Promega, Madison, WI). The reaction was carried out at 42 °C for 40 min, followed by a 5-min phase at 98 °C and then by cooling to 4 °C. Amplification of ACTIN II complementary DNA (cDNA) from Arabidopsis At3g18780 was chosen as a control. At1g54340 (pICDH) and ACTIN II cDNAs were amplified by the PCR as follows: 1 μl of the cDNA produced diluted 1/20 was added to 250 μM dNTPs, 1.5 mM MgCl2, 1× PCR buffer, 1 U of Ampli Taq Gold (PE Applied Biosystems), and 0.5 μM of each primer (ACTIN II, 5′-TCCCTCAGCACATTCCAGCAGAT-3′ and 5′-AACGAT TCCTGGACCTGC CTCATC-3′; At1g54340, 5′-GCGTGA TGTTTGATTTGATGCT-3′ and 5′-CGTAGCCA TTTCTG TTGAT TGGT-3′) in a final volume of 20 μl. Primers used to amplify At1g54340 cDNA are specific and do not amplify other ICDH genes. Reactions were conducted in a Hybaid thermo-cycler. A first phase of 10 min at 94 °C was followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, and 45 s at 72 °C. The expected amplified PCR products were 229 bp for pICDH and 69 bp for ACTIN II which were then detected by electrophoresis in 1 % agarose gels and stained with ethidium bromide. Stomatal index and aperture measurements The stomatal index is defined as the percentage of the number of stomata to the total number of epidermal cells including the stomata, with each stoma being counted as one cell. It was calculated as follows: Stomata index ð%Þ ¼
S 100 ðE þ S Þ
where S corresponds to the number of stomata per unit area, and E is the number of epidermal cells in the same unit area. For aperture measurements, all the experiments were started in the morning after 8 h of the dark cycle. Leaves from a minimum of three independent single plants per genotype and experiment were detached and floated on the incubation medium (50 mM Tris–HCl, pH 7.6, containing 20 mM KCl) in Petri dishes. Light-induced stomatal opening was analyzed after 2 h in the light (20 μE m−2 s−1). Parts of the abaxial epidermis were peeled off, transferred to a drop of incubation medium on a glass slide, and immediately processed for microscopic analysis of stomatal apertures. Bright-field pictures of stomata were taken with a Leica stereomicroscope (M165FC). A concentration of 10 μM ABA, 100 μM ascorbate or 200 μM cPTIO was added to the incubation medium at
the beginning of the treatment in the light. For each experiment and genotype, 30 stomata measurements were taken in three leaves from independent plants using the Adobe Photoshop CS4 ruler tool, and the experiments were repeated twice.
Gas exchange measurements In order to elucidate the photosynthetic behavior and light saturation conditions of Arabidopsis experimental material, photosynthesis-irradiance curves were performed. The curves were obtained by recording the C fixation rate compared with the gradual increase in irradiance on fully expanded leaves from three different plants. Net photosynthetic rates were measured with an LI-6400 Photosynthesis System (Li-Cor Biosciences, Lincoln, USA). This system is equipped with a standard 6 cm2 chamber which allows for regulation of temperature and air flow as well as measurements of light intensity. An external light source was used to increase light intensity from 0 to ca. 2200 μE m−2 s−1. The light–response curve was described by a non-rectangular hyperbola (Hjelm and Ögren 2004):
A¼
h i1 = 2 αI þ Amax − ðαI þ Amax Þ2 −4αθIAmax 2θ
−Rd
where A is the net carbon assimilation rate, Rd is the dark respiration rate, α is carbon assimilation efficiency, I is irradiance, Amax is the light-saturated net assimilation rate, and θ is the convexity (the rate of bending) of the curve. Photosynthetic parameters were calculated by subjecting the equation above to non-linear regression analysis (Curve Expert 1.34). The adjusted expression respiration (Rd) was calculated as the C exchange rate in darkness and the light compensation point (LCP) as the irradiance for a zero net C exchange. After calculating the photosynthesis-irradiance curves, instantaneous rates of net C assimilation (Anet) and stomatal conductance (gs) were measured. During the measurements, the plants were maintained under pre-incubation conditions (120 μE m−2 s−1 and 22 °C and 60 % RH). Intrinsic water use efficiency was calculated as the ratio of net C fixation rate and stomatal conductance to water vapor. The exposed area of leaves used for photosynthetic measurements was determined by image scanning and further digital measurement of leaf area (ImageJ 1.36b). Leaf samples were then dried at 60 °C for 24 h to determine dry mass. Total area of leaves was divided by dry mass to determine leaf mass per total area (LMA). Differences between wild-type and both mutant plants were analyzed by means of one-way ANOVA and a Fisher LSD post-hoc test. The significance of all statistical analyses
M. Leterrier et al.
was tested at α=0.05. Statistical analyses were carried out using STATISTICA 7.0 software (StatSoft Inc). Other assays Protein concentration was determined with the aid of the BioRad protein assay using bovine serum albumin as standard. For an estimate of the statistical significance between means, the data were analyzed by the Student’s t test.
Results In silico analysis of Arabidopsis NADP-ICDH isozymes and gene evolution Analysis of the Arabidopsis database shows that its genome contains three NADP-ICDH genes At1g65930, At5g14590, and At1g54340, coding for the proteins NP_176768, NP_196963, and BT025983, respectively. The three nuclear NADP-ICDH genes show a conserved structure of 15 exons/ 14 introns (Leterrier et al. 2007). Supplemental Table 1 summarizes some molecular properties of each Arabidopsis NADP-ICDH isozyme based on predicted amino-acid sequences in each case. The most remarkable difference was in relation to subcellular localization. This appears to indicate that At1g65930 encodes cytosolic NADP-ICDH and that At1g54340 is expected to encode the peroxisomal NADPICDH, since the deduced protein sequence contains a Cterminal SRL tripeptide which is a variant of a type I peroxisomal targeting (PST1) motif (Reumann 2011; Reumann et al. 2004, 2009). Finally, At5g14590 is expected to encode both chloroplastic and/or mitochondrial isozymes. The alignment of the deduced amino-acid sequence of the three Arabidopsis NADP-ICDHs indicates that the protein sequence of the three isozymes was highly conserved, with 77 % similarity between NP_176768 and NP_196963 and 85 % between NP_176768 and BT025983. This high similarity is also found in highly conserved residues involved in Mg2+-isocitrate and NADP+ binding as well as in the tryptophan residues at the active NADP site that binds adenine (Sankaran et al. 1996). Supplemental Fig. 1 shows the analysis of plant NADPICDH protein sequences using the available complete genomes and their putative ICDH subcellular localization using the WoLF PSORT and the peroxisomal targeting signal 1 predictor programs. As might be expected, NADP-ICDH in green plants was found to have different potential subcellular localizations, including the cytosol, chloroplast, cytoskeleton, peroxisome, and mitochondrion. It is remarkable that the Chlorophyta members corresponding to green algae showed an absence of the peroxisomal NADP-ICDH, even though this isozyme is present in all land plants.
Additionally, microarrays from a public database (Arabidopsis electronic fluorescent pictograph [eFP] browser at www.bar.utoronto.ca; Winter et al. 2007) indicate a higher expression of At1g54340 in guard cells as compared with mesophyll cells (Supplemental Fig. 2). This expression was even higher in the presence of ABA, while the expression of the other NADP-ICDH genes remained unchanged. Identification and characterization of the peroxisomal NADP-ICDH (picdh) Arabidopsis mutant lines In an effort to determine the potential function of the peroxisomal NADP-ICDH, a search of mutants in the At1g54340 gene was carried out in the collection of sequence-tagged TDNA mutants (http://signal.salk.edu/cgibin/tdnaexpress). Two mutants were identified and designated as picdh-1 (SALK_072422) and picdh-2 (SALK_039193C) carrying a T-DNA insertion in exons 9 and in the 5′UTR, respectively (Fig. 1a). The T-DNA insertion in At1g54340 was confirmed by polymerase chain reaction (PCR) of the genomic DNA. Semi-quantitative RT-PCR analysis confirmed that the peroxisomal ICDH transcript was undetectable in both lines (Fig. 1b). To confirm the lack of the peroxisomal NADP-ICDH in both picdh-1 and picdh-2 plants, the immunocytochemical localization of NADP-ICDH in leaves from Wt and both mutant lines was assessed using a well-characterized antibody against NADP-ICDH (Chen et al. 1989; Corpas et al. 1999). Figure 1c shows the subcellular location of NADP-ICDH in Arabidopsis leaves studied with the aid of EM immunocytochemistry, where immunogold particles appeared in the cytosol, chloroplasts, mitochondria, and peroxisomes, which is closely in line with the subcellular prediction in Supplemental Table 1. The immunolocalization in the leaves of picdh-1 (Fig. 1d) and picdh-2 (Fig. 1e) plants shows the absence of immunogold particles in peroxisomes. Thus, the average number of gold particles per peroxisome counted in leaves samples from Wt plants was 15±3 whereas in the case of both mutant lines was 1±1. Phenotypical analysis from seed germination to wholeplant development under standard growth conditions revealed no significant differences between the wild-type (Wt) and the two mutant (picdh-1 and picdh-2) lines (Fig. 2a). Moreover, Fig. 2b–d shows the photosynthetic parameters of Arabidopsis Wt and both picdh mutant lines including the net C assimilation rate (Fig. 2b), stomatal conductance (Fig. 2c), and intrinsic water use efficiency (iWUE) (Fig. 2d). Thus, wild-type plants showed significantly higher C fixation and stomatal conductance rates compared with both mutant (picdh-1 and picdh-2) plants. As incubation irradiance and water availability did not limit the differences in C and water vapor exchange rates, growth rates were not affected. However, these results suggest that the growth rates of both mutant plants could be reduced under stressful environmental
Peroxisomal NADP-isocitrate dehydrogenase Fig. 1 Characterization of peroxisomal NADP-isocitrate dehydrogenase in mutant (picdh1 and picdh-2) lines. a Schematic representation of the At1g54340 gene. Exons are denoted by boxes, with empty boxes representing the untranslated regions and filled boxes representing the coding regions, and introns are represented by a line. The position of T-DNA insertions in picdh-1 and picdh-2 lines are indicated. b RT-PCR analysis demonstrating the absence of picdh transcript in the picdh-1 and picdh-2 mutant lines. c–e Representative micrograph of the electron microscopic immunocytochemical localization of NADP-ICDH in Arabidopsis leaves of wild-type (Wt) and the two mutant (picdh-1 and picdh-2) lines. Ch chloroplasts, M mitochondria, P peroxisomes. Bar= 0.5 μm
conditions. Finally, no differences were found in relation to iWUE since the higher gas exchange rates found in the wildFig. 2 Phenotypic differences in physiological parameters of Arabidopsis wild-type (Wt) and both mutant (picdh-1 and picdh2) lines. a Phenotype Wt and both mutant lines of 3 and 9 weeks. b Net C assimilation rate (Anet). c Stomatal conductance (gs). d Intrinsic water use efficiency (iWUE). Data are the means± SEM (n=6). Different letters indicate significant differences (one-way ANOVA and Fisher LSD post-hoc test, p
ORIGINAL ARTICLE
Peroxisomal NADP-isocitrate dehydrogenase is required for Arabidopsis stomatal movement Marina Leterrier 1 & Juan B. Barroso 2 & Raquel Valderrama 2 & Juan C. Begara-Morales 2 & Beatriz Sánchez-Calvo 2 & Mounira Chaki 1 & Francisco Luque 2 & Benjamin Viñegla 3 & José M. Palma 1 & Francisco J. Corpas 1
Received: 26 February 2015 / Accepted: 8 April 2015 # Springer-Verlag Wien 2015
Abstract Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is
Handling Editor: Liwen Jiang Electronic supplementary material The online version of this article (doi:10.1007/s00709-015-0819-0) contains supplementary material, which is available to authorized users. * Francisco J. Corpas [email protected] 1
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, 18080 Granada, Spain
2
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus BLas Lagunillas^, 23071 Jaén, Spain
3
Departamento de Biología Animal, Biología Vegetal y Ecología (Ecología), Facultad de Ciencias Experimentales, Universidad de Jaén, Jaén, Spain
involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells. Keywords Nitric oxide . Hydrogen peroxide . NADP-isocitrate dehydrogenase . Peroxisomes . Stomata . Guard cells
Introduction Peroxisomes are subcellular organelles characterized by a simple morphological structure and a single boundary membrane but have a complex biochemical machinery involved in nitrooxidative-stress management and signaling (Palma et al. 2009; del Río 2011). In plants, peroxisomes participate in various biochemical pathways such as photorespiration, βoxidation of fatty acids, nitrogen metabolism (Beevers 1979; Hu et al. 2002, 2012), urate catabolism (Corpas et al. 1997), detoxification reactions, synthesis of plant hormones, backconversion of polyamines (Moschou et al. 2008), and the metabolism of reactive oxygen (ROS) and nitrogen species (RNS) (del Río et al. 2006; Corpas et al. 2009; Corpas and Barroso 2014a). Being an essential electron donor in many enzymatic reactions, biosynthetic pathways, and detoxification processes, the reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a key element in cellular redox homeostasis. As a result, NAPDH is a necessary reducing equivalent for the regeneration of ascorbate and reduced glutathione (GSH) by monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) in the ascorbate-glutathione cycle, an important cell antioxidant system against oxidative damage. NADP H is also a cofactor of important enzymes in the RNS and ROS metabolism (Corpas and Barroso 2014b). The main enzymes with the capacity to generate reducing power in the
M. Leterrier et al.
form of NADPH are ferredoxin-NADP reductase and a group of four NADP-dehydrogenases including glucose-6phosphate dehydrogenase (G6PDH) and 6phosphogluconate dehydrogenase (6PGDH), both belonging to the pentose phosphate pathway, the NADP-malic enzyme, and NADP-isocitrate dehydrogenase (NADP-ICDH). NADP-ICDH catalyzes the oxidative decarboxylation of Lisocitrate to 2-oxoglutarate with the production of the reduced coenzyme NADPH (Gálvez and Gadal 1995). In higher plant cells, NADP-ICDH activity has been detected and characterized in the cytosol (Curry and Ting 1976; Chen et al. 1988, 1989; Fieuw et al. 1995; Canino et al. 1996), mitochondria (Rasmusson and Moller 1990; Attucci et al. 1994; Møller and Rasmusson 1998; Gray et al. 2004), chloroplasts (Gálvez et al. 1994; Popova et al. 2002), and peroxisomes (Donaldson 1982; Corpas et al. 1999; Reumann et al. 2007). Significant progress is being made in our understanding of the way in which NADP-ICDH is involved in the nitrogen metabolism, redox regulation, senescence, and responses to oxidative stress (Scheible et al. 2000; Hodges et al. 2003; Sulpice et al. 2010; Mhamdi et al. 2010; Leterrier et al. 2007, 2012; Begara-Morales et al. 2013); however, the specific function of this enzyme in each subcellular compartment under physiological and stress conditions remains unclear. In the present study, Arabidopsis (Arabidopsis thaliana) is used as a model to analyze the potential function of the peroxisomal NADP-ICDH about which very little is known. The results suggest that this peroxisomal enzyme is necessary for stomatal movement.
Materials and methods Plant material A. thaliana wild-type and T-DNA insertion-line seeds SALK_072422 and SALK_039193C (ecotype Columbia) were obtained from the NASC (Nottingham, UK). Plants were grown on soil plus vermiculite (1:3) in a growth chamber at 22 °C under a 16-h photoperiod and a light intensity of 100– 120 μE m−2 s−1. Homozygous seeds from the SALK_072422 line were selected, and the homozygous status of both t-DNA mutants was checked by polymerase chain reaction (PCR) on the genomic DNA. Preparation of crude extracts All operations were carried out at 0–4 °C. Leaves and roots were frozen in liquid N2 and ground to a powder in a mortar with a pestle. The powder was suspended in homogenizing medium containing 50 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, 1 mM MgCl2, 2 mM DTT, 0.2 % (v/v) Triton X-100, and 10 % (v/v) glycerol (1:4, w/v). Homogenates were centrifuged at 13,
000×g for 20 min. Aliquots of supernatants were immediately used for the assays. Electron microscopy and immunocytochemistry Arabidopsis leaf segments of approximately 1 mm2 were fixed, dehydrated and embedded in LR White resin following the technique described by Corpas et al. (1994). Immunolabeling was performed as indicated by Corpas et al. (1999). Ultrathin sections were incubated for 3 h with IgG against pea NADP-ICDH (Chen et al. 1989) diluted 1/500 in TBS plus Tween-20 (TBST) buffer containing 2 % (w/v) BSA and 1 % (v/v) goat normal serum. The sections were then incubated for 1 h with goat anti-rabbit IgG conjugated to 15nm gold particles (Bio Cell, Cardiff, UK) diluted 1:40 in TBST plus 2 % (w/v) bovine serum albumin. Sections were post-stained in 2 % (v/v) uranyl acetate for 3 min and examined under a Zeiss EM 10C transmission electron microscope (Jena, Germany). NADP-ICDH labeling density is given as the number of gold particles per peroxisome. Two separately embedded blocks were used to cut sections of each class of leaves from Wt and both mutant lines. An average of 10 to 15 photographs was used for quantitative analysis for each type of leaves. Sequence analysis, database searches, and subcellular localization predictions NADP-ICDH sequences from completed genomes were retrieved from Plaza 2.0 (http://bioinformatics.psb.ugent.be/ plaza/) (Proost et al. 2009). Blast searches were carried out on the National Center for Biotechnology Information web site (http://www.ncbi.nlm.nih.gov/). Alignments were performed using OMIGA (2.0) and CLUSTAL W 2 (http:// www.ebi.ac.uk/Tools/msa/clustalw2/). Localization predictions were made using WoLF (http://wolfpsort.org/) (Horton et al. 2007) and peroxisomal targeting signal type 1 (PTS1) Predictor (http://mendel.imp.ac.at/mendeljsp/sat/pts1/ PTS1predictor.jsp) (Neuberger et al. 2003a, b). Molecular properties were estimated using the http://www.expasy.org/ cgibin/pi_tool. Phylogenetic analyses were conducted using MEGA software version 3.0 (http://www.megasoftware.net/). The molecular properties of Arabidopsis NADP-ICDH isozymes based on their predicted amino-acid sequences were used for the in silico predictions with the aid of http://www. expasy.org/tools/protparam.html. Microarray data were retrieved from the Arabidopsis eFP Browser (http://bar. utoronto.ca/efp/cgi-bin/efpWeb.cgi). RNA isolation and reverse transcription-PCR Total RNA was isolated from Arabidopsis leaves using the Trizol reagent kit followed by DNnase treatment (New
Peroxisomal NADP-isocitrate dehydrogenase
England Biolab), both according to manufacturer’s instructions. Approximately 2 μg of total RNA from leaves was used as a template for the reverse transcriptase (RT) reaction. This was added to a mixture containing 5 mM MgCl2, 1 mM dNTPs, 0.5 μg oligo (dT23) primers, 1× RT-buffer, 20 U Rnasin ribonuclease inhibitor, and 15 U AMV reverse transcriptase (Promega, Madison, WI). The reaction was carried out at 42 °C for 40 min, followed by a 5-min phase at 98 °C and then by cooling to 4 °C. Amplification of ACTIN II complementary DNA (cDNA) from Arabidopsis At3g18780 was chosen as a control. At1g54340 (pICDH) and ACTIN II cDNAs were amplified by the PCR as follows: 1 μl of the cDNA produced diluted 1/20 was added to 250 μM dNTPs, 1.5 mM MgCl2, 1× PCR buffer, 1 U of Ampli Taq Gold (PE Applied Biosystems), and 0.5 μM of each primer (ACTIN II, 5′-TCCCTCAGCACATTCCAGCAGAT-3′ and 5′-AACGAT TCCTGGACCTGC CTCATC-3′; At1g54340, 5′-GCGTGA TGTTTGATTTGATGCT-3′ and 5′-CGTAGCCA TTTCTG TTGAT TGGT-3′) in a final volume of 20 μl. Primers used to amplify At1g54340 cDNA are specific and do not amplify other ICDH genes. Reactions were conducted in a Hybaid thermo-cycler. A first phase of 10 min at 94 °C was followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, and 45 s at 72 °C. The expected amplified PCR products were 229 bp for pICDH and 69 bp for ACTIN II which were then detected by electrophoresis in 1 % agarose gels and stained with ethidium bromide. Stomatal index and aperture measurements The stomatal index is defined as the percentage of the number of stomata to the total number of epidermal cells including the stomata, with each stoma being counted as one cell. It was calculated as follows: Stomata index ð%Þ ¼
S 100 ðE þ S Þ
where S corresponds to the number of stomata per unit area, and E is the number of epidermal cells in the same unit area. For aperture measurements, all the experiments were started in the morning after 8 h of the dark cycle. Leaves from a minimum of three independent single plants per genotype and experiment were detached and floated on the incubation medium (50 mM Tris–HCl, pH 7.6, containing 20 mM KCl) in Petri dishes. Light-induced stomatal opening was analyzed after 2 h in the light (20 μE m−2 s−1). Parts of the abaxial epidermis were peeled off, transferred to a drop of incubation medium on a glass slide, and immediately processed for microscopic analysis of stomatal apertures. Bright-field pictures of stomata were taken with a Leica stereomicroscope (M165FC). A concentration of 10 μM ABA, 100 μM ascorbate or 200 μM cPTIO was added to the incubation medium at
the beginning of the treatment in the light. For each experiment and genotype, 30 stomata measurements were taken in three leaves from independent plants using the Adobe Photoshop CS4 ruler tool, and the experiments were repeated twice.
Gas exchange measurements In order to elucidate the photosynthetic behavior and light saturation conditions of Arabidopsis experimental material, photosynthesis-irradiance curves were performed. The curves were obtained by recording the C fixation rate compared with the gradual increase in irradiance on fully expanded leaves from three different plants. Net photosynthetic rates were measured with an LI-6400 Photosynthesis System (Li-Cor Biosciences, Lincoln, USA). This system is equipped with a standard 6 cm2 chamber which allows for regulation of temperature and air flow as well as measurements of light intensity. An external light source was used to increase light intensity from 0 to ca. 2200 μE m−2 s−1. The light–response curve was described by a non-rectangular hyperbola (Hjelm and Ögren 2004):
A¼
h i1 = 2 αI þ Amax − ðαI þ Amax Þ2 −4αθIAmax 2θ
−Rd
where A is the net carbon assimilation rate, Rd is the dark respiration rate, α is carbon assimilation efficiency, I is irradiance, Amax is the light-saturated net assimilation rate, and θ is the convexity (the rate of bending) of the curve. Photosynthetic parameters were calculated by subjecting the equation above to non-linear regression analysis (Curve Expert 1.34). The adjusted expression respiration (Rd) was calculated as the C exchange rate in darkness and the light compensation point (LCP) as the irradiance for a zero net C exchange. After calculating the photosynthesis-irradiance curves, instantaneous rates of net C assimilation (Anet) and stomatal conductance (gs) were measured. During the measurements, the plants were maintained under pre-incubation conditions (120 μE m−2 s−1 and 22 °C and 60 % RH). Intrinsic water use efficiency was calculated as the ratio of net C fixation rate and stomatal conductance to water vapor. The exposed area of leaves used for photosynthetic measurements was determined by image scanning and further digital measurement of leaf area (ImageJ 1.36b). Leaf samples were then dried at 60 °C for 24 h to determine dry mass. Total area of leaves was divided by dry mass to determine leaf mass per total area (LMA). Differences between wild-type and both mutant plants were analyzed by means of one-way ANOVA and a Fisher LSD post-hoc test. The significance of all statistical analyses
M. Leterrier et al.
was tested at α=0.05. Statistical analyses were carried out using STATISTICA 7.0 software (StatSoft Inc). Other assays Protein concentration was determined with the aid of the BioRad protein assay using bovine serum albumin as standard. For an estimate of the statistical significance between means, the data were analyzed by the Student’s t test.
Results In silico analysis of Arabidopsis NADP-ICDH isozymes and gene evolution Analysis of the Arabidopsis database shows that its genome contains three NADP-ICDH genes At1g65930, At5g14590, and At1g54340, coding for the proteins NP_176768, NP_196963, and BT025983, respectively. The three nuclear NADP-ICDH genes show a conserved structure of 15 exons/ 14 introns (Leterrier et al. 2007). Supplemental Table 1 summarizes some molecular properties of each Arabidopsis NADP-ICDH isozyme based on predicted amino-acid sequences in each case. The most remarkable difference was in relation to subcellular localization. This appears to indicate that At1g65930 encodes cytosolic NADP-ICDH and that At1g54340 is expected to encode the peroxisomal NADPICDH, since the deduced protein sequence contains a Cterminal SRL tripeptide which is a variant of a type I peroxisomal targeting (PST1) motif (Reumann 2011; Reumann et al. 2004, 2009). Finally, At5g14590 is expected to encode both chloroplastic and/or mitochondrial isozymes. The alignment of the deduced amino-acid sequence of the three Arabidopsis NADP-ICDHs indicates that the protein sequence of the three isozymes was highly conserved, with 77 % similarity between NP_176768 and NP_196963 and 85 % between NP_176768 and BT025983. This high similarity is also found in highly conserved residues involved in Mg2+-isocitrate and NADP+ binding as well as in the tryptophan residues at the active NADP site that binds adenine (Sankaran et al. 1996). Supplemental Fig. 1 shows the analysis of plant NADPICDH protein sequences using the available complete genomes and their putative ICDH subcellular localization using the WoLF PSORT and the peroxisomal targeting signal 1 predictor programs. As might be expected, NADP-ICDH in green plants was found to have different potential subcellular localizations, including the cytosol, chloroplast, cytoskeleton, peroxisome, and mitochondrion. It is remarkable that the Chlorophyta members corresponding to green algae showed an absence of the peroxisomal NADP-ICDH, even though this isozyme is present in all land plants.
Additionally, microarrays from a public database (Arabidopsis electronic fluorescent pictograph [eFP] browser at www.bar.utoronto.ca; Winter et al. 2007) indicate a higher expression of At1g54340 in guard cells as compared with mesophyll cells (Supplemental Fig. 2). This expression was even higher in the presence of ABA, while the expression of the other NADP-ICDH genes remained unchanged. Identification and characterization of the peroxisomal NADP-ICDH (picdh) Arabidopsis mutant lines In an effort to determine the potential function of the peroxisomal NADP-ICDH, a search of mutants in the At1g54340 gene was carried out in the collection of sequence-tagged TDNA mutants (http://signal.salk.edu/cgibin/tdnaexpress). Two mutants were identified and designated as picdh-1 (SALK_072422) and picdh-2 (SALK_039193C) carrying a T-DNA insertion in exons 9 and in the 5′UTR, respectively (Fig. 1a). The T-DNA insertion in At1g54340 was confirmed by polymerase chain reaction (PCR) of the genomic DNA. Semi-quantitative RT-PCR analysis confirmed that the peroxisomal ICDH transcript was undetectable in both lines (Fig. 1b). To confirm the lack of the peroxisomal NADP-ICDH in both picdh-1 and picdh-2 plants, the immunocytochemical localization of NADP-ICDH in leaves from Wt and both mutant lines was assessed using a well-characterized antibody against NADP-ICDH (Chen et al. 1989; Corpas et al. 1999). Figure 1c shows the subcellular location of NADP-ICDH in Arabidopsis leaves studied with the aid of EM immunocytochemistry, where immunogold particles appeared in the cytosol, chloroplasts, mitochondria, and peroxisomes, which is closely in line with the subcellular prediction in Supplemental Table 1. The immunolocalization in the leaves of picdh-1 (Fig. 1d) and picdh-2 (Fig. 1e) plants shows the absence of immunogold particles in peroxisomes. Thus, the average number of gold particles per peroxisome counted in leaves samples from Wt plants was 15±3 whereas in the case of both mutant lines was 1±1. Phenotypical analysis from seed germination to wholeplant development under standard growth conditions revealed no significant differences between the wild-type (Wt) and the two mutant (picdh-1 and picdh-2) lines (Fig. 2a). Moreover, Fig. 2b–d shows the photosynthetic parameters of Arabidopsis Wt and both picdh mutant lines including the net C assimilation rate (Fig. 2b), stomatal conductance (Fig. 2c), and intrinsic water use efficiency (iWUE) (Fig. 2d). Thus, wild-type plants showed significantly higher C fixation and stomatal conductance rates compared with both mutant (picdh-1 and picdh-2) plants. As incubation irradiance and water availability did not limit the differences in C and water vapor exchange rates, growth rates were not affected. However, these results suggest that the growth rates of both mutant plants could be reduced under stressful environmental
Peroxisomal NADP-isocitrate dehydrogenase Fig. 1 Characterization of peroxisomal NADP-isocitrate dehydrogenase in mutant (picdh1 and picdh-2) lines. a Schematic representation of the At1g54340 gene. Exons are denoted by boxes, with empty boxes representing the untranslated regions and filled boxes representing the coding regions, and introns are represented by a line. The position of T-DNA insertions in picdh-1 and picdh-2 lines are indicated. b RT-PCR analysis demonstrating the absence of picdh transcript in the picdh-1 and picdh-2 mutant lines. c–e Representative micrograph of the electron microscopic immunocytochemical localization of NADP-ICDH in Arabidopsis leaves of wild-type (Wt) and the two mutant (picdh-1 and picdh-2) lines. Ch chloroplasts, M mitochondria, P peroxisomes. Bar= 0.5 μm
conditions. Finally, no differences were found in relation to iWUE since the higher gas exchange rates found in the wildFig. 2 Phenotypic differences in physiological parameters of Arabidopsis wild-type (Wt) and both mutant (picdh-1 and picdh2) lines. a Phenotype Wt and both mutant lines of 3 and 9 weeks. b Net C assimilation rate (Anet). c Stomatal conductance (gs). d Intrinsic water use efficiency (iWUE). Data are the means± SEM (n=6). Different letters indicate significant differences (one-way ANOVA and Fisher LSD post-hoc test, p
