J. Plant Biol. (2014) 57:9-19 DOI 10.1007/s12374-013-0350-9
ORIGINAL ARTICLE
Silencing of Tomato Mitochondrial Uncoupling Protein Disrupts Redox Poise and Antioxidant Enzymes Activities Balance under Oxidative Stress Shuangchen Chen1,2*, Airong Liu1*, Degang Ji1, Xiaomin Lin1, Zimei Liu2, Xiaojian Xia2, Dilin Liu3 and Golam Jalal Ahammed2 1
Department of Horticulture, Henan University of Science and Technology, Luoyang 471003, P.R. China Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, P.R. China 3 Institute of Phytopathology and Applied Zoology, Justus-Liebig-University Giessen, Giessen D-35392, Germany 2
Received: August 21, 2013 / Accepted: October 15, 2013 © Korean Society of Plant Biologists 2014
Abstract Plant mitochondrial uncoupling proteins (pUCPs) play important roles in generation of metabolic thermogenesis, response to stress situation, and regulation of energy metabolism. Although the signaling pathways for the pUCPs-regulated plant energy metabolism and thermogenesis are well studied, the role of pUCPs in the regulation of plant stress tolerance has not been fully substantiated. Here we showed that mitochondrial uncoupling protein was required for effective antioxidant enzymes activities, chlorophyll fluorescence and redox poise in tomato under oxidative stress using virusinduced gene silencing approach. Silencing of LeUCP gene reduced maximal quantum yield of PSII (Fv/Fm) and photochemical quenching coefficient (qP), as well as mitigated activation of antioxidant enzymes and related genes expression. The content of reduced ascorbate and reduced glutathione, redox ratio of ascorbate and L-galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) activity were all decreased in the leaves of LeUCP gene-silenced plant. However, malondialdehyde content was increased under methylviologen (MV) stress. ROS accumulation was increased significantly following MV and heat stress treatments. Meanwhile, LeUCP gene silencing aggravated accumulation of H2O2 and O2 − in leaves. Taken together, these results strongly suggest that LeUCP gene plays critical role in maintaining the redox homeostasis and balance in antioxidant enzyme system under oxidative stress. ●
Keywords: Antioxidant enzymes activities, Chlorophyll fluorescence characteristics, Mitochondrial uncoupling protein, Oxidative stress, Redox poise *Corresponding author; Shuangchen Chen; Airong Liu Tel : +86-379-64282345 E-mail :
[email protected];
[email protected]
Introduction Plant uncoupling proteins (pUCPs) are membrane proteins from the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis (Vercesi et al. 2006; Jarmuszkiewicz et al. 2010; Begcy et al. 2011). pUCP mediates a fatty acid (FA)dependent, purine nucleotide-inhibited proton leak across the inner mitochondrial membrane (Echtay et al. 2002; Krauss et al. 2005). Although pUCPs were initially known as key players in thermogenesis (Zhu et al. 2011), their widespread presence in eukaryotes suggests that they are also involved in other processes (Nogueira et al. 2011), such as modulation of mitochondrial membrane potential (Jezek et al. 1996), reactive oxygen species (ROS) production (Kowaltowski et al. 1996; Popov et al. 2011), function as an antioxidant (Vercesi et al. 2006), and tricarboxylic acid cycle flux (Smith et al. 2004), thus modulating the energy balance within the mitochondria. Expression analyses and functional studies on pUCPs under normal and stress conditions suggest that the physiological role lies most likely in tuning up the mitochondrial energy metabolism in response of cells to stress situations (Borecky and Vercesi 2005). The induction of pUCPs can be a consequence of increased cellular ROS production under biotic and abiotic stressful conditions, such as pathogenic attack (Maxwell et al. 2002), drought and salt stress (Begcy et al. 2011), low or high temperature (Ozawa et al. 2006; Popov et al. 2011), and hyperosmotic stress (Trono et al. 2004). Induction of pUCPs gene expression by cold stress was observed in Arabidopsis (Maia et al. 1998), rice (Ozawa et al. 2006) and tomato (Popov et al. 2011). AtPUMP5 is also up-regulated by
10
wounds caused by abiotic stress factors such as wind, rain, and hail, and by biotic factors, particularly insect feeding (Cheong et al. 2002), indicating a possible physiological role of At-PUMP5 in plant response to stress. Again, expression of AtPUMP1 and -2 is increased in heatinduced programmed cell death but not in senescence, implying that some of the At-PUMP gene products may play a role in limiting ROS formation following exposure to heat (Swidzinski et al. 2002). Previous reports indicate that pUCP-mediated mitochondrial uncoupling controls mitochondrial ROS formation through a negative-feedback mechanism (Begcy et al. 2011). Furthermore, overexpression of AtPUMP1 in transgenic tobacco plants led to a significant increase in tolerance against H2O2-induced oxidative stress (Brandalise et al. 2003). Mitochondrial preparations from wheat seedlings exposed to salt (NaCl) or osmotic (mannitol) stress had increased pUCPs activities, suggesting that pUCP plays a role in ROS detoxification (Pastore et al. 2007). Thus, these studies suggest that pUCPs contribute to plant antioxidant defense by reducing mitochondrial ROS production in response to cellular stress. Several lines of evidence suggest that the role of pUCPs of unicellular eukaryotes is related to both the capacity of mitochondrial uncoupling and the metabolic status of the cell (Jarmuszkiewicz et al. 2010). All pUCP1 homologues may play a role in the mitochondrial redox balance in limiting the production of active oxygen species according to the Goglia and Skulachev (2003) hypothesis. However, the signaling cascades mediating pUCPs action in plant stress tolerance have not yet been fully substantiated. To obtain insights into the redox status in pUCPs-regulated abiotic stress tolerance, in this study, we examined the changes in H2O2 and O2·– accumulation, transcripts of stress responsive and defense related genes, antioxidant enzymes activities and redox poise in LeUCPs-silenced tomato plants under oxidative stress. We provided strong evidence that mitochondrial uncoupling proteins silencing negatively regulated redox poise, defense related genes expression, antioxidant enzymes activities and involved in ROS overproduction.
J. Plant Biol. (2014) 57:9-19
Fig. 1. Efficiency of gene silencing of LeUCP gene in the leaves of tomato plants. (A) The expression of LeUCP via qRT-PCR. (B) Immunoblotting analysis for LeUCP. After about 6 weeks, leaf samples were taken for quantitative RT-PCR to determine the gene-silencing efficiency. Data are expressed as means of triplicates with standard errors shown by vertical bars. Values with different letters are significantly different among the treatments (P < 0.05, Tukey’s test). Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
LeUCP transcript was observed in pTRV-LeUCP plants. Transcript level for LeUCP was reduced by 83.85% in the plants infected with pTRV-LeUCP VIGS vectors compared with mock plants (Fig. 1A). Meanwhile, no distinct difference for LeUCP transcripts was detected between mock and pTRV plants. As revealed in immunoblotting, decreased synthesis of LeUCP was marked in mitochondria isolated from pTRV-LeUCP plants (Fig. 1B). Hence, the VIGS was quite effective in silencing the target gene LeUCP. In addition, there were no significant differences bewteen pTRV plants and the mock plants.
Results Efficient Silencing of LeUCP in pTRV-LeUCP Plants
Effects of LeUCP Silencing on Chlorophyll Fluorescence Parameters under MV Stress
We first confirmed the stable integration of the transgene by PCR in the empty vector transformant and LeUCP-VIGS plants. Then we examined the expression of LeUCP in the plants infected with infiltration buffer (mock), empty vector (pTRV) and pTRV-LeUCP vector. In comparison with that in mock and pTRV plants, a dramatically decreased level of the
To examine how uncoupling proteins regulated photosynthetic capacity, we determined the effects of LeUCP gene absence on Chl fluorescence quenching. As shown in Fig. 2, LeUCP gene silencing did not cause any visible stress symptoms or changes in the maximum quantum yield of PSII (Fv/Fm). While an obvious damage in Fv/Fm in the leaves was
J. Plant Biol. (2014) 57:9-19
11
observed under methylviologen (MV) stress (Fig. 2), which indicated severe oxidative stress in the leaves. Compared with mock plants, the value of Fv/Fm was decreased and spot areas indicating injury to Fv/Fm were distinctly increased in LeUCP-silenced plants under MV stress. Similarly, photochemical quenching coefficient (qP) and electron transport rate (ETR) were remarkably declined (Fig. 3B-C), while non-photochemical quenching coefficient (NPQ) was significantly increased in LeUCP-silenced plants under MV stress (Fig. 3D). These results strongly suggested that LeUCP gene was important in regulating photosynthetic capacity through redistribution of the proportion of PSII electron flow. Effects of LeUCP Silencing on Antioxidant Enzymes Capacity and Genes Expression under MV Stress
Fig. 2. Methylviologen (MV)-induced damage in tomato leaves visualized by chlorophyll fluorescence imaging. Six leaves of each treatment were used to detect the changes in the maximum quantum yield of PSII (Fv/Fm). These six leaves were randomly selected as samples. Representative images of Fv/Fm indicated photooxidative injury. The false color code depicted at the bottom of the image ranges from 0.0 (black) to 1.0 (purple). Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
To determine whether a decrease in LeUCP results in the modulation of antioxidant enzymes under MV stress, we examined activities of the antioxidant enzymes. Regardless of MV treatment, the activities of APX, CAT, GPOD and SOD were all suppressed in the leaves of LeUCP-silenced plants (Fig. 4A-D). Furthermore, MV treatment decreased the antioxidant enzymes activities to a certain degree, which was decreased by 25.57%, 23.63%, 35.74% and 32.53%, respectively. LeUCP gene silencing also induced the activities or activation states of antioxidant enzymes under MV stress
Fig. 3. Effects of LeUCP gene silencing on maximum quantum yield of PSII (Fv/Fm) (A), photochemical quenching coefficient (qP) (B), non-photochemical quenching coefficient (NPQ) (C) and electron transport rate (ETR) (D) in the leaves of tomato plants under MV stress. Means ± s.e., n = 5. Means followed by different letters are significantly different at P0.05 as determined by Tukey’s test. Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
12
J. Plant Biol. (2014) 57:9-19
Fig. 4. Effects of LeUCP gene silencing on antioxidative enzymes activities and gene transcripts in the leaves of tomato plants under MV stress. Means ± s.e., n = 5. Means followed by different letters are significantly different at P0.05 as determined by Tukey’s test. Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
J. Plant Biol. (2014) 57:9-19
13
results, the content of H2O2 (Fig. 6C) and O2 − (Fig. 6D) was induced significantly in the leaves of LeUCP gene-silenced plants. In agreement with MV treatment, intensive H2O2 (Fig. 6E) and O2 − (Fig. 6F) accumulation as dark brown spots in leaves of LeUCP-silenced plants were also observed under high temperature. Using the CeCl3-based procedure, we observed that H2O2 was predominantly accumulated on the cell walls of mesophyll cells facing intercellular spaces but was hardly detectable in the cytosol or intracellular organelles, such as chloroplasts, mitochondria, nuclei, or vacuoles (Fig. 7A-F). ●
●
Fig. 5. Effects of LeUCP gene silencing on MDA content in the leaves of tomato plants under MV stress. Means ± s.e., n = 5. Means followed by different letters are significantly different at P0.05 as determined by Tukey’s test. Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
compared with mock, while the activities were lower than Mock+MV and pTRV+MV treatment. Consistent with the changes in enzyme activity, LeUCP gene silencing significantly reduced the transcript level of stress responsive and antioxidant genes in tomato leaves (Fig. 4E-J). Interestingly, low gene expression was observed in pTRV-LeUCP+MV treatment compared with Mock+MV and pTRV+MV treatments. The expression levels of CAT1, cAPX, CAT2, Fe-SOD, G-POD, GR1 genes in the pTRVLeUCP + MV treatment were 0.319, 0.327, 0.436, 0.626, 0.351 and 0.545 folds, respectively compared with Mock+MV treatment. By contrast, no significant effect was detected in the above gene expression between mock and pTRV plants. These results strongly suggest that LeUCP gene is essential for effective antioxidant enzymes capacity. Both LeUCP silencing and MV treatment increased the MDA content in a dose dependent manner in the leaves compared with the control. Importantly, LeUCP silencing remarkably simulate MDA levels under MV stress, which was three times than that observed following treatment with pTRV-LeUCP alone (Fig. 5). Effects of LeUCP Silencing on the Change of Reactive Oxygen Species under MV and Heat Stress To determine a possible role of ROS in LeUCP-silenced plants under MV stress, we attempted to detect in situ accumulation of H2O2 (Fig. 6A) and O2 − (Fig. 6B) using 3,3'diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining procedures, respectively. Little or only slight differences were observed in the staining of the leaves between mock and pTRV treatment. However, an increased staining was detected in the leaves under MV treatment. The leaves of LeUCP-silenced plants showed even more severe damage under MV stress. Consistent with histochemical staining ●
Effects of LeUCP Silencing on Changes of Cellular Redox Status under MV Stress To determine the involvement of altered cellular redox status in response to LeUCP gene silencing, we examined the redox state on the accumulation of AsA and GSH pool under MV stress (Fig. 8A-F). The content of AsA and GSH was decreased in the leaves of LeUCP gene-silenced plants compared with mock, and the decline was 24.68% and 39.74%, respectively. By contrast, no major differences on AsA or GSH contents were observed between mock and pTRV treatment. In addition, MV stress resulted in greater decreases in the level of AsA, GSH and redox ratio of ascorbate, but led to equivalent degree of dehydroascorbate (DHA) increase in mock and LeUCP gene-silenced plants. In comparison, the content of AsA and GSH of pTRVLeUCP+MV was still lower than Mock+MV and pTRV+MV treatment. LeUCP gene silencing also resulted in the reduction of GaILDH activity, and MV stress resulted in greater decreases in the level of GaILDH activity compared with mock (Fig. 9). Taking all these data together, we showed that LeUCP gene was associated with the cellular redox homeostasis under MV stress.
Discussion Uncoupling protein (pUCP)-like activities have been detected in mitochondria from unicellular organisms, higher eukaryotes and plants (Jarmuszkiewicz et al. 2010). It is proposed that pUCP1 has a specialized physiological role in generation of metabolic (non-shivering) thermogenesis in hibernating mammals, in cold-adaptation of newborn mammals, and in diet-induced thermogenesis in small rodents (Fávaro et al. 2007). Additionally, in unicellular organisms resistance to exogenous ROS is enhanced by pUCP activity probably as a result of pUCP-induced reduction in endogenous ROS production (Krauss et al. 2005). However, the precise mechanism of superoxide activation and the exact function of pUCP in plants are not yet fully established. In this study,
14
J. Plant Biol. (2014) 57:9-19
Fig. 6. Effects of LeUCP gene silencing on H2O2 and O2 − accumulation in leaves of tomato plants under MV and heat stress. (A-B) The in situ detection of H2O2 and O2.- in leaves. The leaf segments were loaded with DAB and NBT, and then incubated for 8 hr and 6 hr, respectively. These leaves were randomly selected as samples. The accumulation of H2O2 and O2 − was observed by an Olympus motorized system microscope (BX61, Olympus Co., Tokyo, Japan). Bar = 1.0 mm. (C-F) Quantification of H2O2 and O2 . Means ± s.e., n = 5. Means followed by different letters are significantly different at P0.05 as determined by Tukey’s test. MV: methyl viologen; NT: normal temperature; HT: high temperature (42°C). Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector. ●
●
●
we demonstrated a possible role of pUCPs, which was essential for the effectiveness of chlorophyll fluorescence parameters, redox poise and antioxidant enzymes activities in response to oxidative stress, thereby reducing ROS
overproduction. Some studies have shown that mitochondrial uncoupling protein is required for efficient photosynthesis in Arabidopsis (Sweetlove et al. 2006). Absence of pUCP1 in Arabidopsis
J. Plant Biol. (2014) 57:9-19
Fig. 7. Cytochemical detection of H2O2 accumulation in mesophyll cells of tomato leaves with CeCl3 staining and transmission electron microscopy. Arrows, CeCl3 precipitates; C, chloroplast; CW, cell wall; V, vacuole; IS, intercellular space. Scale bars = 1 µm. Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’vector.
resulted in altered mitochondrial electron transport chain. We also suggested that LeUCP gene in tomato leaves is crucial in maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism (Chen et al. 2013). In the present study, we provided strong evidence that LeUCP gene regulated chlorophyll fluorescence characteristics, to a large extent, by affecting capacity of Fv/ Fm, qP, ETR and NPQ (Fig. 2 & 3). qP reflects the share of light energy absorbed by PSII antenna pigments which are used for photochemical electron transfer. NPQ reflects the part of light energy absorbed by PSII antenna pigments which can not be used for photosynthetic electron transport and dissipated in the form of heat (Zai et al. 2012). In this study, Fv/Fm, ETR and qP in the leaves of LeUCP genesilenced plants under MV stress were much lower than that in mock plants. This observation, together with higher NPQ value in the leaves of LeUCP gene-silenced plants implies that the efficiency of PSII photochemistry of LeUCP genesilenced plants is lower than that of normal plants. We proposed that inactivation of LeUCP gene could restrict electron transport, which increases ROS production by the respiratory chain. The chloroplast redox state, manifested through the plastquinone pool, is involved in the regulation of several
15
important biological processes including nuclear and plastid gene expression, hormone signaling and stress responses. Expression of nuclear genes encoding plastid proteins has been shown to correlate with redox shifts in the plastquinone pool, glutathione and ascorbate pools in coordination with chloroplast development and light signals (Foyer and Noctor 2009; Suzuki et al. 2012). Under high light or drought and salt stress, changes in the redox state of the plastquinone pool are known to be correlated with expression of antioxidant and defense genes, including pathogen defense genes, and phosphorylation of thylakoid proteins (Karpinski et al. 1999; Li et al. 2009; Begcy et al. 2011). In the present study, we showed that the AsA pool, glutathione content and redox ratio of ascorbate were all significantly reduced accompanied by increased ROS accumulation in LeUCP gene-silenced plants under oxidative stress. In turn, interaction of ROS with membrane lipids leads to the change of redox status. Unlike chloroplasts and mitochondria, the apoplast has a low antioxidant-buffering capacity. So it showed a marked accumulation of H2O2 on the cell walls of mesophyll cells. ROS network genes and redox regulatory enzymes such as CAT, SOD, GPOD and APX, GalLDH and related genes were also lower expressed in the LeUCP gene-silenced plants. This is in agreement with earlier studies on higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. These genes could be considered as functional redox state reporters that would indicate the oxidative status under different physiological conditions (Mittler et al. 2004). In plant cells, antioxidants provide essential information on cellular redox state, and they influence gene expression associated with biotic and abiotic stress responses to maximize defense (Foyer and Noctor 2005). Thus, low levels of ascorbate in LeUCP-silenced plants can act as an elicitor of oxidative stress resistance responses, as can changes to the cellular glutathione pool (Barth et al. 2004). As well as their established roles in the regulation of the enzymes of carbon metabolism, LeUCP may also act to modulate the activity of key antioxidative enzymes, such as APX, CAT, GPOD and SOD. Our data highlight the protective role of pUCPs in vivo and provide a new approach for manipulating plants with regulated tolerance to various abiotic stresses. In addition, our results suggest that LeUCP silencing decreased maximal quantum yield of PSII accompanied with enhanced ROS accumulation and imbalanced redox signals under oxidative stress. Therefore, it is likely that the LeUCP gene play an important role for establishing extensive tolerance.
16
J. Plant Biol. (2014) 57:9-19
Fig. 8. Effects of LeUCP gene silencing on ascorbate-glutatione pool in the leaves of tomato plants under MV stress. Means ± s.e., n = 5. Means followed by different letters are significantly different at P0.05 as determined by Tukey’s test. Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
Materials and Methods Plant Materials and Growth Conditions One-week-old tomato (Solanum lycopersicon L. cv. Zhongza 9) seedlings were transplanted into a growth medium with a mixture of peat and vermiculite (2:1, v:v) in 15 cm diameter pots. Plants were maintained in growth chambers under a 16 h light (200 µmol m−2 s−1) at 25°C and an 8 h dark at 22°C, respectively. Plants were watered daily and fertilized with Hoagland’s nutrition solution every two days. When cotyledons were fully expanded and the true leaves have not yet appeared, they were used for the experiment.
Fig. 9. Effects of LeUCP gene silencing on GaILDH activity in the leaves of tomato plants under MV stress. Means ± s.e., n = 5. Means followed by different letters are significantly different at P0.05 as determined by Tukey’s test. Mock: the plants infected with infiltration buffer; pTRV: the control transformed by ‘empty’ vector; pTRV-LeUCP: the plants transformed by ‘pTRV-LeUCP’ vector.
Virus-induced Gene Silencing (VIGS) Constructs and Agrobacteriummediated Virus Infection VIGS was performed by infiltration on 15-d-old tomato seedlings with a mix of pTRV1- and pTRV2-carrying Agrobacteria tumefaciens according to Ekengren et al. (2003). The empty vectors of pTRV1 and pTRV2 were obtained from Dr. Song Fengming (Zhejiang University, Hangzhou, China). The pTRV2-LeUCP VIGS constructs were generated
J. Plant Biol. (2014) 57:9-19
17
as described previously (Kandoth et al. 2007). For generating LeUCPsilencing construct, a 425-bp fragment of the LeUCP (accession: AF472619.1) was PCR-amplified (forward primer: GGATTGGGATGTATGAAC; reverse primer: GGACCCTATGCAAACTG; the two primers contain EcoRI and XhoI restriction sites, respectively). The PCR fragment was digested with EcoRI and XhoI and cloned into the same sites of pTRV2. All the constructs were confirmed by sequencing and then transformed to Agrobacteria tumefaciens strain GV3101. After the virus infection, plants were kept at 23oC/21oC under 200±20 µmol m−2 s−1 PPFD. After 6 weeks, quantitative RTPCR and LeUCP Western blot in isolated mitochondria were performed to determine the gene silencing efficiency before using the plants for further research (Panda et al. 2013). Mitochondria were isolated in accordance with standard differential centrifugation procedures as previously described (Vianello et al. 1995).
the case of O2 −, whole leaves were vacuum infiltrated directly with 0.5 mg mL−1 NBT in 25 mM K-Hepes buffer (pH 7.8) and incubated at 25°C in the light for 6 h (light intensity of 600 µmol m−2 s−1). In the case of H2O2, whole leaves were vacuum infiltrated with 1 mg mL−1 2,3’-diaminobenzidine (DAB) in 50 mM Tris-acetate (pH 3.8) and incubated at 25°C in light for 8 h. In both cases, leaves were rinsed in 80% (v/v) ethanol for 10 min at 90°C, mounted in lactic acid/phenol/ water (1:1:1; v/v), and photographed by microscope at ×4 magnification. H2O2 was visualized at the subcellular level using CeCl3 for localization (Bestwick et al. 1997). Samples were fixed, embedded, sectioned, and stained for conventional electron microscopy (Xia et al. 2009). Sections were examined using a transmission electron microscope (JEM-1200EX, JEOL, Japan) at an accelerating voltage of 75 kV.
Experimental Design and Stress Tolerance Measurement
For the enzyme assays, 0.3 g of frozen leaves were ground with 3 mL ice-cold 25 mM PBS buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM AsA (ascorbic acid) and 2% PVP (polyvinylpolypyrrolidone). The homogenates were centrifuged at 4°C for 20 min at 12,000×g, and the resulting supernatants were used for the determination of enzymatic activity. Superoxide dismutase (SOD) activity was assayed by measuring the ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT) as described by Stewart and Bewley (1980). The activity of CAT was measured using the method of Cakmak and Marschner (1992). Ascorbate peroxidase (APX) activity was measured by a decrease in A290 according to Nakano and Asada (1981). The method published by Cakmak and Marschner (1992), with some modifications, was used to determine guaiacol peroxidase (GPOD) activity. The contents of the reaction mixture were as follows: 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 10 mM H2O2, and enzyme extract. The increase in absorbance at 470 nm, caused by guaiacol oxidation (E = 26.6 mM cm−1), was used to measure activity. Lipid peroxidation was estimated by measuring the content of MDA in leaves using the method of Hodges et al. (1999).
VIGS plants of mock, pTRV and pTRV- LeUCP at the 6-leaf stage were sprayed with 50 µM MV, 1000 µmol m−2 s−1 PPFD for 4 h or exposed to heat stress at 42oC, 200±20 µmol m−2 s−1 PPFD for 24 h. Stress tolerance was measured based on changes in Fv/Fm at 4 h after MV or 10 h after heat treatment. The 5th leaves were sampled at 6 h after MV and heat stress respectively for analysis of H2O2 and O2 − content, redox status, gene expression and antioxidative enzyme activities. ●
Analysis of Chlorophyll Fluorescence Chlorophyll fluorescence was measured using an Imaging-PAM Chlorophyll Fluorometer attached a computer-operated PAM-control unit (Walz, Germany). The seedlings were kept in dark for about 30 min before measurement. The intensities of actinic light and saturating light setting were 280 µmol mol−2 s−1 and 2500 µmol mol−2 s−1 PAR, respectively. The maximum quantum yield of PSII (Fv/Fm) and nonphotochemical quenching coefficient (NPQ) were measured and calculated according to van Kooten & Snel (1990): NPQ = (Fm – F’m)/F’m; Fv/Fm = (Fm – Fo)/Fm; ΦPSII = (Fm’ – Fs)/Fm’, where Fm is the maximum chlorophyll fluorescence from dark-adapted leaves, F’m the maximum chlorophyll fluorescence under actinic light exposition, and Fs the stationary fluorescence during illumination. Quantitative Assay and Histochemical Detection of H2O2 and O2 − ●
H2O2 was extracted from leaf tissue according to Doulis et al. (1997). Leaf material (0.5 g) was ground in liquid nitrogen and 2 mL of 0.2 M HClO4. After thawing, the mixture was transferred to a 10 mL plastic tube and another 2 mL of 0.2 M HClO4 was added. The homogenate was centrifuged at 2,700×g for 30 min at 4°C and the supernatant was collected, adjusted to pH 6.0 with 4 M KOH and centrifuged at 110×g for 1 min at 4°C. The supernatant was placed onto a AG1x8 prepacked column (Bio-Rad, Hercules, CA) and H2O2 was eluted with 4 mL double-distilled H2O. Recovery efficiencies of H2O2 from different samples were determined by analysing duplicate samples to which H2O2 was added during grinding at a nal concentration of 50 µM. H2O2 was determined by a spectrophotometric assay (Willekens et al. 1997). The sample (800 µL) was mixed with 400 µL reaction buffer containing 4 mM 2,2’-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 µL deionized water, and 0.25 U of horseradish peroxidase. H2O2 content was measured at OD412. O2 − was measured as described by Elstner and Heupel (1976) by monitoring the nitrite formation from hydroxylamine in the presence of O2 −. The absorbance in the aqueous solution was recorded at 530 nm. A standard curve with NO2- was used to calculated the production rate of O2 − from the chemical reaction of O2 − and hydroxylamine. The histochemical staining of O2 − and H2O2 was performed as previously described (Xia et al. 2009) with minor modifications. In ●
●
●
●
●
●
Antioxidant Enzymes Activities and MDA Assay
RNA Extraction and qRT-PCR for Gene Expression Analysis Total RNA was isolated from tomato young leaves in different treatments using Trizol reagent (Sangon, China) according to the manufacturer’s instruction. Genomic DNA was removed with RNeasy Mini Kit (Qiagen, Germany). Total RNA (1 µg) was reverse-transcribed using ReverTra Ace qPCR RT Kit (Toyobo, Japan) following the manufacturer’s instruction. Quantitative real-time PCR was performed using the iCycler iQTM real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Each reaction (25 µL) consists of 12.5 µL SYBR Green PCR Master Mix (Takara, Japan), 1 µL of diluted cDNA and 0.1 µmol of forward and reserve primers. PCR cycling conditions were as follows: 95oC for 3 min and 40 cycles of 95oC for 10 s 58oC for 45 s. On the basis of EST sequences, the following gene-specific primers were designed and used for amplification: CAT1 (encoding catalase 1), 5'-TTCGTGCATTGAAACCAAAT-3' and 5'-TGTAATCCGTTGGGAGACAA-3'; CAT2 (encoding catalase 2), 5'-ACCCAGAGGTATCGACTTGG-3' and 5'-GTCGATCTCCTCATCCCTGT 3'; GR1 (encoding glutathione reductase 1), 5'- GCCACTCTTTCTGGTCTTCC-3' and 5'-TGCTGTAGGGTGAATACCCA-3'; cAPX (encoding ascorbate peroxidase 1), 5'-GACTCTTGGAGCCCATTAGG-3' and 5'AGGGTGAAAGGGAACATCAG-3'; Fe-SOD (encoding Fe superoxide dismutase), 5'-TTGTGGAGCCAATTGAACAT-3' and 5'-TAACCACGATGATGCTCTCC-3'; G-POD (encoding glutathione peroxidase), 5'-GACAGGAGCCTGGAAACATT-3'; 5'-CATCTCCAAAGAACCCACCT-3'; actin, 5'-TGGTCGGAATGGGACAGAAG-3' and 5'CTCAGTCAGGAGAACAGGGT-3'. The quantification of mRNA levels is based on the method of Livak and Schmittgen (2001). The threshold cycle (Ct) value of actin was subtracted from that of the
18
gene of interest to obtain a ∆Ct value. The Ct value of untreated control sample was subtracted from the ∆Ct value to obtain a ∆∆Ct value. The fold changes in expression level relative to the control were expressed as 2−∆∆Ct. Determination of Non-enzymatic Antioxidants in Leaves For the measurement of AsA, DHA, reduced glutathione (GSH) and oxidized glutathione (GSSG) content, leaf tissue (0.2 g) was homogenized in 2 mL of 2% metaphosphoric acid containing 2 mM EDTA and centrifuged at 4°C for 10 min at 14,000×g. AsA and DHA were measured following Law et al. (1983). The total AsA was determined by initially incubating the extract for 50 min with 200 mM phosphate buffer solution (pH 7.4) and 1.5 mM DTT to reduce all DHA to AsA. After incubation, 200 µL of 0.5% (w/v) N-ethylmaleimide (NEM) was added to remove excess DTT. AsA was analyzed in a similar manner except that 400 µL deionized H2O was substituted for DTT and NEM. Color was developed in both series of reaction mixtures (total and reduced ascorbate) with the addition of 400 µL 10% (w/v) trichloroacetic acid, 400 µL 44% ophosphoric acid, 4% α-dipyridyl in 70% ethanol and 200 µL 3% (w/ v) FeCl3. The reaction mixtures were then incubated at 40°C for 40 min in a water bath and the absorbance was recorded at 525 nm. The DHA concentration was obtained by subtracting the AsA concentration from the total concentration. GSH and GSSG content was measured according to Rao and Ormrod (1995). After neutralization with 0.5 M phosphate buffer (pH 7.5), 0.1 mL of the supernatant was added to a reaction mixture containing 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.5), 5 mM EDTA and 0.6 mM 5,5’-dithio-bis (2-nitrobenzoic acid). The reaction was started by adding 3 U of GR and was monitored by measuring the changes in absorbance at 412 nm for 1 min. For the GSSG assay, GSH was masked by adding 20 µL of 2-vinylpyridine to the neutralized supernatant, whereas 20 µL of water was added for the total glutathione assay. The GSH concentration was obtained by subtracting the GSSG concentration from the total concentration. L-galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3) was extracted and measured by the method of Shan and Liang (2010). Statistical Analyses All data presented are averages of five repetitions of each treatment. Data were statistically analyzed using analysis of variance (AVONA), and tested for significant (P0.05) treatment differences using Tukey's test. Orgin pro 7.5 version was used to prepare graphs.
Acknowledgments This work was supported by National Natural Science Foundation of China (31101536), Science Development Plan Project of Shandong Province (2012GNC011111), Outstanding Young Teacher Project in Henan Province (2011GGJS-075), the National Key Technology R&D Program of China (2011BAD12B03) and the National Scholar Foundation for Studying Abroad (2011841011, 2011841012).
Author's Contributions SC, AL and XX designed the research; ZL and DJ performed the research and biochemical analysis; SC, ZL, DL and GJA analyzed the data and wrote the paper. All the authors agreed on the contents of the paper and post no conflicting interest.
J. Plant Biol. (2014) 57:9-19
References Barth C, Moeder W, Klessig DF, Conklin PL (2004) The timing of senescence and response to pathogens is altered in the ascorbatedeficient Arabidopsis mutant vitamin c-1. Plant Physiol 134:1784− 1792 Begcy K, Mariano ED, Mattiello L, Nunes AV, Mazzafera P, Maia IG, Menossi M (2011) An Arabidopsis mitochondrial uncoupling protein confers tolerance to drought and salt stress in transgenic tobacco plants. PLoS One 6:e23776 Bestwick CS, Brown IR, Bennett MHR, Mansfield JW (1997) Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. Plant Cell 9:209−221 Borecky J, Vercesi AE (2005) Plant uncoupling mitochondrial protein and alternative oxidase: Energy metabolism and stress. Bioscience Rep 25:271−286 Brandalise M, Maia IG, Borecký J, Vercesi AE, Arruda P (2003) Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress. J Bioenerg Biomembr 35:203−209 Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of super oxide dismutase ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol 98:1222−1227 Cheong YH, Chang HS, Gupta R, Wang X, Zhu T, Luan S (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol 129:661−677 Chen SC, Jin WJ, Liu AR, Liu ZM, Liu DL, Xia XJ, Lin XM. (2013)Mitochondrial uncoupling protein silence is compromised in photosynthesis and redox poise. Acta Physiol Plant 35:2547− 2558 Doulis AG, Debian N, Kingston-Smith AH, Foyer CH (1997) Differential localization of antioxidants in maize leaves. Plant Physiol 114:1031−1037 Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD. (2002) Superoxide activates mitochondrial uncoupling proteins. Nature 415:96−99 Ekengren SK, Liu Y, Schiff M, Dinesh-Kumar SP, Martin GB (2003) Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato. Plant J 36:905−917 Elstner EF, Heupel A (1976) Inhibition of nitrite formation from hydroxylammonium-chloride-simple assay for superoxide dismutase. Anal Biochem 70:616−620 Fávaro RD, Borecký J, Colombi D, Vercesi AE, Maia IG (2007) Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein. BBA-Bioenergetics 1767:1412−1417 Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866−1875 Foyer CH, Noctor G (2009) Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxid Redox Sign 11:861−905 Goglia F, Skulachev VP (2003) A function for novel uncoupling proteins: antioxidant defense of mitochondrial matrix by translocating fatty acid peroxides from the inner to the outer membrane leaflet. FASEB J 12:1585−1591 Hodges DM, Delong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactivesubstances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604−611 Jarmuszkiewicz W, Woyda-Ploszczyca A, Antos-Krzeminska N, Sluse FE (2010) Mitochondrial uncoupling proteins in unicellular
J. Plant Biol. (2014) 57:9-19
eukaryotes. BBA-Bioenergetics 1797:792−799 Jezek P, Costa AD, Vercesi AE (1996) Evidence for aniontranslocating plant uncoupling mitochondrial protein in potato mitochondria. J Biol Chem 271:32743−48 Kandoth PK, Ranf S, Pancholi SS, Jayanty S, Walla MD, Miller W, Howe GA, Lincoln DE, Stratmann JW (2007) Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systeminmediated defense response against herbivorous insects. Proc Natl Acad Sci USA 104:12205−12210 Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P (1999) Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 284:654−657 Kowaltowski AJ, Castilho RF, Vercesi AE (1996) Opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of Ca2+ is dependent on mitochondrial-generated reactive oxygen species. Febs Lett 378:150−152 Krauss S, Zhang CY, Lowell BB (2005) The mitochondrial uncouplingprotein homologues. Nat Rev Mol Cell Biol 6:248−261 Law MY, Charles SA, Halliwell B (1983) Glutathione and ascorbicacid in spinach (Spinacia oleracea) chloroplasts - the effect of hydrogen-peroxide and of paraquat. Biochem J 210:899–903 Li Z, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and responding to excess light. Annu Rev Plant Biol 60:239−260 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods 25:402−408 Maia IG, Benedetti CE, Leite A, Turcinelli SR, Vercesi AE, Arruda P (1998) AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein. Febs Lett 429:403−406 Maxwell DP, Nickels R, Mcintosh L (2002) Evidence of mitochondrial involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence. Plant J 29:269−279 Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490−498 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867−880 Nogueira FTS, Sassaki FT, Maia IG (2011) Arabidopsis thaliana uncoupling proteins (AtUCPs): insights into gene expression during development and stress response and epigenetic regulation. J Bioenerg Biomembr 43:71–79 Ozawa K, Murayama S, Kobayashi-Uehara A, Handa H (2006) Overexpression of wheat mitochondrial uncoupling protein in rice plants confers tolerances to oxidative stresses promoted by exogenous hydrogen peroxide and low temperature. Mol Breeding 18:51−56 Pastore D, Trono D, Laus MN, Di Fonzo N, Flagella Z (2007) Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria. J Exp Bot 58:195−210 Panda SK, Sahoo L, Katsuhara M, Matsumoto H (2013) Overexpression of alternative oxidase gene confers aluminum tolerance by altering the respiratory capacity and the response to oxidative
19
stress in tobacco cells. Mol Biotechnol 54:551−563 Popov VN, Eprintsev AT, Maltseva EV (2011) Activation of genes encoding mitochondrial proteins involved in alternative and uncoupled respiration of tomato plants treated with low temperature and reactive oxygen species. Russ J Plant Physiol 58:914−920 Rao MV, Ormrod DP (1995) Impact of UVB and O3 on the oxygenfree radical scavenging system in Arabidopsis thaliana genotypes differing in flavonoid biosynthesis. Photochem Photobiol 62:719− 726 Shan CJ, Liang ZS (2010) Jasmonic acid regulates ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress. Plant Sci 178:130−139 Smith AMO, Ratcliffe RG, Sweetlove LJ (2004) Activation and function of mitochondrial uncoupling protein in plants. J Bioll Chem 279:51944−51952 Stewart RRC, Bewley JD (1980) Lipid-peroxidation associated with accelerated aging of soybean axes. Plant Physiol 65:245–248 Suzuki N, Koussevitzky S, Mittler R, Miller G (2012) ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ 35:259−270 Sweetlove LJ, Lytovchenko A, Morgan M, Nunes-Nesi A, Taylor NL, Baxter CJ, Eickmeier I, Fernie AR (2006) Mitochondrial uncoupling protein is required for efficient photosynthesis. Proc Natl Acad Sci USA 103:19587−19592 Swidzinski JA, Sweetlove LJ, Leaver CJ (2002) A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana. Plant J 30:431−446 Trono D, Flagella Z, Laus MN, Di Fonzo N, Pastore D (2004) The uncoupling protein and the potassium channel are activated by hyperosmotic stress in mitochondria from durum wheat seedlings. Plant Cell Environ 27:437−448 van Kooten O, Snel JFH (1990) The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res 25:147− 150 Vercesi AE, Borecky J, Godoy Maia ID, Arruda P, Cuccovia IM, Chaimovich H (2006) Plant uncoupling mitochondrial proteins. Annu Rev Plant Biol 57:383−404 Vianello A, Macri F, Braidot E, Mokhova EN (1995) Effect of 6ketocholestanol on FCCP- and DNP-induced uncoupling in plant mitochondria. FEBS Lett 365:7–9 Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, Van Montagu M, InzéD, Van Camp W. (1997) Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. EMBO J 16:4806−4816 Xia XJ, Wang YJ, Zhou YH, Tao Y, Mao WH, Shi K, Asami T, Chen ZX, Yu JQ. (2009) Reactive oxygen species are involved in Brassinosteroid-induced stress tolerance in cucumber. Plant Physiol 150:801−814 Zai XM, Zhu SN, Qin P, Wang XY, Che L, Luo FX (2012) Effect of Glomus mosseae on chlorophyll content, chlorophyll fluorescence parameters, and chloroplast ultrastructure of beach plum (Prunus maritima) under NaCl stress. Photosynthetica 50:323−328 Zhu Y, Lu JF, Wang J, Chen F, Leng FF, Li HY (2011) Regulation of thermogenesis in plants: the interaction of alternative oxidase and plant uncoupling mitochondrial protein. J Integr Plant Biol 53:7−13
