DOI: 10.1007/s10535-012-0115-1
BIOLOGIA PLANTARUM 56 (4): 635-640, 2012
Modified alternative oxidase expression results in different reactive oxygen species content in Arabidopsis cell culture but not in whole plants V.I. TARASENKO*, E.Y. GARNIK, V.N. SHMAKOV and Y.M. KONSTANTINOV Siberian Institute of Plant Physiology and Biochemistry, Siberian Division of the Russian Academy of Sciences, Lermontova 132, Irkutsk 664033, Russia
Abstract Alternative oxidase (AOX) transfers electrons from ubiquinone to oxygen in the respiratory chain of plant mitochondria. It is widely accepted that AOX functions as a mechanism decreasing the formation of reactive oxygen species (ROS) produced during respiratory electron transport. However, there are no experimental data to provide unambiguous proof of this hypothesis. We have studied growth characteristics, ROS content, and stress sensitivity in Arabidopsis transgenic lines with reduced or increased levels of AOX. We demonstrated that AOX-deficient plants grown in soil had an extended reproductive phase. Changes in AOX activity did not affect ROS content or stress sensitivity in the whole plants. However in the suspension culture, cells overexpressing AOX had significantly lower ROS content, whereas the AOX-deficient cells had higher ROS content compared to the wild-type (WT) cells. Prooxidant treatment led to the increase in ROS content and to the reduction of viability more in the cells overexpressing AOX than in WT and AOX-deficient cells. Thus, we demonstrated that differences in the metabolism of whole plants and cultured cells might affect AOX functioning. Additional key words: antimycin A, hydrogen peroxide, menadione, oxidative stress, superoxide, suspension culture.
Introduction The electron transport chain (ETC) of plant mitochondria has unique features compared with that of other eukaryotes, including the presence of alternative electron transfer pathway. This pathway is represented by the single AOX protein, which is capable of accepting electrons from ubiquinone and transferring them directly to oxygen, thereby bypassing 2 of 3 oxidation and phosphorylation coupling sites in the ETC (Moller 2001). It is widely accepted that the alternative pathway functions as a mechanism to prevent the formation of ROS when the ubiquinone pool is over-reduced (Vanlerberghe and McIntosh 1997). Thus, AOX may be involved in plant acclimation to a number of abiotic stresses. Numerous data on AOX induction upon cytochrome pathway inhibition (Karpova et al. 2002, Clifton et al. 2005) and under diverse stress treatments (Ferreira et al. 2008, Hu et al. 2010) serve as arguments in favor of this hypothesis. However, there is still no direct proof of this role of AOX. Transgenic plants or cell cultures with modified expression of genes encoding components of the
respiratory chain are useful tools to study functions of these proteins. Thus, the use of tobacco transgenic cells has provided evidence in favor of the important role of AOX in preventing of ROS formation in the respiratory chain. Tobacco suspension cells that overexpress or underexpress AOX had significantly lower and higher ROS content, respectively (Maxwell et al. 1999). However, similar research (Umbach et al. 2005) done on transgenic Arabidopsis plants has not revealed significant differences in ROS content. Altered ROS content was observed only upon chemical inhibition of the ETC. Treatment with the cytochrome pathway inhibitor potassium cyanide resulted in increased ROS content in leaves and roots of AOX-deficient plants. Additionally, slower growth of antisense plants and accelerated growth of plants over-expressing AOX was demonstrated at lower temperatures (Fiorani et al. 2005). In another study, authors have analyzed the Arabidopsis plants with the genetic knockout of aox1a gene. Similar to Arabidopsis antisense plants, these plants did not differ phenotypically from the wild-type (WT)
⎯⎯⎯⎯ Received 30 May 2011, accepted 15 November 2011. Abbreviations: AOX - alternative oxidase; DAB - 3,3′-diaminobenzidine; DCF-DA - 2′,7′-dichlorofluorescein diacetate; ETC - electron transport chain; NBT - nitroblue tetrazolium; ROS - reactive oxygen species; WT - wild-type. Acknowledgements: The study was financially supported by the Integration project SB RAS № 59, by RFBR (12-04-01148-а) and by Ministry of Education and Science of the Russian Federation. * Corresponding author; fax: (+7) 3952 510754, e-mail:
[email protected]
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plants when growing under normal conditions. However, the authors demonstrated higher sensitivity of the AOX1a knockout plants to a combination of high irradiance and drought (Giraud et al. 2008). Study of tobacco plants with reduced or increased AOX activity has resulted in a paradoxical conclusion: AOX overexpressors were more sensitive to oxidative stress caused by ozone treatment (Pasqualini et al. 2007). As compared to the WT plants, the ozone-treated plants
overexpressing AOX had higher content of H2O2 in leaves and displayed signs of leaf damage. Thereby, published data on the relationship between AOX activity and ROS content remain controversial. To further investigate the reason for such contradictions, we compared growth characteristics, ROS content, and stress sensitivity in Arabidopsis plants with altered levels of AOX1a and in the suspension cultures obtained from these plants.
Materials and methods Seeds of Arabidopsis thaliana (L.) Heynh line Col-0 (WT plants, ecotype Columbia), line AS-12 (plants transformed with the construct carrying the aox1a gene under control of the CAMV 35S promoter in the antisense orientation), and line XX-2 (plants transformed with the aox1a gene construct in the sense orientation) (Umbach et al. 2005) were obtained from Nottingham Arabidopsis Stock Centre (UK). Plants were grown at temperature of 23 ºС, 16-h photoperiod and irradiance of 100 μmol m-2 s-1. A mixture of compost and Vermiculite (1:1) was used as a substrate. At least 30 plants were used to estimate growth characteristics. Seeds were firstly sterilized by immersion into 0.1 % HgCl2 solution for 15 min, then they were washed three times with sterile water. Vertically mounted Petri dishes with 1/2 Murashige and Skoog (MS) salts and 0.8 % Phytagel were used to grow seedlings. Stress agents (180 mM NaCl, 3 mМ H2O2, 25 µМ menadione) were added into the medium before pouring. At least 100 seedlings were tested in each of five biological replicates. Superoxide content in leaves was determined by nitroblue tetrazolium (NBT) staining according to Meyer et al. (2009). Leaves were incubated with NBT solution (1 mg cm-3) in 10 mМ KH2PO4 (pH 7.8) in dark for 1 h and then were placed in 70 % ethanol solution and incubated at 80 °С until the chlorophyll was completely removed. Formazan blue precipitate formed during the reaction was extracted through leaf grinding in a mixture of 2 M KOH and DMSO (1:1) according to Myouga et al. (2008). After centrifugation for 10 min at 12 000 g, the absorbance of the supernatant was measured at 700 nm. The specificity of the reaction was assessed by adding MnCl2 (final concentration 10 mМ) and superoxide dismutase (final concentration 10 U cm-3) in the incubation solution. Leaves were not stained in the presence of these agents. Hydrogen peroxide content in leaves was determined by 3,3′-diaminobenzidine (DAB) staining according to Ramel et al. (2009). Leaves were incubated with DAB (2 mg cm-3) in 10 mМ Tris-acetate (pH 5.0) in dark for 5 h. Chlorophyll was removed as described above. Brown precipitate formed during DAB polymerization was extracted by HClO4 solution (0.2 M). After centrifugation for 10 min at 12 000 g, the absorbance of the supernatant was measured at 450 nm. The specificity of DAB staining
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was assessed by adding 10 mM ascorbic acid. Leaf coloring was not observed in the presence of this agent. Each experiment was performed in at least four biological replicates. In each of five biological replicates, at least 30 rosette leaves of similar age were collected from at least 15 individual plants at the age of 3 - 4 weeks. Suspension culture was obtained from 7-d-old sterile Arabidopsis seedlings. Seedlings (10 - 15) were placed in 70 cm3 of nutrient medium based on MS salts with addition of thiamine (1 mg dm-3), pyridoxine (0.5 mg dm-3), nicotinic acid (0.5 mg dm-3), inositol (100 mg dm-3), sucrose (30 g dm-3), and 2,4-dichlorophenoxyacetic acid (0.3 mg dm-3) and cultivated in dark at 25 ºС. After 3 to 4 weeks, the cells were placed into 70 cm3 of fresh medium and further subcultivations were performed every 12 d. Experiments were carried out on the 6th day of 3rd subcultivation. Viability of suspension cultures was assessed by absorption of Evans Blue dye by dead cells (Baker and Mock 1994). ROS content in Arabidopsis suspension culture cells was measured according to Maxwell et al. (1999) using 2′,7′-dichlorofluorescein diacetate (DCF-DA). Antimycin А and menadione were dissolved in ethanol. In control samples, ethanol was present at the same concentration as in experimental samples (< 0.2 %). After treatment was complete, DCF-DA was added to cells at a final concentration of 5 µМ and the suspension was incubated for 40 min in the dark at 25 ºС. The samples were centrifuged for 1 min at 10 000 g, and the supernatant was collected and diluted 5-fold. Fluorescence was measured using a Shimadzu (Kyoto, Japan) RF 5301 PC fluorescence spectrophotometer with excitation and emission wavelengths 488 and 520 nm, respectively. Screening for the presence of the genetic constructs in the plants and cells was conducted by PCR as previously described (Umbach et al. 2005). Total DNA of an individual plant or culture cells was acquired according to Doyle and Doyle (1987) and used as the PCR substrate. To extract total RNA, suspension culture cells were homogenized with liquid nitrogen, then 0.45 cm3 of TE buffer, 0.05 cm3 SDS (10 %), and 0.5 cm3 of phenol were added. Further extraction was performed as previously described (Garnik et al. 2006). Total RNA from plants was extracted by TRI reagent (Sigma-Aldrich, St. Louis, USA) according to manufacturer’s instructions. Total RNA was separated in a 1.2 % agarose gel in dena-
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turating conditions and transferred onto nylon membrane Hybond N (GE Healthcare, Piscataway, USA). Hybridization and DNA probe labeling were performed as previously described (Tarasenko et al. 2009).
All data were statistically analyzed using SPSS11.5 for Windows (SPSS Inc., Chicago, USA). Differences between genotypes were determined by an unpaired t-test (P ≤ 0.05 or 0.01).
Results and discussion Arabidopsis lines transformed by the constructs carrying the aox1a gene under control of the CAMV 35S promoter in the sense or antisense orientation have been previously established (Umbach et al. 2005). It should be noted that although there are five genes encoding different isoforms of AOX in the Arabidopsis genome, only the AOX1a isoform is expressed at a high level and is most actively induced by stresses or upon inhibition of the cytochrome pathway (Cliffton et al. 2006). The plants of aforementioned lines (T4 generation) and suspension cell cultures obtained from those plants were used in all further experiments. PCR carried out with primers specific to each of genetic constructs confirmed that these constructs were present in plants and suspension cultures under study (data not shown). To verify the transgene expression, we analyzed the level of аох1а transcript in three lines by Northern hybridization. The аох1а transcript content in leaves of individual plants and suspension culture cells of the XX-2 line was significantly increased compared to the WT line (Fig. 1). In plants and cells of the AS-12 line, the transcript corresponding in size to the aox1a transcript was not found. A larger transcript corresponding to the antisense construct expression product, as well as smear corresponding probably to products of AOX transcript degradation in vivo, were present. On the basis of these data, we concluded that a high level of transgene expression is preserved in both whole plants and suspension cells.
Fig.1. Northern blot analysis of AOX1a expression in AOXoverexpressing (XX-2), AOX-deficient (AS-12), and wild-type (Col-0) plants and cell suspension cultures.
Soil-grown plants did not show any differences during the initial development stages defined according to Boyes et al (2001). This result correlates with previously published reports on these plant lines (Umbach et al. 2005, Fiorani et al. 2005). However, our data indicated that AOX-deficient plants has an increased duration of the reproductive growth phase (Table 1), which has not previously been reported for these plants (Fiorani et al. 2005). Whereas Fiorani et al. used a soil-less mix
fertilized with Hoagland solution, we used a mixture of soil and Vermiculite with no additional fertilizer. As such, differences in the phenotypic appearance of plants with reduced АОX levels might be due to different growth conditions. Duration of reproductive phase in the XX-2 line did not differ from that of the WT. Analysis of productivity demonstrated that plants formed a similar number of siliques, with a similar number of seeds. Therefore, later senescence is the only and, as far as we know, previously unreported, phenotypic feature of the AOX-deficient plants. Plants overexpressing AOX did not show any phenotypic differences from the wild-type plants. It seems that a lack of phenotypic differences under normal conditions is characteristic of virtually all plants with altered АОX activity (Vanlerberghe et al. 1994, Gilliland et al. 2003, Strodtkotter et al. 2009). Table 1. The length of different growth stages [d] of AOXoverexpressing (XX-2), AOX-deficient (AS-12), and wild-type (Col-0) plants. Values are means ± SD, n = 30 - 50, ** - significant difference from other genotypes at P ≤ 0.01. Genotype Germination Seedling growth Col-0 XX-2 AS-12
7.1 ± 1.0 7.1 ± 1.2 7.2 ± 1.1
11.2 ± 1.5 11.0 ± 1.7 11.4 ± 1.5
Rosette growth
Flowering
12.1 ± 1.3 13.5 ± 1.8 13.0 ± 1.6
40.6 ± 4.9 42.2 ± 5.8 59.2 ± 5.6**
Previously, the correlation between the reduced AOX levels and the increase in the cells susceptibility to apoptosis has been demonstrated (Robson and Vanlerberghe 2002). However, our data indicated that reduced AOX activity resulted in plant life cycle extension. Since data of Robson et al. were acquired in cultured cells, it may not be extrapolated to the whole plant. Interestingly, an extension of the life cycle was previously observed upon the knockout of the cytochrome pathway components, in particular, subunits of complex I (Lee et al. 2002, Tarasenko et al. 2010). This extension could be related to both the reduced ATP production (Meyer et al. 2009) and the changes in cell redox status (Noctor et al. 2004). For the plants of AS-12 and XX-2 lines, no changes in ROS content have been previously reported (Umbach et al. 2005). As ROS content in that study was determined only in roots, we decided to compare the ROS content in leaves of plants of these lines. Histochemical staining with NBT and DAB has demonstrated the similar ROS content in leaves (Table 2). To study ROS content
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in plants subjected to oxidative stress, the harvested leaves were treated with menadione, a redox cycling quinone that generates superoxide in vivo. ROS content in menadione-treated leaves of all three lines increased equally (Table 2). Table 2. ROS content in untreated and menadione-treated (100 μM; 1 h) leaves of WT, XX-2, and AS-12 Arabidopsis plants. NBT staining followed by formazane quantification was used to measure superoxide. DAB staining was used to measure hydrogen peroxide. Values are means ± SD, n = 5. Genotype Formazane [a.u. g-1(f.m.)] DAB [a.u. g-1(f.m.)] no treatment menadione no treatment menadione Col-0 XX-2 AS-12
2.45 ± 0.24 4.41 ± 0.25 4.45 ± 0.46 7.51 ± 0.54 2.33 ± 0.32 4.46 ± 0.27 4.56 ± 0.42 7.46 ± 0.41 2.36 ± 0.29 4.29 ± 0.27 4.49 ± 0.51 7.40 ± 0.55
Table 3. Cell viability [% of living cells] determined by Evans Blue staining of untreated or menadione-treated (100 μM; 3 and 24 h) suspension cells of WT, XX-2, and AS-12 lines. Values are means ± SD, n = 5. ** - significant difference from other genotypes at P ≤ 0.01. Genotype
No treatment
Menadione, 3 h Menadione, 24 h
Col-0 XX-2 AS-12
86.1 ± 5.0 84.5 ± 5.8 86.9 ± 5.1
85.0 ± 5.4 86.3 ± 5.7 86.8 ± 4.6
68.6 ± 4.8 31.0 ± 5.4** 71.4 ± 5.5
Next, we compared the resistance of plants subjected to various abiotic stresses. Growing of seedlings on the media containing 180 mМ NaCl, 3 mМ H2O2, or 25 µМ menadione showed identical resistance of all three lines (data not shown). Seed germination and seedling viability were at similar levels for all treatments. Taken together, these data indicate that altered AOX levels do not affect plant sensitivity to the applied stresses. Since altered AOX activity had no effect on ROS content or stress sensitivity in plants, we investigated suspensions culture cells. The obtained cell suspension of three lines did not differ in growth rate and had similar viability (Table 3), so they could be used to compare
ROS content. To measure intracellular ROS in suspension cultures, we used the fluorescent dye DCF-DA, which is specific for H2O2 and peroxyl radicals (Maxwell et al. 1999), and the superoxide-specific dye NBT. Intracellular ROS contents identified by both DCF-DA fluorescence and NBT staining were significantly reduced in cells overexpressing AOX and were increased in cells deficient in AOX (Table 4). Therefore, unlike the plant leaves, culture cells with altered AOX activity had considerably different ROS content. The respiratory inhibitor antimycin А blocks electron transport downstream of the ubiquinone pool, which results in an over-reduced state of the upstream segment of the respiratory chain and increased ROS production (Moller 2001). To investigate the effect of AOX activity on response of Arabidopsis cells when the cytochrome pathway is inhibited, we treated the cells with antimycin A. This treatment resulted in an increase in ROS content in the WT cells and even more in the AS-12 line (Table 4). ROS content in the XX-2 cells did not change upon the antimycin A treatment. Therefore, overexpression of the aox1a gene in Arabidopsis cells is sufficient to prevent an increase in ROS content during an inhibition of the ETC. Next, we measured ROS content in the cells of the three lines subjected to treatment with menadione and H2O2. As expected, the treatment with pro-oxidants resulted in the increase of ROS content in WT cells (Table 5). ROS content in AOX-deficient cells increased to a similar extent as in WT cells. However, overTable 4. ROS content in suspension cells of WT, XX-2, and AS-12 lines. DCF-DA fluorescence assay was used to measure H2O2 in untreated and antimycin-treated (10 μM; 1.5 and 3 h) cells. NBT staining was used to measure superoxide. Means ± SD, n = 4. ** - significant difference between genotypes at P ≤ 0.01. Line
Formazane DCF-DA [a.u. g-1(f.m.)] [a.u. g-1(f.m.)] no treatment antimycin A, antimycin A, no treatment 1.5 h 3h
Col-0 50.0±6.4 XX-2 18.2±2.5** AS-12 93.9±7.6**
108.5± 6.3 294.1± 8.7 1.18±0.09 19.6± 2.9** 19.7± 4.2** 0.53±0.06** 237.9±14.5** 369.7±17.4** 2.01±0.13**
Table 5. ROS content in suspension cells of WT, XX-2, and AS-12 lines treated with menadione (100 μM) and H2O2 (5 mM) for 1 h. DCF-DA assay was used to measure H2O2 and NBT staining was used to measure superoxide. Values are means ± SD, n = 5, *,** - significant difference between genotypes at P ≤ 0.05 or P < 0.01, respectively. Genotype
Col-0 XX-2 AS-12
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DCF-DA fluorescence [% of control] no treatment menadione H2O2
Formazane [% of control] no treatment menadione
H2O2
100 ± 6.3 100 ± 6.7 100 ± 7.6
100 ± 6.9 100 ± 7.4 100 ± 7.6
181.1 ± 10.1 463.5 ± 33.2** 156.5 ± 16.7*
245.5 ± 9.1 535.0 ± 36.7** 217.9 ± 13.7*
184.1 ± 8.6 478.2 ± 26.9** 136.6 ± 7.6**
198.5 ± 14.8 440.5 ± 28.9** 190.9 ± 13.8
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expression of AOX1a resulted in a significantly higher ROS increase. Determination of superoxide content in the cells subjected to the same treatment revealed similar differences between the lines (Table 5). Thus, suspension cells that overexpress AOX had a decreased ability to scavenge ROS induced by menadione treatment. Determination of the cell viability also revealed differences between suspension cultures of the three lines subjected to menadione treatment (Table 3). Viability of the cells after 24 h of menadione treatment was decreased, which was much more evident in cells overexpressing AOX. Thus, AOX-overexpressing cells were more sensitive to the menadione-induced oxidative stress. Despite numerous attempts, there is still no unambiguous connection between the АОX activity and ROS content. Studies conducted on different species, organs, and plant tissues often lead to opposite results. Plants or cell cultures with reduced AOX activities showed both significantly increased (Maxwell et al. 1999), unchanged (Umbach et al. 2005) or reduced ROS content (Amirsadeghi et al. 2006, Watanabe et al. 2008). The results of our study indicated that altered AOX activity in whole plants had no effect on intracellular ROS formation, under both normal conditions and upon the pro-oxidant treatment. Contrary, cell suspension cultures revealed differences between the lines with different AOX activities. This finding correlates with the results of a study done on tobacco suspension cell cultures (Maxwell et al. 1999), although in latter case the amplitude of shown differences was higher. It should be noted that the cells overexpressing AOX had reduced ROS content under normal conditions, but they responded to menadione treatment by the largest ROS increase, as well as by a reduction in viability. Similar data were obtained for tobacco plants, where АОX overexpression resulted in increased leaf sensitivity to ozone treatment (Pasqualini et al. 2007). It was
suggested that these results are due to a reduced antioxidant defense systems in cells with enhanced АОX activity. A similar explanation is likely to be valid for the paradoxical result acquired on tobacco suspension cultures, when the line with inactivated АОX had reduced ROS content (Amirsadeghi et al. 2006). In this case, the stress caused by a lack of АОX could induce antioxidant defenses, thereby resulting in overcompensation and reduced ROS content. According to a recent hypothesis, AOX activity under stress participates in so-called “signalling homeostasis”, controlling not only mitochondrial ROS production, but also the antioxidant system response (Vanlerberghe et al. 2009). As such, the effect of altered AOX activity on intracellular ROS content might vary depending on which functional role of AOX prevails in given metabolic conditions. Such a situation could take place during the transition from whole plant to cell culture when significant metabolic changes are evident. In recent years, the significant role of АОX in chloroplast-mitochondria interactions, particularly in utilization of reduced equivalents produced during photosynthesis, has been reported (Yoshida et al. 2007, 2008). It is possible that the absence of photosynthetic activity and related changes in cell redox status have an effect on AOX functioning in heterotrophic cell suspension. Thus, for the first time, we have shown that the same Arabidopsis transgenic lines with the altered AOX activity behave in a different way with respect to ROS content, depending on whether they are represented by whole plants or by cell cultures. Therefore, we suggest that differences in results of studies on the AOX role might be, at least in part, due to the use of different model systems. Our data indicate considerable differences in AOX functioning under different metabolic conditions which take place in photosynthetic tissues of green plant and heterotrophic culture cells.
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