Overexpression of the CBF 2 transcriptional Activator Enhances Oxidative Stress Tolerance in Arabidopsis Plants

The C-repeat/dehydration-responsive-element binding factor genes (CBF1-3) are transcriptional activators involved in governing plant responses to low temperatures; their overexpression enhances plant frost tolerance. We found that overexpression of CBF2 in Arabidopsis enhanced oxidative stress tolerance as compared with wild-type plants, an effect that was manifested in: increased seed germination rates on Petri dishes containing H2O2; delayed leaf senescence following incubation with H2O2; and delayed wilting and senescence after spraying whole plants with paraquat, a generator of superoxide radicals. Transcript profiling analysis using the Affymetrix ATH1 genome array revealed that overexpression of CBF2 did not affect expression of reactive oxygen-scavenging genes but rather, remarkably enhanced expression of oxidative-stress-responsive transcription factor genes. Overall, the present findings suggest that overexpression of CBF2 in Arabidopsis enhances oxidative stress tolerance, most likely via activation of a network interaction among stress-related transcription-factor genes.


Introduction
Aerobic organisms utilize molecular oxygen as a terminal oxidant during respiration, and thus consistently generate reactive oxygen species (ROS) as a normal by-product of aerobic respiration (Finkel & Holbrook, 2000;Martin et al., 1996).ROS include singlet oxygen ( 1 O 2 ), superoxide radical (O 2 -), hydroperoxyl radical (HO 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (•OH) -all of which are extremely reactive and capable of oxidizing biological molecules such as DNA, proteins, or lipids (Alscher et al., 1997;Schopfer et al., 2001).In addition to generating ROS in mitochondria during oxidative phosphorylation, plants also produce large amounts of ROS in the chloroplasts during photosynthesis and CO 2 fixation (Arora et al., 2002;Mittler et al., 2004;Munne-Bosch & Alegre, 2002).Furthermore, because plants are immobile and fixed in the soil they are constantly exposed to environmental stresses such as unfavorable temperatures (heating, chilling, and freezing), drought, salinity, flooding, pathogen attack, etc. Exposures to such stresses result in massive generation of ROS that interfere with the delicate balance of cellular redox homeostasis (Bolwell et al., 2002;J. Dat et al., 2000;Foyer & Noctor, 2005).Overall, their status as photosynthesizing organisms and their constant exposure to stresses make plants especially vulnerable to oxidative stresses.Because of the vital importance to control intercellular ROS levels, all aerobic organisms, including plants, developed enzymatic systems to detoxify excessive accumulation of ROS, mainly ROS scavenging enzymes such as catalase and superoxide dismutase (Mittler et al., 2004).In addition, many plant transcription factor genes are also regulated by ROS accumulation (Gadjev et al., 2006).
Many plants, including Arabidopsis, increase their frost tolerance in response to low nonfreezing temperatures; a phenomenon known as "cold acclimation" (M.F. Thomashow, 1999).Transcript profiling experiments revealed that multiple regulatory pathways are activated during cold acclimation, and that one such important pathway involves the c-repeat binding factor (CBF) regulon (M.F. Thomashow, 1999;M.F. Thomashow, 2001).The CBF genes are members of a small family of three AP2 domain transcriptional activators, comprising CBF1, CBF2 and CBF3 (Gilmour et al., 2004;Shinwari et al., 1998).Ectopic expression of CBF1 in Arabidopsis induced expression of cold-regulated (COR) genes and significantly enhanced freezing tolerance even without cold acclimation (Gilmour et al., 2004;Jaglo-Ottosen et al., 1998).In addition to frost tolerance, overexpression of CBF genes also induced plant tolerance towards other environmental stresses, such as drought and salinity (Kasuga et al., 1999;Shinozaki & Yamaguchi-Shinozaki, 2000).Furthermore, it was reported that ectopic expression of CBF1 also induced tolerance to water deficit, chilling, and salt stress in tomato plants (Hsieh et al., 2002a;Lee et al., 2003).Moreover, Hsieh et al. (2002b) suggested that overexpression of CBF1 increased chilling tolerance in tomato by enhancing CATALASE1 gene expression and enzyme activity, and oxidative stress tolerance (Hsieh et al., 2002b).In addition to its effects on induction of plant stress tolerance, we recently reported that overexpression of CBF2 in Arabidopsis also considerably delayed leaf senescence and extended the life span of the plants by approximately 2 weeks as compared with wild-type plants (Sharabi-Schwager et al., 2010).In this study, we show that overexpression of CBF2 enhanced oxidative stress tolerance in Arabidopsis, as manifested in: increased seed germination rates on Petri dishes containing H 2 O 2 ; delayed leaf senescence following incubation with H 2 O 2 ; and delayed wilting and senescence following spraying of whole plants with paraquat, a generator of superoxide radicals.Also, during growth and development the CBF2-overexpressing plants accumulated much lower levels of H 2 O 2 and O 2 -radicals than wild-type plants.Moreover, transcriptome analysis with the Affymetrix ATH1 genome array revealed that overexpression of CBF2 may have enhanced oxidative stress tolerance via activation of a network of oxidative-stress-responsive transcription factor genes.

Plant Material and Growth Conditions
Seeds of Arabidopsis thaliana (L.) Heynh.ecotype Wassilewskija (WS-2) and of transgenic plants overexpressing the CBF2 gene in a WS-2 background were obtained from Prof. M. Thomashow of Michigan State University, MI, USA (Gilmour et al., 2004).Before sowing, seeds were sterilized in 5% bleach and immersed in water at 4°C for 48 h to ensure uniform germination.The plants were grown in 7 × 7 × 8 cm plastic pots filled with a commercial growing-soil mix, at a constant temperature of 22°C, and illuminated by cool-white fluorescent lamps at approximately 100 µmol m -2 s -1 , with a photoperiod of 16 h.Plants were grown at a density of four plants per pot.In some experiments, seeds were grown on Petri dishes containing 0.8% agar and 0.5 × Murashige and Skoog (MS) medium including Gamborg B5 vitamins (Duchefa Biochemie, Haarlem, the Netherlands) at pH 5.7, as described (Weigal & Glazebrook, 2002).

Chlorophyll Content
Chlorophyll content was measured in 5-mm-diameter leaf disc samples.Chlorophyll was extracted from two leaf discs placed in a microtube containing 1 mL of 80% acetone.The discs were homogenized with a fitted pestle and incubated overnight at 4°C.Chlorophyll content in the acetone extracts was measured spectrometrically according to Porra et al. (Porra et al., 1989).Each measurement included four replications.

Electrolyte Leakage
Electrolyte leakage was measured by placing entire rosettes in scintillation vials containing 10 mL of double-distilled water.The first reading was taken after 2 h of incubation at room temperature with gentle agitation, and afterwards the rosettes were exposed to a high level of microwave radiation for 2 min, to destroy all living cells.The vials were then cooled to room temperature, and second readings were taken.Electrolyte leakage data are presented as percentages of the total amount of electrolytes present in the tissue.

Exposure to Oxidative Stress
Oxidative stress tolerance of wild-type and CBF2-overexpressing plants was evaluated by three different means.First, we examined seed germination rates following sowing on MS medium containing various concentrations (0-10 mM) of H 2 O 2 in Petri dishes.The seed germination rate was determined as the percentage of seeds that survived with each concentration of H 2 O 2 after 7 days at 22°C.Second, we evaluated the degree of yellowing of detached leaves following incubation in 0-10 mM H 2 O 2 solutions: leaves numbers 5 and 6 were detached from 36-day-old plants, and incubated for 72 h at 22°C, adaxial side up, in Petri dishes containing H 2 O 2 at 0-10 mM.Third, 36-day-old wild-type and CBF2-overexpressing plants were sprayed with various concentrations (0-30 M) of paraquat (Sigma, St Louis, MO, USA), a generator of superoxide radicals.In all these experiments, seeds, detached leaves, and whole plants were kept at 22°C under a 16-h photoperiod of illumination at ~100 µmol m -2 s -1 .

NBT and DAB Staining
To evaluate H 2 O 2 and O 2 -levels in plant tissues we used 3,3'-diaminobenzidine (DAB) (J.F. Dat et al., 2003), and nitroblue tetrazolium (NBT) (Le Deunff et al., 2004) staining procedures, respectively.Briefly, leaves numbers 5 and 6 were detached from wild-type and CBF2-overexpressing plants at various development stages and were vacuum infiltrated for 20 min in 2.5 mM DAB or 500 mM NBT solutions in citrate buffer (10 mM citrate, pH 6.0).Afterwards leaves were washed several times with 80% ethanol in order to remove the green chlorophyll, and the intensity of DAB or NBT staining was assessed visually by photography.In one experiment, entire rosettes of 36-day-old plants were stained in DAB and NBT solutions.

Transcript Profiling Analysis
Total RNA was isolated from leaves numbers 5 and 6 of 40-day-old wild-type and CBF2-overexpressing plants by phenol/chloroform extraction and precipitation with LiCl, according to standard procedures (Sambrook et al., 1992).For each treatment, we performed three separate RNA extractions, each involving leaves from 5 to 10 different plants.The RNA samples were prepared for hybridization according to the protocols outlined in the Affymetrix GeneChip Expression Analysis Technical Manual, and were hybridized to the Affymetrix Arabidopsis ATH1 Genome Array representing ~24,000 genes (Affymetrix, Santa Clara, CA, USA).Hybridizations were performed at the Department of Biological Services in the Weizmann Institute of Science, Rehovot, Israel.Data were analyzed with the Affymetrix Microarray Suite 5.0 (MAS5.0)statistical algorithms.Further advanced data analyses, including background subtraction, normalization and elimination of false positives, were performed with the Partek Genomics Suite (Partek GS) statistical and data visualization program.One-way analysis of variance was used to identify probe sets that exhibited significant changes in signal levels at P ≤ 0.05.

Effects of CBF2-Overexpression on Oxidative Stress Tolerance
We evaluated the effects of CBF2-overexpression on oxidative stress tolerance by three different means: a) evaluation of seed germination on MS medium containing various concentrations of 0-10 mM H 2 O 2 ; b) evaluation of leaf yellowing following incubation in 0-10 mM H 2 O 2 solutions; and c) evaluation of whole plant wilting following sprays with 0-30 M paraquat.It can be seen that in all cases, CBF2-overexpressing plants were more tolerant to the imposed oxidative stresses than wild-type plants.
In the seed germination assay, we found that seeds of both wild-type and CBF2-overexpressing plants had high germination rates (~95%) on MS agar media containing up to 4 mM H 2 O 2 .However, at higher concentrationsof 6 and 8 mM H 2 O 2 -germination rates were significantly higher in seeds of the transgenic line: 68 and 38%, respectively, in CBF2-overexpressing plants as compared with just 32 and 13%, respectively, in those of wild-type plants (Figure 1).At the high concentration of 10 mM H 2 O 2 seed germination was very low (below 14%) in both wild-type and CBF2-overexpressing plants (Figure 1).
In the leaf-yellowing assay, we found that leaves of wild-type plants lost chlorophyll following incubation at the lowest concentration of 2.5 mM H 2 O 2 , whereas those of CBF2-overexpressing plants began to loose chlorophyll only after incubation at the higher concentration of 5 mM H 2 O 2 (Figure 2).Overall, leaves of wild-type plants lost 50% of their chlorophyll content, as compared with leaves incubated in water alone, after incubation in 5 mM H 2 O 2 , whereas leaves of CBF2-overexpressing plants lost this proportion only after incubation in H 2 O 2 at the highest concentration of 10 mM (Figure 2B).It can be seen (Figure 2A) that leaves of wild-type plants became transparent and lost almost all of their chlorophyll following incubation at 7.5 and 10 mM H 2 O 2 , whereas leaves of CBF2-overexpressing plants remained green and viable even after incubation at the highest H 2 O 2 concentration of 10 mM.
In the paraquat spray assay, we found that CBF2-overexpressing plants were much more tolerant than wild-type plants to the generated superoxide.It can be seen (Figure 3) that wild-type rosettes suffered from slight necrosis and increased electrolyte leakage rates already after being sprayed with paraquat at the lowest concentration of 10 M, whereas the CBF2-overexpressing plants still remained green and healthy.Furthermore, rosettes of wild-type plants wilted completely after receiving 20-30 M paraquat, whereas those of CBF2-overexpressing plants showed necrosis symptoms mainly at the high paraquat concentration of 30 M (Figure 3A).Electrolyte leakage rates in rosettes of wild-type plants increased continuously from 33% without paraquat to 42, 56, and 76% following paraquat sprays at 10, 20, and 30 M, respectively, whereas electrolyte leakage rates of CBF2-overexpressing plants increased above base level only after exposure to paraquat at the highest concentration of 30 M (Figure 3B).

Effects of CBF2-Overexpression on H 2 O 2 and O 2 -Contents in Leaves and Rosettes
We used DAB and NBT staining to evaluate the accumulation of H 2 O 2 and O 2 -in leaves of wild-type and CBF2-overexpressing plants during plant development.Figures 4 and 5, respectively, show that in wild-type plants both H 2 O 2 and O 2 -began to accumulate in leaf tissue during initiation of flowering, peaked at mid-flowering, and then declined.In contrast, we did not detect any accumulation of either H 2 O 2 or O 2 -in leaves of CBF2-overexpressing plants at any stage of plant development (Figures 4 and 5). Figure 6 shows ROS accumulation in rosettes of wild-type and CBF2-overexpressing plants 36 days after sowing.Once again, it can be seen that rosettes of wild-type plants accumulated high levels of H 2 O 2 and O 2 -, whereas those of CBF2-overexpressing plants contained only minor levels of these radicals (Figure 6).

Effects of CBF2 Overexpression on Transcript Levels of ROS-Scavenging and Oxidative-Stress-Responsive Transcription Factor Genes
In order to identify the molecular mechanisms that might be involved in governing the enhanced tolerance of CBF2-overexpressing plants to oxidative stress we analyzed transcript profiles with the Affymetrix ATH1 genome array, and examined the expression patterns of ROS-scavenging and oxidative-stress-responsive transcription factor genes.The list of Arabidopsis ROS-scavenging genes was taken from the review article by Mittler et al. (Mittler et al., 2004).Table 1 shows that none of the Arabidopsis ROS-scavenging genes, including superoxide dismutase (SOD), catalase (Cat), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), peroxiredoxin (PrxR) and ferritin were induced or repressed by more than a factor of 2. Thus, overexpression of CBF2 did not have any direct effect on ROS-scavenging transcript levels.
In addition to the ROS-scavenging system, (Gadjev et al., 2006), on the basis of various ROS-related microarray experiments, defined a group of 32 regulatory Arabidopsis transcription-factor genes whose expression levels were elevated at least fivefold following exposure to various ROS, and Table 2 shows that the transcript levels of 18 out of these 32 oxidative-stress-responsive transcription factor genes were up-regulated at least threefold in CBF2-overexpressing plants.Furthermore, the transcript levels of 10 of these ROS-responsive transcription factor genes were remarkably up-regulated, by more than fivefold, in CBF2-overexpressing plants (Table 2).The ROS-responsive transcription-factor genes induced by CBF2 overexpression belong to various families, including WRKY, NAM, C 2 H 2 zinc finger, CCCH-type zinc finger AP2, HSF, MYB, ZAT (Table 2).

Discussion
In the present study, we showed that overexpression of CBF2 further increased oxidative-stress tolerance in Arabidopsis plants, and we demonstrated this by various assays, including evaluation of seed germination, leaf yellowing and whole-plant wilting, in response to various ROS treatments (Figures 1-3).Although not reported before, the finding that overexpression of CBF2 enhanced oxidative-stress tolerance in Arabidopsis is not very surprising; it is known that overexpression of CBF genes enhanced plant tolerance towards various environmental stresses, such as freezing, drought, and salinity (Kasuga et al., 1999;Shinozaki & Yamaguchi-Shinozaki, 2000).Furthermore, it was previously reported that ectopic expression of CBF1 increased tolerance of chilling and oxidative stress also in tomato plants (Hsieh et al., 2002b).
Since generation of ROS and activation of oxidative processes are known to provide integral components of the senescence syndrome in all aerobic organisms, including plants (Finkel & Holbrook, 2000;Martin et al., 1996;Prochazkova et al., 2001;Zimmermann & Zentgraf, 2005), we hypothesize that overexpression of CBF2 might have delayed leaf and whole-plant senescence, at least in part, by increasing oxidative-stress tolerance and/or by reducing ROS accumulation during plant development.Indeed, our present findings clearly demonstrate that overexpression of CBF2 increased oxidative stress tolerance and remarkably reduced accumulation of H 2 O 2 and O 2 -radicals in leaf tissue during development, as compared with that in wild-type plants (Figures 4-6).
Therefore, by analogy with previous studies, which reported that the Arabidopsis delayed-leaf-senescence mutants ore1, ore3, and ore9, and the long-living mutant gigantea exhibited enhanced tolerance to oxidative stresses (Kurepa et al., 1998;Woo et al., 2004), we hereby suggest that ectopic expression of CBF2 also might have delayed senescence and extended plant longevity via enhancement of oxidative stress tolerance.Similar correlations between oxidative stress resistance and extension of life span were reported in various aerobic organisms, ranging from yeasts to mammals (Johnson et al., 1996;Kapahi et al., 1999;Orr & Sohal, 1994).
Finally, we do not yet know for certain the exact mechanism by which overexpression of CBF2 might have increased oxidative stress tolerance.In tomato, it was reported that overexpression of CBF1 increased chilling tolerance by enhancing CATALASE1 gene expression and enzyme activity (Hsieh et al., 2002b).However, in the current study, we could not detect any induction of genes involved in ROS-scavenging, but rather detected a remarkable up-regulation of transcript levels of 18 out of 32 Arabidopsis ROS-responsive transcription-factor genes (Tables 1-2) (Gadjev et al., 2006).Therefore, we suggest that overexpression of CBF2 might have enhanced oxidative stress tolerance in Arabidopsis via activation of a network of oxidative-stress-responsive transcription-factor genes.
Table 1.Expression levels of reactive oxygen species (ROS) scavenging network genes in mature leaves of wild-type (WS ecotype) and CBF2-overexpressing plants.The list of Arabidopsis ROS-scavenging genes was taken from Mittler et al. (2004).For gene expression analysis, RNA was isolated from leaves numbers.5 and 6 of wild-type and CBF2-overexpressing plants 40 days after sowing.Expression values are from gene profiling experiment using the Affymetrix ATH1 genome array.nd, not detected (expression level below background).

Enzyme and Reaction
Table 2. Expression levels of oxidative stress-responsive transcription factor genes in mature leaves of wild-type (WS ecotype) and CBF2-overexpressing plants.CCCH-type zinc finger 5.10 £ At2g38470 AtWRKY33 6.13 £ The list of transcription factors commonly up-regulated (at least fivefold) in Arabidopsis by oxidative stress is according to Gadjev et al. (2006).Changes by factors of 3-4, 4-5, and >5 are marked by ¥, § and £, respectively.For gene expression analysis, RNA was isolated from leaves numbers 5 and 6 of wild-type and CBF2-overexpressing plants 40 days after sowing.Expression values are from gene profiling experiment with the Affymetrix ATH1 genome array.nd, not detected (expression level below background).

FigureFigure 2 .
Figure 1.E plants.Seeds w of H 2 O 2 A, Ph presence of v

Figure
Figure 3. Effe Photographs the plant