Heme oxygenase-1 is involved in the cytokinin-induced alleviation of senescence in detached wheat leaves during dark incubation

Journal of Plant Physiology 168 (2011) 768–775

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Heme oxygenase-1 is involved in the cytokinin-induced alleviation of senescence in detached wheat leaves during dark incubation Jingjing Huang a,b , Bin Han a,b , Sheng Xu a,b , Meixue Zhou c , Wenbiao Shen a,b,∗ a b c

College of Life Sciences, Cooperative Demonstration Laboratory of Centrifuge Technique, Nanjing Agricultural University, Nanjing 210095, PR China Beckman Coulter Ltd. Co., Nanjing Agricultural University, Nanjing 210095, PR China Tasmanian Institute of Agricultural Research, University of Tasmania, Kings Meadows, TAS 7249, Australia

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Article history: Received 19 June 2010 Received in revised form 25 October 2010 Accepted 28 October 2010 Keywords: Cytokinin Dark-induced senescence Detached wheat leaves Heme oxygenase-1 Lipid peroxidation

a b s t r a c t This study tested whether an inducible isoform of heme oxygenase (HO, EC 1.14.99.3), HO-1, is involved in the cytokinin (CTK)-induced alleviation of senescence in detached wheat (Triticum aestivum L.) leaves during dark incubation. We discovered that exogenous supplement of 6-benzylaminopurine (6-BA) at 10 ␮M for 48 h not only delayed the dark-induced loss of chlorophyll and protein contents in detached wheat leaves, but also significantly increased HO activity in a time-dependent manner. This induction reached a maximum within 3 h of 6-BA supply, which was further confirmed by using semi-quantitative RT-PCR and protein gel blot analysis. Furthermore, the decreases in intracellular thiobarbituric acid reactive substances (TBARS) content, and the increases in the transcript level, total and isozymatic activities of some important antioxidant enzymes, such as catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), and ascorbate peroxidase (APX, EC 1.11.1.11), were observed. Reversed responses of chlorophyll, protein and TBARS contents, HO activity, and the expression of above antioxidant enzymes were observed when zinc protoporphyrin-IX (ZnPPIX), a potent HO-1 inhibitor, was added together with 6-BA. In contrast, HO-1 inducer hemin could partially mimic the effects of 6-BA. Together, the results suggest that HO-1 might be involved in the CTK-induced alleviation of senescence and lipid peroxidation in detached wheat leaves. © 2010 Elsevier GmbH. All rights reserved.

Introduction Cytokinins (CTKs) play a major role in many different developmental and physiological processes in plants, such as cell division, root and shoot growth and branching, chloroplast development, stress response and pathogen resistance. In particular, it has been proposed that CTK induces the delay of dark-associated leaf senescence. For example, the application of CTK usually slows senescence-induced decreases in protein and chlorophyll contents, and photosynthetic parameters such as the rate of CO2 assimilation (Selivankina et al., 2001; Vlˇcková et al., 2006), and delays the senescence-induced increase in lipid peroxidation by the up-

Abbreviations: ABA, abscisic acid; APX, ascorbate peroxidase; 6-BA, 6benzylaminopurine; BV, biliverdin-IX␣; CAT, catalase; cGMP, cyclic guanosine monophosphate; CO, carbon monoxide; CTK, cytokinin; H2 O2 , hydrogen peroxide; HO, heme oxygenase; HO-1, heme oxygenase-1; NO, nitric oxide; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; ZnPPIX, zinc protoporphyrin IX. ∗ Corresponding author at: Department of Biochemistry and Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China. Tel.: +86 258 439 6542; fax: +86 258 439 6542. E-mail address: [email protected] (W. Shen). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.10.010

regulation of antioxidative enzyme expression (Zavaleta-Mancera et al., 2007). However, the molecular mechanisms of these CTK responses are poorly understood. In animals, heme oxygenase (HO, EC 1.14.99.3) is known to catalyze the oxidative cleavage of the ␣-mesocarbon of Feprotoporphyrin-IX yielding equimolar amounts of biliverdin-IX␣ (BV), free divalent iron, and carbon monoxide (CO). Among the three isoforms identified to date, only HO-1 is a stress-responsive protein induced by hypoxia, endotoxic shock, atherosclerosis, inflammation, and other oxidative stimuli, conferring protection against oxidative stress in a variety of tissues (Bauer and Bauer, 2002). Previous results have illustrated that the antioxidant mechanism of HO-1 might involve its enzymatic reaction products, including BV and CO. In plants, interestingly, the first role attributed to HOs was their participation in the biosynthetic pathway leading to phytochrome chromophore formation or functioning in light signaling (Muramoto et al., 1999; Davis et al., 2001; Shekhawat and Verma, 2010). Furthermore, the involvement of HO-1, BV, and CO in the antioxidant defense system was confirmed in soybean and alfalfa plants (Noriega et al., 2004; Balestrasse et al., 2005; Han et al., 2008). Recently, we provided a series of pharmacological, physiological, and molecular evidence that HO-1 was involved in the auxin-induced adventitious rooting in cucumber

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explants (Xuan et al., 2008). The induction of HO-1 by osmotic stress, glutathione (GSH) depletion, cobalt chloride (CoCl2 ), abscisic acid (ABA), nitric oxide (NO), and hydrogen peroxide (H2 O2 ) was also confirmed (Yannarelli et al., 2006; Cao et al., 2007; Noriega et al., 2007; Chen et al., 2009; Cui et al., 2010; Liu et al., 2010; Xu et al., 2010). For example, HO-induced CO production was involved in ABA-induced stomatal closure in Vicia faba, and NO and cyclic guanosine monophosphate (cGMP) might function as downstream intermediates in the CO signaling responsible for stomatal closure (Cao et al., 2007). However, the role of HO in CTK-induced cytoprotective effects is still unclear, and there is no published study on CTK-induced up-regulation of HO isoforms in wheat plants. In fact, detached wheat leaf is a good biological system for senescence bioassays of CTK activity. In this context, detached wheat leaves were treated with 6-benzylaminopurine (6-BA), HO-1 inducer hemin, or the potent inhibitor of HO-1 zinc protoporphyrin IX (ZnPPIX) alone, or 6-BA plus ZnPPIX. Thus, the relationship between CTK-induced HO-1 expression and the CTK responses, such as the delay of leaf senescence and the alleviation of lipid peroxidation in detached wheat leaves, were studied. The results suggest that HO-1 might be involved in CTK responses by the modulation of antioxidative enzyme expression, such as catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), and ascorbate peroxidase (APX, EC 1.11.1.11). Materials and methods Plant material and growth condition Seeds of wheat (Triticum aestivum L., Yangmai 13) were surfacesterilized with 2% NaClO for 10 min, rinsed extensively in distilled water and germinated at 25 ◦ C in the darkness for 1 d. Then, the uniform buds were selected. Roots were gently washed and transferred to separate containers for hydroponics. Plants were germinated and grown in a controlled climate chamber (12 h light period, 25 ◦ C, humidity 50 ± 4%; 12 h dark period, 18 ◦ C, humidity 56 ± 5%, MGC-300B, Shanghai Yiheng Technology Co., Ltd., Shanghai, China) with modified Hoagland solution containing 3 mM KNO3 , 1 mM NH4 H2 PO4 , 0.5 mM MgSO4 , 5.5 mM Ca(NO3 )2 , 50 mg of FeEDTA/l (10% iron), 25 ␮M KCl, 12.5 ␮M H3 BO3 , 1 ␮M MnSO4 , 1 ␮M ZnSO4 , 0.25 ␮M CuSO4 , and 2 ␮M H2 MoO4 (Gulick and Dvoˇrák, 1987). The irradiance was approximately 300 ␮mol m−2 s−1 provided by fluorescent lamps. The culture solution was renewed every other day until two fully expanded leaves appeared. Chemicals All chemicals were obtained from Sigma (St Louis, MO, USA). 6-BA was used at a concentration of 10 ␮M. ZnPPIX, and a specific inhibitor of HO-1 (Lamar et al., 1996; Xuan et al., 2008), was used at 100 ␮M. Hemin was used at 1 ␮M as the HO-1 inducer (Xuan et al., 2008). Treatments The first fully expanded leaves, which have exposed ligulae, were cut off 1 cm below the leaf tips. Leaf segments (4 cm) were detached and then floated abaxial side down in a plastic chamber containing 100 mL of distilled water and kept in darkness at 25 ◦ C for 72 h to induce senescence. Subsequently, wheat leaf segments were exposed to 100 mL of distilled water (Con), 100 ␮M ZnPPIX, 10 ␮M 6-BA, 1 ␮M hemin alone, or 6-BA plus ZnPPIX respectively, for another 48 h in the dark. After various treatments, leaf segments were immediately frozen in liquid nitrogen, and stored at −80 ◦ C until further analysis. At least three replicates were carried out for

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each treatment, and at least fifteen detached leaves were in each replicate. Determination of chlorophyll, soluble protein, and thiobarbituric acid reactive substances (TBARS) content Chlorophyll content was determined according to the method described by Wintermans and De Mots (1965). Soluble protein content was determined according to the method of Bradford (1976) using bovine serum albumin (BSA) as the standard. Lipid peroxidation was estimated by measuring the amount of TBARS as described previously by Liu et al. (2007). HO activity determination Heme oxygenase (HO, EC 1.14.99.3) activity was analyzed following the method described by Xuan et al. (2008). For the HO activity assay, the concentration of BV was estimated using a molar absorption coefficient at 650 nm of 6.25 mM−1 cm−1 in 0.1 M HEPES–NaOH buffer (pH 7.2). One unit of activity (U) was calculated by taking the quantity of the enzyme to produce 1 nmol BV per 30 min. Protein gel blot of HO-1 Rabbit polyclonal antibody against the mature wheat HO-1 (TaHO1) expressed in Escherichia coli was used (Wu et al., 2011). Homogenates obtained for HO activity assays were also analyzed by protein gel blot analysis. Sixty micrograms of protein from homogenates were subjected to SDS-PAGE using a 12.5% acrylamide resolving gel (Mini Protean II System, Bio-Rad, Hertz, UK). Separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes, and non-specific binding of antibodies was blocked with 5% non-fat dried milk in phosphate-buffered saline (PBS, pH 7.4) for 2 h at room temperature. Membranes were then incubated overnight at 4 ◦ C with primary antibodies diluted 1:200 in PBS buffer plus 1% non-fat dried milk. Immune complexes were detected using horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G. The color was developed with a solution containing 3,3 -diaminobenzidine tetrahydrochloride (DAB) as the HRP substrate. Additionally, the films were scanned (Uniscan B700+ , Tsinghua Unigroup Ltd., Beijing, China) and analyzed using Quantity One v4.4.0 software (Bio-Rad, USA). Antioxidative enzyme activity assays Frozen wheat leaf segments (0.5 g) were homogenized in 10 mL of 50 mM PBS (pH7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1% polyvinylpyrrolidone (PVP) for catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) total activity assays, or combinations with the addition of 1 mM ascorbic acid (ASC) in the case of ascorbate peroxidase (APX) determination. The homogenate was centrifuged at 10,000 × g for 30 min at 4 ◦ C, and the supernatant was used for the four antioxidative enzyme determinations. CAT activity was spectrophotometrically measured by monitoring the consumption of H2 O2 (extinction coefficient 39.4 mM−1 cm−1 ) at 240 nm. POD was determined by measuring the oxidation of guaiacol (extinction coefficient 26.6 mM−1 cm−1 ) at 470 nm. Total SOD activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) according to the method described by Beauchamp and Fridovich (1971). One unit of SOD (U) was defined as the amount of crude enzyme extract required to inhibit the reduction rate of NBT by 50%. APX activity was determined by monitoring the

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decrease at 290 nm (extinction coefficient 2.8 mM−1 cm−1 , Nakano and Asada, 1981). Gel electrophoresis Native polyacrylamide gel electrophoresis (PAGE) was performed for 2.5 h at 4 ◦ C in the stacking gel (5%, 12 mA) followed by 18 mA in the separating gel (8% for POD isozymes, 10% for SOD and APX isozymes) in Tris–Gly buffer (pH 8.3) for POD and SOD isozymatic activity assays, or combinations with the addition of 2 mM ASC in the case of APX analysis. For each lane, equal fresh weights (20 mg) of wheat leaf segments from different treatments were extracted and loaded. POD isozymes were stained according to the method described by Janda et al. (1999). SOD isozymatic activity was localized in the gel as described by Beauchamp and Fridovich (1971). APX isozymatic activity was determined according to the procedure described by Pinhero et al. (1997). Semi-quantitative RT-PCR analysis Total RNA was extracted by grinding 100 mg of fresh leaf tissue in liquid nitrogen using Trizol reagent (Invitrogen) according to the manufacturer’s instructions (Wu et al., 2011). DNA-free total RNA (5 ␮g) from different treatments was used for first-strand cDNA synthesis in a 20-␮L reaction volume containing 2.5 units of avian myeloblastosis virus reverse transcriptase XL (TaKaRa) and 2.5 ␮M random primer. PCR was performed using 2 ␮L of a 20-fold dilution of the cDNA, 10 pmol of each oligonucleotide primer, and 1 unit of Taq polymerase (TaKaRa) in a 25-␮L reaction volume. Primers used were as follows: for HO-1 (accession number HM014348), forward (5 -CCGTTCGTGGACGAGATGAGG-3 ) and reverse (5 -CAGTTCAGACCTGACGGAGCATGT-3 ), amplifying a 768-bp fragment; for CAT (accession number D86327), forward (5 -CTTCTCCTACTCCGACACGC-3 ) and reverse (5 TGTTGATGAATCGCTCTTGC-3 ), amplifying a 311-bp fragment; for POD (accession number X85228), forward (5 -ACTTCCACGACTGCTTTGT-3 ) and reverse (5 -ACTGGGCCTTCCCGATG3 ), amplifying a 643-bp fragment; for Cu/Zn-SOD (accession number U69632), forward (5 -GTCGTCACGCTCACCCA-3 ) and reverse (5 -GCTCATGCCCACCTTTT-3 ), amplifying a 346-bp fragment; for Mn-SOD (accession number AF092524), forward (5 -CGTCGCCCACTACAACA-3 ) and reverse (5 -TCACAAGAGGGTCCTGATT-3 ), amplifying a 347-bp fragment; for APX (accession number EF184291), forward (5 -TGCGGATTTGTTCCAGTT-3 ) and reverse (5 -GGCTTCGGCGTAGTCTTT-3 ), amplifying a 496-bp fragment; for 18s rRNA (accession number AJ272181), forward (5 -CAAGCCATCGCTCTGGATACATT-3 ) and reverse (5 CCTGTTATTGCCTCAAACTTCC-3 ), amplifying a 658-bp fragment. To standardize the results, the relative abundance of 18s rRNA was determined and used as the internal standard. The cycle numbers of the PCR reactions were adjusted for each gene to obtain visible bands on agarose gels. Aliquots (5 ␮L) from the PCR were loaded on 2% agarose gels and stained with ethidium bromide. Specific amplification products of the expected size were observed, and their identities were confirmed by sequencing. Ethidium bromide stained gels were scanned and analyzed using TotalLab v1.10 software (Nonlinear Dynamics, Newcastleupon-Tyne, UK). Statistical analysis Where indicated, results are expressed as the means ± SE of three independent experiments. Statistical analysis was performed using SPSS 16.0 software. Differences among treatments were analyzed by one-way ANOVA, taking P < 0.05 as significant according to Duncan’s multiple range test.

Fig. 1. Time course of chlorophyll and soluble protein contents in detached wheat leaves floating on water or 6-BA in darkness. Detached wheat leaves were pretreated in darkness for 72 h, and then exposed to distilled water (Con) or 10 ␮M 6-BA, respectively, for another 48 h. Chlorophyll (A) and soluble protein (B) levels were measured at the indicated times. Means and SE values were calculated from at least three independent experiments. FW, Fresh weight.

Results Effects of 6-BA on chlorophyll and protein levels during dark-induced senescence The dark-induced senescence of detached wheat leaves was evaluated by measuring the decrease of chlorophyll and protein contents. Fig. 1 shows the time courses of chlorophyll and protein levels of wheat leaf segments floating on water or 6-BA (10 ␮M) in the dark. It was clear that 6-BA significantly delayed dark-induced senescence of detached wheat leaves, which is consistent with earlier reports of other investigators in plant tissues (Vlˇcková et al., 2006; Zavaleta-Mancera et al., 2007; Zubo et al., 2008). Responses of HO activity, HO-1 transcript and its protein level to 6-BA Time course experiments for 48 h (Fig. 2A) showed that, in comparison with the control sample (Con), HO activity in wheat leaf segments abruptly increased at 3 h after 6-BA treatment, which was about 1.4-fold higher than that of the control. A gradual decrease followed, the HO activity level was still higher than that of the control. Fig. 2B further illustrates that 6-BA treatment for 3 h resulted in an induction of HO-1 gene expression. Densitometric analysis revealed that the steady-state level of HO-1 mRNA increased by 85.0%. Protein gel blot analysis for HO-1 (Fig. 2C) showed only a single band with a molecular mass of about 26 kDa, determined by using molecular mass markers (data not shown), a mass consistent approximately to that previously reported for mature HO-1

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to increase HO activity at 3 h after treatment, which is consistent with our previous results (Xuan et al., 2008). TBARS measurement was used as an assay for lipid peroxidation in vitro. To confirm the cytoprotective role of HO-1 in CTK-induced alleviation of oxidative stress during dark incubation, we treated wheat leaf segments with hemin alone, 6-BA and/or ZnPPIX for 48 h and measured the content of TBARS. Compared with the control sample, treatment with 6-BA or hemin alone caused a significant decrease in levels of TBARS (19.6 and 5.5%, respectively), and the combination with ZnPPIX blocked the responses of 6-BA. Additionally, a significant increase in TBARS (P < 0.05) was observed when ZnPPIX was applied alone (Fig. 3E). Responses of antioxidant enzyme activities and their transcripts to 6-BA, ZnPPIX, and hemin

Fig. 2. Comparison of HO activity (A), HO-1 transcript (B), and HO-1 protein level (C) in detached wheat leaves floating on water or 6-BA in darkness. Wheat leaf segments were pretreated in darkness for 72 h, and then exposed to distilled water (Con) or 10 ␮M 6-BA, respectively, for another 48 h. HO activity (A) was measured at the indicated times. Means and SE values were calculated from three independent experiments. HO-1 mRNA expression (B) and its protein level (C) were analyzed by semi-quantitative RT-PCR and protein gel blot after 3 h of treatment. Relative HO-1 transcript and its protein level taking control as 100%. The values below the band (B and C) represent relative levels with respect to the control samples. The RT-PCR and protein gel blot results of three independent experiments show similar trends of mRNA or protein level, respectively.

of pea and Arabidopsis (Muramoto et al., 1999; Linley et al., 2006). This assay also demonstrated approximate relatively positive correlation among HO enzyme activity, HO-1 transcript and its protein level (Fig. 2). Taken together, these results clearly suggest a possible interrelationship between CTK and HO-1 expression during the delay of dark-induced senescence in detached wheat leaves. ZnPPIX inhibited 6-BA-induced alleviation of senescence and lipid peroxidation We also aimed to confirm that the nature of 6-BA-induced delay of dark-induced senescence was related to HO activity. In the subsequent experiment, the combination of 6-BA with the potent HO-1 inhibitor ZnPPIX, proven recently in plant tissues (Xuan et al., 2008), was used. This treatment blocked the alleviation of dark-induced senescence (Fig. 3A) because chlorophyll and soluble protein contents were decreased significantly to below the levels of 6-BA treatment alone at 48 h of treatment (P < 0.05, Fig. 3B and C). Furthermore, wheat leaf segments treated with the HO-1 inducer hemin (Fig. 3D) exhibited notable increases in the chlorophyll and soluble protein contents, but to a lower level than those induced by 6-BA. Meanwhile, the administration of ZnPPIX alone resulted in a slight, but non-significant reduction in chlorophyll and soluble protein levels, further suggesting that endogenous HO-1 might play a partial role in the delay of dark-induced senescence elicited by 6-BA. In the absence or presence of 6-BA, incubation with ZnPPIX produced a significant decrease in enzyme activities (P < 0.05, Fig. 3D). However, the application of hemin alone was able

In the following experiments, analysis of four antioxidant enzymes revealed that activities of CAT, POD, SOD, and APX increased and peaked after 24 h of 6-BA treatment, being 12.1, 13.9, 14.1, and 16.4% higher than that of controls, respectively (P < 0.05, Fig. 4A–D, some data not shown). The transcript levels of several antioxidant genes, such as CAT, POD, Cu/Zn-SOD, Mn-SOD, and APX were also induced (Fig. 4E). By using native PAGE and activity staining, five clear POD or SOD isozymes, and eleven APX isozymes were detected in wheat leaf segments during dark incubation. 6BA treatment generally induced increases in the band size of some isozymes, and no band appeared or disappeared. We also noted that enhancement of HO-1 expression induced by CTK preceded the upregulation of antioxidant enzyme expression and cytoprotective functions in detached wheat leaves. To establish a link between the cytoprotective role of HO-1 and the antioxidant defense systems in CTK signaling, wheat leaf segments were treated with 6-BA, ZnPPIX, and hemin alone or the combination treatments. Further experimental results showed that ZnPPIX partly blocked the 6-BA-induced enhancements in enzyme activities (P < 0.05) and corresponding transcripts (Fig. 4). Hemin applied alone led to significant increases (P < 0.05), except for the SOD enzyme, in comparison with the control plants. Interestingly, the increased amounts of POD, SOD, and APX isoforms induced by 6-BA were partially inhibited by the ZnPPIX treatment (Fig. 5). We assumed that the POD-II, POD-IV, SOD-IV, SOD-V, and APX-IX induced by 6-BA might be responsible for the CTK-induced alleviation of oxidative stress, because in the samples treated with ZnPPIX alone, isozymatic activities decreased significantly. These results clearly suggest that HO-1 might be partially involved in CTKinduced up-regulation of antioxidant defense in detached wheat leaves. Discussion Senescence is a sequence of structural, biochemical and physiological events under a well regulated genetic program. There is evidence that natural endogenous CTK levels decreased during dark-induced senescence in Cucurbita cotyledons and natural senescence in maize leaves (He et al., 2005). In this study, as expected (Zavaleta-Mancera et al., 2007), we demonstrated that 6BA delays the dark-induced senescence in detached wheat leaves (Fig. 1), which has been demonstrated in rice (Ookawa et al., 2004) and maize (He et al., 2005). Further data illustrated a linear signal transduction cascade involving up-regulation of HO-1 downstream of CTK responses. Several results supported these conclusions. First, HO activity increased in a time-dependent manner after 6-BA treatment for a 48-h period under dark incubation, reaching a maximum within 3 h of 6-BA supply, which was consistent with the changes of HO-1

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Fig. 3. Effects of ZnPPIX, 6-BA, and hemin on phenotype of leaf color (A, 48 h), chlorophyll (B, 48 h) and soluble protein (C, 48 h) contents, HO activity (D, 3 h), and TBARS level (E, 48 h) in detached wheat leaves during dark incubation. Detached wheat leaves were pretreated in darkness for 72 h, and then exposed to distilled water (Con), 100 ␮M ZnPPIX, 10 ␮M 6-BA, 1 ␮M hemin alone, or 6-BA plus ZnPPIX respectively, for another 48 h. Values are means ± SE of three independent experiments. Bars denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. FW, Fresh weight.

transcript and corresponding protein levels (Fig. 2). Second, CTKinduced responses, including the alleviation of decay of soluble protein and chlorophyll contents (Fig. 1), were reversed partially in the presence of the potent inhibitor of HO-1, ZnPPIX (Fig. 3A–C). Third, the HO-1 inducer hemin (Fig. 3D) could produce weaker but significant effects on the delay of dark-induced effects, in comparison with strong inducible effects of 6-BA. In addition, the increase of HO activity in 6-BA-treated detached wheat leaves was suppressed by ZnPPIX (Fig. 3D). Finally, inhibition of HO activity with ZnPPIX alone resulted in the marked inhibition of soluble protein content (P < 0.05), whereas the chlorophyll level was decreased slightly, but not significantly. Thus, this pharmacological and physiological evidence supports the idea that HO-1 might, at least partially, mediate CTK-induced alleviation of senescence in detached wheat leaves during dark incubation. It has been demonstrated that, in Arabidopsis, a small family of HOs with four members clusters into two subfamilies: HY1 (HO1), HO3, and HO4 belong to the HO1 subfamily (Emborg et al., 2006), whereas HO2 is the only member of the HO2 subfamily, which was reported to be unable to bind or degrade heme and therefore not a true HO (Gisk et al., 2010). Previous results have also shown that the hy1 mutant displays long hypocotyls, early flowering, and decreased chlorophyll accumulation, all of which might be caused by the stabilization of heme feedback inhibiting 5-aminolevulinic acid (ALA) synthesis (Muramoto et al., 1999; Emborg et al., 2006). Some hy1-conferred phenotypes were able to be rescued by BV

(Parks and Quail, 1991; Emborg et al., 2006). Similarly, chlorophyll fluorescence of ptr116 cells, a HO-deficient mutant of the moss Ceratodon purpureus, was clearly elevated with the addition of phycocyanobilin in comparison with the non-treated samples (Brücker et al., 2000). These genetic results further suggest that plant HO-1 might be related to chlorophyll metabolism, which could explain the reversal effect of the inhibitor of HO-1 ZnPPIX on CTK-induced delay of chlorophyll content (Fig. 3A and B). It is well known that the increased levels of ROS that appear during senescence are caused not only by the enhanced production of free radicals, but also by a loss in antioxidant capacity (Srivalli and Khanna-Chopra, 2004). We suggest that CTK might induce HO-1 expression, and then the CTK-induced up-regulation of HO-1 stimulate the activities of antioxidant enzymes. Figs. 2–5 support this assumption. For example, CTK treatment induced a decrease in the TBARS content (Fig. 3E), an indicator of lipid peroxidation and free radical generation, and enhanced the expression of the antioxidant genes CAT, POD, Cu/Zn-SOD, Mn-SOD, and APX, and the total activities of the antioxidant enzymes CAT, POD, SOD, and APX (Fig. 4). Furthermore, POD-II, POD-IV, SOD-IV, SOD-V, and APX-IX induced by 6-BA might be responsible for the CTK-induced alleviation of oxidative stress (Fig. 5). These results are consistent with the observations that 6-BA enhanced the activities of CAT and APX, and reduced the level of H2 O2 in the delayed-senescence wheat leaves (Zavaleta-Mancera et al., 2007). However, such enhancements were blocked partially by the simultaneously applied ZnPPIX (Fig. 4),

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Fig. 4. Effects of ZnPPIX, 6-BA, and hemin on the activities and expression of antioxidant enzymes in detached wheat leaves during dark incubation. Wheat leave segments were pretreated in darkness for 72 h, and then exposed to distilled water (Con), 100 ␮M ZnPPIX, 10 ␮M 6-BA, 1 ␮M hemin alone, or 6-BA plus ZnPPIX, respectively. Activities of antioxidant enzymes such as catalase (CAT, A), guaiacol peroxidase (POD, B), superoxide dismutase (SOD, C), ascorbate peroxidase (APX, D) were determined after various treatments for 24 h. Values are means ± SE of three independent experiments. Bars denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test. Simultaneously, transcript levels of antioxidant genes CAT, POD, Cu/Zn-SOD, Mn-SOD, and APX were analyzed by semi-quantitative RT-PCR (E). The 18s rRNA amplification band is shown to confirm equal loading of RNA and RT efficiency. These data, which represent relative transcript levels with respect to that of control sample (Con, 100%), were obtained by densitometric analysis. The RT-PCR results of three independent experiments show similar trends of mRNA. FW, Fresh weight.

which is consistent with the former result that 6-BA triggered the up-regulation of HO-1 (Fig. 2). In comparison with the effects of 6-BA, treatment with hemin induced significant increases in the transcript levels and the total activities of antioxidant enzymes, except for SOD activity (Fig. 4), and the increases were substantially arrested by the application of ZnPPIX (data not shown). In animals, the fact that HO-1 is strongly induced by oxidant stress and its substrate heme, in conjunction with the robust ability of HO-1 to guard against oxidative insult, suggests a countervailing system to oxidative stress injury (Motterlini et al., 1996). By using an HO-1 inducer and its potent inhibitor, previous results illustrated that the vascular cytoprotective mechanism of HO-1 against oxidative stress required an increase in extracellular SOD (EC-SOD) and CAT expression (Turkseven et al., 2005). Therefore, combined with former results, we deduced that the antioxidant

effects of wheat HO-1 derived from its capacity to form BV, since BV can not only act as an efficient ROS scavenger, but also as an inducer for the enhancement of SOD and guaiacol peroxidase (GPOX) activity in soybean leaves (Noriega et al., 2004). Our recent results also suggested that bilirubin (BR), another catalytic by-product of HO-1, was able to significantly suppress the increased H2 O2 generation in GA-treated wheat aleurone layers by enhancing APX and CAT activities (Wu et al., 2011). In addition, although CO is not an antioxidant, it does induce antioxidative enzyme expression in rice and wheat plants (Liu et al., 2007; Xie et al., 2008). Our results confirmed a novel role for HO in the CTK-induced delay of senescence and alleviation of lipid peroxidation in detached wheat leaves. It was also suggested that the above cytoprotective roles of HO are at least partially involved in the enhancement of antioxidative enzyme activity.

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Fig. 5. Effects of ZnPPIX, 6-BA, and hemin on the in-gel activities of guaiacol peroxidase (POD, T = 8%, A), superoxide dismutase (SOD, T = 10%, B), and ascorbate peroxidase (APX, T = 10%, C) in detached wheat leaves during dark incubation. Wheat leave segments were pretreated in darkness for 72 h, and then exposed to distilled water (Con), 100 ␮M ZnPPIX, 10 ␮M 6-BA, 1 ␮M hemin alone, or 6-BA plus ZnPPIX respectively, for another 24 h. Then, the extracts corresponding to the equal fresh weight of plant tissues were loaded onto the non-denaturing polyacrylamide gel electrophoresis (PAGE). The arrows point to the bands corresponding to various isozymes.

Acknowledgements This work was supported by the Program for New Century Excellent Talents in University (grant no. NCET-07-0441), the Fundamental Research Funds for the Central Universities (grant no. KYZ200905), and the 111 Project (grant no. B07030). We also thank Dr. Evan Evans from the University of Tasmania, Australia, for his kind help in writing the manuscript. References Balestrasse KB, Noriega GO, Batlle AMC, Tomaro ML. Involvement of heme oxygenase as antioxidant defence in soybean nodules. Free Rad Res 2005;39: 145–51. Bauer M, Bauer I. Heme oxygenase-1: redox regulation and role in the hepatic response to oxidative stress. Antioxid Redox Signal 2002;4:749–58. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276–87.

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