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Decline in leaf photooxidative-stress tolerance with age in tobacco
Plant Science 168 (2005) 1487–1493 www.elsevier.com/locate/plantsci
Decline in leaf photooxidative-stress tolerance with age in tobacco Mieko Ohe, Madhusudhan Rapolu, Takahiro Mieda, Yoshiko Miyagawa, Yukinori Yabuta, Kazuya Yoshimura, Shigeru Shigeoka * Graduate School of Advanced Bioscience, Kinki University, Nakamachi 3327-204, Nara 631-8505, Japan Received 27 September 2004; received in revised form 27 January 2005; accepted 28 January 2005 Available online 17 March 2005
Abstract To clarify the mechanism of the decline in oxidative stress tolerance of leaves during the course of senescence, we studied the differences in the levels of various antioxidants and antioxidant enzymes in leaves of different ages. In 8-week-old tobacco plants, even though the chlorophyll content had not changed, the protein content and the photosynthetic capacity were significantly decreased in the older leaves compared to the younger leaves. The older leaves had constitutively higher levels of H2O2. Analyses of the components of the active oxygen species-scavenging system revealed that older leaves had significantly lower levels of ascorbate, glutathione and most of the antioxidant enzymes, especially catalase, Cu/Zn-superoxide dismutase, and ascorbate peroxidase (APX). When leaves of different ages were treated with 0.5 mM methylviologen under moderate light intensity at 150 mmol photons m2 s1, the older leaves lost more chlorophyll and sustained ion leakage. During the induced photooxidative stress, chloroplastic APX, a key component of the water–water cycle, was particularly destroyed in the older leaves. These findings suggest that the decline in the levels of antioxidants and antioxidant enzymes associated with leaf senescence lead to lower photooxidative-stress tolerance, which might in turn accelerate the propagation of senescence. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Active oxygen species; Antioxidants; Antioxidant enzymes; Leaf age; Nicotiana tabacum; Photooxidative stress
1. Introduction Leaf senescence, the final stage of leaf development, involves an array of physiological and metabolic changes that lead to the eventual leaf death. Although senescence is initiated by programmed differential regulation of a set of senescence-related genes, at the metabolic level senescence is an oxidative process, and most of the catabolic processes involved in senescence are propagated irreversibly once initiated [1–4]. Active oxygen species (AOS) such as the superoxide radical (O2), H2O2, and hydroxyl radical (OH ) that are generated by the normal metabolic processes in various subcellular organelles have been shown to play an important role in the dynamics of senescence. Programmed cell death can be triggered by a variety of stimuli, including AOS [2]. Pastori and del Rı´o [5] proposed that AOS generated in the peroxisomes mediate the dark-induced * Corresponding author. Tel.: +81 742 43 8083; fax: +81 742 43 2252. E-mail address: [email protected] (S. Shigeoka).
senescence of pea leaves. A later study revealed a substantial decrease in all the components of the mitochondrial ascorbate–glutathione (AsA–GSH) cycle, suggesting that mitochondria may be affected earlier than peroxisomes [6]. Little is known about the role of photooxidative stress in leaf senescence. In photosynthetic organisms, chloroplasts are the major source of AOS because even under optimal conditions the photosynthetic electron transport generates AOS [7,8]. It has been well documented that chloroplasts undergo the earliest changes during senescence. One of the most conspicuous features of senescence is the loss of chlorophyll and the associated decline in the photosynthetic activity [9]. In chloroplasts, even a low level of H2O2 (10 mM) can inhibit CO2 fixation by up to 50% through oxidative inactivation of the photosynthetic carbon-reduction-cycle enzymes [10,11]. Ascorbate peroxidase (APX; EC 1.11.1.11), which occurs as two isoenzymes in the chloroplast (as a soluble enzyme in the stroma and a thylakoid membrane-bound enzyme), is a key component of the water–water cycle, which is essential for photosynthesis
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.020
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[12]. Transgenic tobacco plants expressing bacterial catalase in the chloroplast have been shown to have higher tolerance to photooxidative stress [13]. Recently we demonstrated that the thylakoid membrane-bound APX is the limiting factor in plant tolerance to photooxidative stress [14]. The global changes that are associated with senescence, especially any decline in the cellular antioxidant capacity, could have important effects on the AOS-scavenging mechanism in the chloroplast. In order to understand the differences in the photooxidative-stress tolerance of leaves with age, in the present study we analyzed the levels of antioxidants and antioxidant enzymes in leaves of different ages in tobacco plants. Our findings suggest that leaf photooxidative-stress tolerance declines with age.
2. Materials and methods 2.1. Plant material and growth conditions Nicotiana tabacum cv. Xanthi was cultured for 8 weeks in a growth chamber under a 12 h light/12 h dark regime with a moderate light intensity of 400 mmol m2 s1, 60% relative humidity, and day/night temperatures of 25/20 8C [15]. 2.2. Measurement of antioxidants The levels of AsA and dehydroascorbate (DHA) in leaf tissues (1.1 cm2 10 discs) were determined spectrophotometrically using AsA oxidase (AOX) as described previously [13]. Levels of total glutathione were determined using a glutathione reductase (GR) recycling system coupled to 5-50 -dithiobis(2-nitrobenzoic acid) (DTNB). The reaction mixture (1 ml) contained 100 mM sodium phosphate buffer (pH 7.5), 5 mM EDTA, 0.2 mM NADPH, 0.6 mM DTNB, and the sample. The reaction was started by adding of 0.5 unit of GR, and the rate of reduction of DTNB was monitored at 412 nm for 3 min. The level of oxidized glutathione (GSSG) was selectively measured by derivatizing GSH with 2-vinylpyridine [16]. The difference between the total glutathione and GSSG contents was taken as the content of GSH. 2.3. Enzyme assays Leaf tissues (1.1 cm2 10 discs) were ground to a fine powder in liquid N2 and then homogenized in 1 ml of 50 mM potassium phosphate buffer (pH 7.6) with 0.3 M sorbitol, 10 mM KCl, 5 mM MgCl2, 1 mM AsA and 2% (w/v) polyvinylpyrrolidone using a mortar and pestle on ice. The homogenate was centrifuged for 15 min at 100,000 g at 4 8C. The soluble fraction was assayed for the activities of stromal and cytosolic APX isozymes, while the membrane fraction was used for assaying thylakoid and microbody membrane-bound APX isozymes. APX isoenzymes were assayed by following the oxidation of AsA at 290 nm
(2.8 mM1 cm1) according to the method of Shigeoka et al. [17]. Differential assays of cytosolic APX and stromal APX in the soluble fraction, and thylakoid membrane-bound APX and microbody membrane-bound APX, were carried out by a method that exploits the differences in the inactivation kinetics of APX isoenzymes as described previously [18]. The sum of thylakoid membrane-bound APX activity and stromal APX activity was taken as the activity of chloroplastic APX. Dehydroascorbate reductase (DHAR) activity was assayed by measuring the formation of AsA at 290 nm according to Shigeoka et al. [16] with some modifications. The reaction mixture contained 10 mM potassium phosphate buffer (pH 6.3), 0.5 mM NADH, 1 mM DHA, 1 mM GSH, and the sample in a total volume of 1 ml. The reaction was initiated by the addition of the sample. For the monodehydroascorbate reductase (MDAR) assay, the reaction mixture contained 100 mM Tris–HCl buffer (pH 7.2), 0.2 mM NADH, 1 mM AsA, 0.2 units of AOX, and the sample in a total volume of 1 ml. The reaction was started by adding AOX, which generates saturating concentrations of MDA, and the oxidation of NADH was followed by monitoring the decrease in absorbance at 340 nm. Glutathione reductase (GR) activity was determined by measuring the rate of NADPH oxidation as the decrease in absorbance at 340 nm (6.22 mM1 cm1) according to the method of [19]. Total superoxide dismutase (SOD) activity was assayed by the extent of inhibition of ferricytochrome c reduction by measuring the O2 generated in a xanthine–xanthine oxidase system according to the method of McCord and Fridovich [20]. Individual activities of Cu/Zn-SOD, FeSOD, and Mn-SOD isoenzymes were estimated from the inhibition by H2O2 or KCN as described previously [21]. 2.4. Methylviologen treatment Methylviologen (MV) treatment was carried out according to Shikanai et al. [22]. Leaf discs (1 cm diameter) were punched from the leaves using a cork borer and floated on a solution containing 0.5 or 2 mM MV with 0.1% Tween-20 in a Petri plate. After preincubation in the dark for 12 h at 25 8C, the Petri plates were exposed to light (150 mmol m2 s1) for 1 h at 25 8C. Then the leaf discs were collected, frozen in liquid N2 and stored at 80 8C until used for analysis of the levels of antioxidants and antioxidant enzymes. 2.5. Gas-exchange measurement Net CO2 assimilation rates were measured using a portable photosynthesis system LI-6400 (Li-Cor, Lincoln, NE) on fully expanded leaves as described previously [15] under the following conditions: 1000 mmol photons m2 s1, 360 ppm CO2, 60% relative humidity at 25 8C.
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2.6. Ion leakage Leaf discs were treated with MV as described above except that the MV concentration was 1.0 mM. After the treatment, the conductivity of the floatation solution was measured with a conductivity meter (HORIBA ES-12). The leaf discs along with the floatation solutions were autoclaved and the conductivity of the solution was measured again and taken as an index for total ion leakage. 2.7. Other methods Total protein in leaves was measured as follows. Leaf tissues (0.5 g) were harvested at the sixth hour in the light regime and ground to a fine powder in liquid N2 using a mortar and pestle in 2 ml of 1% (w/v) SDS. The extract was allowed to thaw and then was heated at 100 8C for 5 min. The suspension was centrifuged at 12,000 g for 5 min. The pellet was re-extracted as above and the supernatant was used for the determination of total protein by the method of Lowry et al. [23] using bovine serum albumin as a standard. Chlorophyll content was measured by the method of Arnon [24]. H2O2 was measured by the homovanillic acid method with some modifications as described previously [18].
3. Results 3.1. Photosynthetic capacity and photooxidative-stress tolerance of leaves of different ages In order to examine whether there are any differences in the photosynthetic capacity of leaves of different ages, we measured the chlorophyll content and the rate of photosynthesis in leaves at different heights in tobacco plants, as leaves at different heights can represent a continuous spectrum of different stages of senescence. In 8-week-old tobacco plants, the chlorophyll content was scarcely different among leaves of different ages (Fig. 1a). However, the total protein content of older leaves was significantly lower than that of younger leaves (Fig. 1b). Furthermore, the photosynthetic activity of leaves of different ages was negatively correlated with leaf age, with the net carbon fixation rate of older leaves being less than one-fourth that of the younger leaves (Fig. 1c). These data suggest that the biochemical changes associated with senescence affect the process of photosynthesis in the older leaves of 8-week-old tobacco plants. The tobacco plants described above were grown in a growth chamber with a daily photoperiod of 12 h under a moderate light intensity at 400 mmol photons m2 s1. However, even under these mild growth conditions, the steady-state levels of H2O2 in the older leaves were approximately 40% higher than those in the younger leaves (Fig. 2), suggesting that the capacity of the AOS-scavenging system in the older leaves is much lower than that in the younger leaves.
Fig. 1. Photosynthetic activity and protein content in leaves of different ages. L2, L3, L4, L7 and L8 were the second, third, fourth, seventh, and eighth leaves from the top in 8-week-old tobacco plants. Net CO2 assimilation rates were measured on fully expanded leaves under the following conditions: 1000 mmol photons m2 s1, 360 ppm CO2, 60% relative humidity. Data are means S.D. from three replicate experiments. Different letters indicate significant differences (P < 0.05).
To simulate the field conditions of high light intensity, which causes photooxidative stress, we treated the leaves with MV. In chloroplasts, MV takes electrons from PSI and transfers them to dioxygen, generating O2 radicals, effectively simulating the photooxidative stress. When leaves of different age were treated with 0.5 mM MV and then exposed to light with a moderate intensity at
Fig. 2. Steady-state levels of H2O2 in leaves of different ages. L2, L3, L4, L7 and L8 were the second, third, fourth, seventh, and eighth leaves from the top in 8-week-old tobacco plants. Leaves at mid-daylight were extracted with perchloric acid and the H2O2 levels were quantified by the homovanillic acid method. Data are means S.D. from three replicate experiments. Different letters indicate significant differences (P < 0.05).
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150 mmol photons m2 s1, the older leaves blanched, losing 68% of their chlorophyll during 24 h of the treatment, whereas the younger leaves lost only 21% chlorophyll (Fig. 3). Furthermore, the older leaves sustained significantly higher cellular damage as a result of the MV treatment compared to the younger leaves, as evident from ion leakage (Fig. 3). 3.2. Differences in levels of antioxidants and antioxidant enzymes of leaves of different ages In order to understand the differences in the photosynthetic capacity and the tolerance to photooxidative stress with leaf age, we determined the levels of various antioxidants and antioxidant enzymes in tobacco leaves of
different ages. As shown in Fig. 4, the levels of almost all the analyzed antioxidant enzymes and the antioxidants AsA and GSH were significantly lower in the older leaves than those in the younger leaves. Especially, the activities of catalase, Cu/Zn-SOD, and cytosolic APX in the older leaves were 14, 25, and 54%, respectively, of those in the younger leaves. However, there was no significant difference in the activities of Fe- and Mn-SOD in younger versus older leaves. The total AsA and glutathione pool sizes in older leaves were 44 and 33%, respectively, of those in the younger leaves. Further, the proportions of DHA and GSSG in the total AsA and glutathione pools, respectively, were higher in the older leaves. The low levels of antioxidants and antioxidant enzymes in the older leaves may account for the higher steady-state levels of H2O2 in the older leaves (Fig. 2). In contrast to the significantly lower activities of catalase and Cu/Zn-SOD, chloroplastic APX activity in the older leaves was only 35% lower than that in the younger leaves (Fig. 4). However, we recently demonstrated that chloroplastic APX, especially the thylakoid membrane bound APX, is a limiting factor in tolerance to photooxidative stress [14]. 3.3. Effect of photooxidative stress on levels of antioxidants and antioxidant enzymes in leaves of different ages
Fig. 3. Photooxidative-stress tolerance of leaves of different ages. (A) Chlorosis under induced photooxidative stress. Leaf discs were floated on a 0.1% Tween-20 solution containing the indicated concentrations of MV and preincubated in the dark for 12 h. They were then exposed to light of 150 mmol photons m2 s1 for 24 h at 25 8C. (B) Residual chlorophyll. The 0.5 mM MV-treated samples were extracted with acetone and the chlorophyll contents were measured. The residual chlorophyll contents are expressed as percentages of those of samples that were not exposed to light. (C) Ion leakage. Leaf discs were treated as described above at 1.0 mM MV for 12 h. Conductivities of the flotation solutions after the treatment were measured and expressed as percentages of the conductivity values obtained with total cell lysis attained by autoclaving the samples. L3, L4, L7 and L8 were the third, fourth, seventh, and eighth leaves from the top in 8week-old tobacco plants.
To clarify the role of chloroplastic APX in older leaves, we analyzed the levels of AsA and chloroplastic APX under the photooxidative stress induced by the MV treatment (Fig. 5). During the course of the induced photooxidative stress, both the younger and older leaves increasingly lost their chloroplastic APX with the increase in MV concentration in a dose-dependent manner, but the destruction of chloroplastic APX in the older leaves was much more marked at higher MV concentration. At 2 mM MV, 75% of the chloroplastic APX in the older leaves was destroyed, whereas the extent of the destruction in the younger leaves was only 31%. Furthermore, the total AsA level in the older leaves markedly decreased to 42% of that before the MV treatment, while it decreased to only 59% in the younger leaves. On the other hand, the activities of cytosolic APX, MDAR, DHAR, and GR were scarcely changed in both younger and older leaves, suggesting that among the antioxidant enzymes, chloroplastic APX is particularly destroyed during photooxidative stress.
4. Discussion In order to clarify the mechanism of the oxidative stress tolerance of leaves during the course of senescence, in this work we studied the differences in the levels of various antioxidant enzymes and antioxidants in leaves of different ages in tobacco plants. Chloroplasts are one of the earliest sites of catabolism in the senescence process, while mitochondria remain intact until late in the process [9]. In
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Fig. 4. Levels of antioxidants and antioxidant enzymes in leaves of different ages. L2, L3, L4, L7, and L8 were the second, third, fourth, seventh, and eighth leaves from the top of 8-week-old tobacco plants. cAPX, cytosolic APX; chlAPX, chloroplastic APX; mAPX, microbody APX; GR, glutathione reductase; DHAR, dehydroascorbate reductase; MDAR, monodehydroascorbate reductase; AsA, ascorbate; DHA, dehydroascorbate; GSH, reduced glutathione; GSSG, oxidized glutathione.
8-week-old tobacco plants, the total protein content was already significantly decreased in the older leaves (Fig. 1). Leaf senescence involves the degradation of proteins [25], nucleic acids [2] and membranes [26], and in advanced stages, the loss of chlorophyll [9]. Even though the chlorophyll content had not changed in the leaves of 8week-old tobacco plants, the photosynthetic capacity decreased with increasing leaf age. During senescence,
the transcript levels of several photosynthesis-related genes such as rbcS (small subunit of Rubisco) and cab (chlorophyll a/b-binding protein) decline [27,9]. The older leaves had constitutively higher levels of H2O2 (Fig. 2). H2O2 is not toxic by itself, but can be detrimental to plant metabolism by forming the highly reactive hydroxyl radical through the metal-catalyzed Haber–Weiss reaction [28]. The high level of H2O2 in the older leaves was clearly due to low levels of
Fig. 5. Effect of photooxidative stress on the levels of antioxidants and antioxidant enzymes in leaves of different ages. Leaf discs were floated on a 0.1% Tween-20 solution containing the indicated concentrations of MV, preincubated in the dark for 12 h, and then exposed to light of 150 mmol photons m2 s1 for 1 h at 25 8C. After the treatment, the leaf tissues were extracted and assayed for the levels of the indicated antioxidants and antioxidant enzymes. ‘Younger leaves’ were the third and fourth leaves, and ‘older leaves’ were the seventh and eighth leaves from the top in 8-week-old tobacco plants. See the legend of Fig. 4 for abbreviations. Data are means S.D. from three replicate experiments.
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antioxidant enzymes, particularly catalase, Cu/Zn-SOD, and cytosolic APX, and low levels of the antioxidants AsA and GSH (Fig. 4). Pastori and del Rı´o [5] reported that during senescence in pea leaves, peroxisomes generate AOS due to elevated levels of xanthine oxidase, urate oxidase, and MnSOD activities coupled with reduced catalase activity. Arabidopsis leaves have been found to loose the stressinducibility of catalase expression with senescence [29]. Similarly, the SOD expression in mature barley leaves was less sensitive to oxidative stress than that in the young leaves [30]. Thus, it seems that the dynamics of AOS regulation during senescence include both the proactive generation of AOS by up-regulation of certain enzymes as well as finetuned attenuation of AOS-scavenging mechanisms [31]. Photooxidative stress contributes to senescence through even more complex mechanisms, because photosynthetic electron transport is the major source of AOS even under optimal conditions [7,8]. Under the induced photooxidative stress conditions, the older leaves lost more chlorophyll and sustained cellular damage (Fig. 3). AOS can cause lipid peroxidation and membrane permeability, which can lead to decreased photosynthetic capacity and increased cellular damage [7]. During the induced photooxidative stress, chloroplastic APX in the older leaves was lost to a much greater extent than that in the younger leaves (Fig. 5). Even though there was no significant difference in the loss of total GR activity in the older and younger leaves during the induced photooxidative stress (Fig. 5), Casano et al. [32] reported that in barley, chloroplastic GR was inactivated more in the mature leaves than in the younger leaves. Chloroplastic APX, the key enzyme responsible for scavenging H2O2 generated in chloroplasts, is paradoxically sensitive to H2O2, with a half-life of only 15–20 s under limiting AsA concentration [33]. Both the chloroplastic APX activity and AsA content in the older leaves were significantly decreased (Fig. 4). Of all the APX isoenzymes, chloroplastic stromal and thylakoid-membrane bound APX isoenzymes are the most labile to H2O2, and are primary targets under photooxidative stress [34]. Taken together, these data suggest that the combination of low levels of AsA, catalase, Cu/Zn-SOD, and cytosolic APX in the older leaves (Fig. 4) might lead to a catastrophic state resulting in rapid inactivation of the already lowered level of chloroplastic APX during photooxidative stress (Fig. 5), and a resultant loss of chlorophyll and increased cellular damage.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (no. 15380078 to S.S.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by the Japan Society for the Promotion of Science Research for the Future Program grant (no. JSPS-RFTF 00L01604 to S.S.). This work was also supported in part by ‘‘Academic Frontier’’ Project for Private Universities:
matching fund subsidy from MEXT, 2004–2008. R.M. gratefully acknowledges a postdoctoral fellowship (ID no. P03127) from the Japan Society for the Promotion of Science.
References [1] K. Krupinska, J. Falk, Jon, K. Humbeck, Genetic, metabolic and environmental factors associated with aging in plants, in: S.I.S. Rattan (Ed.), Biology of Aging and its Modulation, vol. 4, Kluwer Academic Publishers, Dordrecht, 2003, pp. 55–78. [2] V. Buchanan-Wollaston, The molecular biology of leaf senescence, J. Exp. Bot. 48 (1997) 181–199. [3] A.M. Jones, J.L. Dangl, Logjam at the Styx: programmed cell death in plants, Trends Plant Sci. 1 (1996) 114–119. [4] L.M. Weaver, E. Himelblau, R.M. Amasino, Leaf senescence: gene expression and regulation, in: J.K. Setlow (Ed.), Genetic Engineering, vol. 19, Plenum Press, New York, 1997, pp. 215–234. [5] G.M. Pastori, R.A. del Rı´o, Natural senescence of pea leaves: an activated oxygen-mediated function for peroxisomes, Plant Physiol. 113 (1997) 411–418. [6] A. Jime`nez, J.A. Herna`ndez, G. Pastori, R.A. del Rı`o, F. Sevilla, Role of the ascorbate–glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves, Plant Physiol. 118 (1998) 1327–1335. [7] K. Asada, The water–water cycle as alternative photon and electron sinks, Phil. Trans. Roy. Soc. Lond. B 355 (2000) 1419–1431. [8] K. Asada, The water–water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601–639. [9] C.M. Smart, Gene expression during leaf senescence, New Phytol. 126 (1994) 419–448. [10] T. Takeda, A. Yokota, S. Shigeoka, Resistance of photosynthesis to hydrogen peroxide in algae, Plant Cell Physiol. 36 (1995) 1089–1095. [11] W.M. Kaiser, The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts, Biochim. Biophys. Acta 440 (1976) 476–482. [12] S. Shigeoka, T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta, K. Yoshimura, Regulation and function of ascorbate peroxidase isoenzymes, J. Exp. Bot. 53 (2002) 305–1319. [13] Y. Miyagawa, M. Tamoi, S. Shigeoka, Evaluation of the defense system in chloroplasts to photooxidative stress caused by paraquat using transgenic tobacco plants expressing catalase from Escherichia coli, Plant Cell Physiol. 41 (2000) 311–320. [14] Y. Yabuta, T. Motoki, K. Yoshimura, T. Takeda, S.T. Ishikawa, S. Shigeoka, Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress, Plant J. 32 (2002) 915–925. [15] Y. Miyagawa, M. Tamoi, S. Shigeoka, Overexpression of cyanobacterial fructose-1,6-/sedoheptulose-1,7-biphosphatase in tobacco enhances photosynthesis and growth, Nature Biotechnol. 19 (2001) 965–969. [16] S. Shigeoka, R. Yasumoto, T. Onishi, Y. Nakano, S. Kitaoka, Properties of monodehydroascorbate reductase and dehydroascorbate reductase and their participation in the regeneration of ascorbate in Euglena gracilis, J. Gen. Microbiol. 133 (1987) 227–232. [17] S. Shigeoka, Y. Nakano, S. Kitaoka, Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase, Biochem. J. 186 (1980) 377–380. [18] K. Yoshimura, Y. Yabuta, T. Ishikawa, S. Shigeoka, Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses, Plant Physiol. 123 (2000) 223–233. [19] B. Halliwell, C.H. Foyer, Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography, Planta 139 (1978) 9–17.
M. Ohe et al. / Plant Science 168 (2005) 1487–1493 [20] J.M. McCord, I. Fridovich, Superoxide dismutase, an enzymatic function for erythrocuprein (hemocuprein), J. Biol. Chem. 244 (1969) 6049–6055. [21] K. Asada, M. Takahashi, M. Nagata, Assay and inhibitors of spinach superoxide dismutase, Agric. Biol. Chem. 38 (1974) 471–473. [22] T. Shikanai, T. Takeda, H. Yamauchi, S. Sano, K. Tomizawa, A. Yokota, S. Shigeoka, Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts, FEBS Lett. 428 (1998) 47–51. [23] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [24] D.I. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris, Plant Physiol. 24 (1949) 1–15. [25] S. Lutts, J.M. Kinet, J. Bouharmont, NaCl-induced senescence in leaves of rice (Oryza sativa L) cultivars differing in salinity resistance, Ann. Bot. 78 (1996) 389–398. [26] S. Trippi, K.V. Thimann, The exudation of solutes during senescence of oat leaves, Physiol. Plant. 58 (1983) 21–28. [27] N.J. Bate, S.J. Rothstein, J.E. Thompson, Expression of nuclear and chloroplast photosynthesis-specific genes during leaf senescence, J. Exp. Bot. 42 (1991) 801–811. [28] M.L. Salin, Toxic oxygen species and protective systems of chloroplast, Physiol. Plant. 72 (1988) 681–689.
1493
[29] G. Orendi, P. Zimmermann, C. Baar, U. Zentgraf, Loss of stressinduced expression of catalase 3 during leaf senescence in Arabidopsis thaliana is restricted to oxidative stress, Plant Sci. 161 (2001) 301– 314. [30] L.M. Casano, M. Martı´n, B. Sabater, Sensitivity of superoxide dismutase transcript levels and activities to oxidative stress is lower in mature-senescent than in young barley leaves, Plant Physiol. 106 (1994) 1033–1039. [31] M.A. Longa, L.A. del Rı´o, J.M. Palma, Superoxide dismutase of chestnut leaves Castanea sativa: characterization and study of their involvement in natural leaf senescence, Physiol. Plant. 92 (1994) 227– 232. [32] L.M. Casano, M. Martı´n, J.M. Zapata, B. Sabater, Leaf age- and paraquat concentration-dependent effects on the levels of enzymes protecting against photooxidative stress, Plant Sci. 149 (1999) 13–22. [33] C. Miyake, K. Asada, Inactivation mechanism of ascorbate peroxidase at low concentrations of ascorbate: hydrogen peroxide decomposes compound I of ascorbate peroxidase, Plant Cell Physiol. 37 (1996) 423–430. [34] J. Mano, C. Ohno, Y. Domae, K. Asada, Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR, Biochim. Biophys. Acta 1504 (2001) 275–287.
Decline in leaf photooxidative-stress tolerance with age in tobacco Mieko Ohe, Madhusudhan Rapolu, Takahiro Mieda, Yoshiko Miyagawa, Yukinori Yabuta, Kazuya Yoshimura, Shigeru Shigeoka * Graduate School of Advanced Bioscience, Kinki University, Nakamachi 3327-204, Nara 631-8505, Japan Received 27 September 2004; received in revised form 27 January 2005; accepted 28 January 2005 Available online 17 March 2005
Abstract To clarify the mechanism of the decline in oxidative stress tolerance of leaves during the course of senescence, we studied the differences in the levels of various antioxidants and antioxidant enzymes in leaves of different ages. In 8-week-old tobacco plants, even though the chlorophyll content had not changed, the protein content and the photosynthetic capacity were significantly decreased in the older leaves compared to the younger leaves. The older leaves had constitutively higher levels of H2O2. Analyses of the components of the active oxygen species-scavenging system revealed that older leaves had significantly lower levels of ascorbate, glutathione and most of the antioxidant enzymes, especially catalase, Cu/Zn-superoxide dismutase, and ascorbate peroxidase (APX). When leaves of different ages were treated with 0.5 mM methylviologen under moderate light intensity at 150 mmol photons m2 s1, the older leaves lost more chlorophyll and sustained ion leakage. During the induced photooxidative stress, chloroplastic APX, a key component of the water–water cycle, was particularly destroyed in the older leaves. These findings suggest that the decline in the levels of antioxidants and antioxidant enzymes associated with leaf senescence lead to lower photooxidative-stress tolerance, which might in turn accelerate the propagation of senescence. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Active oxygen species; Antioxidants; Antioxidant enzymes; Leaf age; Nicotiana tabacum; Photooxidative stress
1. Introduction Leaf senescence, the final stage of leaf development, involves an array of physiological and metabolic changes that lead to the eventual leaf death. Although senescence is initiated by programmed differential regulation of a set of senescence-related genes, at the metabolic level senescence is an oxidative process, and most of the catabolic processes involved in senescence are propagated irreversibly once initiated [1–4]. Active oxygen species (AOS) such as the superoxide radical (O2), H2O2, and hydroxyl radical (OH ) that are generated by the normal metabolic processes in various subcellular organelles have been shown to play an important role in the dynamics of senescence. Programmed cell death can be triggered by a variety of stimuli, including AOS [2]. Pastori and del Rı´o [5] proposed that AOS generated in the peroxisomes mediate the dark-induced * Corresponding author. Tel.: +81 742 43 8083; fax: +81 742 43 2252. E-mail address: [email protected] (S. Shigeoka).
senescence of pea leaves. A later study revealed a substantial decrease in all the components of the mitochondrial ascorbate–glutathione (AsA–GSH) cycle, suggesting that mitochondria may be affected earlier than peroxisomes [6]. Little is known about the role of photooxidative stress in leaf senescence. In photosynthetic organisms, chloroplasts are the major source of AOS because even under optimal conditions the photosynthetic electron transport generates AOS [7,8]. It has been well documented that chloroplasts undergo the earliest changes during senescence. One of the most conspicuous features of senescence is the loss of chlorophyll and the associated decline in the photosynthetic activity [9]. In chloroplasts, even a low level of H2O2 (10 mM) can inhibit CO2 fixation by up to 50% through oxidative inactivation of the photosynthetic carbon-reduction-cycle enzymes [10,11]. Ascorbate peroxidase (APX; EC 1.11.1.11), which occurs as two isoenzymes in the chloroplast (as a soluble enzyme in the stroma and a thylakoid membrane-bound enzyme), is a key component of the water–water cycle, which is essential for photosynthesis
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.020
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[12]. Transgenic tobacco plants expressing bacterial catalase in the chloroplast have been shown to have higher tolerance to photooxidative stress [13]. Recently we demonstrated that the thylakoid membrane-bound APX is the limiting factor in plant tolerance to photooxidative stress [14]. The global changes that are associated with senescence, especially any decline in the cellular antioxidant capacity, could have important effects on the AOS-scavenging mechanism in the chloroplast. In order to understand the differences in the photooxidative-stress tolerance of leaves with age, in the present study we analyzed the levels of antioxidants and antioxidant enzymes in leaves of different ages in tobacco plants. Our findings suggest that leaf photooxidative-stress tolerance declines with age.
2. Materials and methods 2.1. Plant material and growth conditions Nicotiana tabacum cv. Xanthi was cultured for 8 weeks in a growth chamber under a 12 h light/12 h dark regime with a moderate light intensity of 400 mmol m2 s1, 60% relative humidity, and day/night temperatures of 25/20 8C [15]. 2.2. Measurement of antioxidants The levels of AsA and dehydroascorbate (DHA) in leaf tissues (1.1 cm2 10 discs) were determined spectrophotometrically using AsA oxidase (AOX) as described previously [13]. Levels of total glutathione were determined using a glutathione reductase (GR) recycling system coupled to 5-50 -dithiobis(2-nitrobenzoic acid) (DTNB). The reaction mixture (1 ml) contained 100 mM sodium phosphate buffer (pH 7.5), 5 mM EDTA, 0.2 mM NADPH, 0.6 mM DTNB, and the sample. The reaction was started by adding of 0.5 unit of GR, and the rate of reduction of DTNB was monitored at 412 nm for 3 min. The level of oxidized glutathione (GSSG) was selectively measured by derivatizing GSH with 2-vinylpyridine [16]. The difference between the total glutathione and GSSG contents was taken as the content of GSH. 2.3. Enzyme assays Leaf tissues (1.1 cm2 10 discs) were ground to a fine powder in liquid N2 and then homogenized in 1 ml of 50 mM potassium phosphate buffer (pH 7.6) with 0.3 M sorbitol, 10 mM KCl, 5 mM MgCl2, 1 mM AsA and 2% (w/v) polyvinylpyrrolidone using a mortar and pestle on ice. The homogenate was centrifuged for 15 min at 100,000 g at 4 8C. The soluble fraction was assayed for the activities of stromal and cytosolic APX isozymes, while the membrane fraction was used for assaying thylakoid and microbody membrane-bound APX isozymes. APX isoenzymes were assayed by following the oxidation of AsA at 290 nm
(2.8 mM1 cm1) according to the method of Shigeoka et al. [17]. Differential assays of cytosolic APX and stromal APX in the soluble fraction, and thylakoid membrane-bound APX and microbody membrane-bound APX, were carried out by a method that exploits the differences in the inactivation kinetics of APX isoenzymes as described previously [18]. The sum of thylakoid membrane-bound APX activity and stromal APX activity was taken as the activity of chloroplastic APX. Dehydroascorbate reductase (DHAR) activity was assayed by measuring the formation of AsA at 290 nm according to Shigeoka et al. [16] with some modifications. The reaction mixture contained 10 mM potassium phosphate buffer (pH 6.3), 0.5 mM NADH, 1 mM DHA, 1 mM GSH, and the sample in a total volume of 1 ml. The reaction was initiated by the addition of the sample. For the monodehydroascorbate reductase (MDAR) assay, the reaction mixture contained 100 mM Tris–HCl buffer (pH 7.2), 0.2 mM NADH, 1 mM AsA, 0.2 units of AOX, and the sample in a total volume of 1 ml. The reaction was started by adding AOX, which generates saturating concentrations of MDA, and the oxidation of NADH was followed by monitoring the decrease in absorbance at 340 nm. Glutathione reductase (GR) activity was determined by measuring the rate of NADPH oxidation as the decrease in absorbance at 340 nm (6.22 mM1 cm1) according to the method of [19]. Total superoxide dismutase (SOD) activity was assayed by the extent of inhibition of ferricytochrome c reduction by measuring the O2 generated in a xanthine–xanthine oxidase system according to the method of McCord and Fridovich [20]. Individual activities of Cu/Zn-SOD, FeSOD, and Mn-SOD isoenzymes were estimated from the inhibition by H2O2 or KCN as described previously [21]. 2.4. Methylviologen treatment Methylviologen (MV) treatment was carried out according to Shikanai et al. [22]. Leaf discs (1 cm diameter) were punched from the leaves using a cork borer and floated on a solution containing 0.5 or 2 mM MV with 0.1% Tween-20 in a Petri plate. After preincubation in the dark for 12 h at 25 8C, the Petri plates were exposed to light (150 mmol m2 s1) for 1 h at 25 8C. Then the leaf discs were collected, frozen in liquid N2 and stored at 80 8C until used for analysis of the levels of antioxidants and antioxidant enzymes. 2.5. Gas-exchange measurement Net CO2 assimilation rates were measured using a portable photosynthesis system LI-6400 (Li-Cor, Lincoln, NE) on fully expanded leaves as described previously [15] under the following conditions: 1000 mmol photons m2 s1, 360 ppm CO2, 60% relative humidity at 25 8C.
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2.6. Ion leakage Leaf discs were treated with MV as described above except that the MV concentration was 1.0 mM. After the treatment, the conductivity of the floatation solution was measured with a conductivity meter (HORIBA ES-12). The leaf discs along with the floatation solutions were autoclaved and the conductivity of the solution was measured again and taken as an index for total ion leakage. 2.7. Other methods Total protein in leaves was measured as follows. Leaf tissues (0.5 g) were harvested at the sixth hour in the light regime and ground to a fine powder in liquid N2 using a mortar and pestle in 2 ml of 1% (w/v) SDS. The extract was allowed to thaw and then was heated at 100 8C for 5 min. The suspension was centrifuged at 12,000 g for 5 min. The pellet was re-extracted as above and the supernatant was used for the determination of total protein by the method of Lowry et al. [23] using bovine serum albumin as a standard. Chlorophyll content was measured by the method of Arnon [24]. H2O2 was measured by the homovanillic acid method with some modifications as described previously [18].
3. Results 3.1. Photosynthetic capacity and photooxidative-stress tolerance of leaves of different ages In order to examine whether there are any differences in the photosynthetic capacity of leaves of different ages, we measured the chlorophyll content and the rate of photosynthesis in leaves at different heights in tobacco plants, as leaves at different heights can represent a continuous spectrum of different stages of senescence. In 8-week-old tobacco plants, the chlorophyll content was scarcely different among leaves of different ages (Fig. 1a). However, the total protein content of older leaves was significantly lower than that of younger leaves (Fig. 1b). Furthermore, the photosynthetic activity of leaves of different ages was negatively correlated with leaf age, with the net carbon fixation rate of older leaves being less than one-fourth that of the younger leaves (Fig. 1c). These data suggest that the biochemical changes associated with senescence affect the process of photosynthesis in the older leaves of 8-week-old tobacco plants. The tobacco plants described above were grown in a growth chamber with a daily photoperiod of 12 h under a moderate light intensity at 400 mmol photons m2 s1. However, even under these mild growth conditions, the steady-state levels of H2O2 in the older leaves were approximately 40% higher than those in the younger leaves (Fig. 2), suggesting that the capacity of the AOS-scavenging system in the older leaves is much lower than that in the younger leaves.
Fig. 1. Photosynthetic activity and protein content in leaves of different ages. L2, L3, L4, L7 and L8 were the second, third, fourth, seventh, and eighth leaves from the top in 8-week-old tobacco plants. Net CO2 assimilation rates were measured on fully expanded leaves under the following conditions: 1000 mmol photons m2 s1, 360 ppm CO2, 60% relative humidity. Data are means S.D. from three replicate experiments. Different letters indicate significant differences (P < 0.05).
To simulate the field conditions of high light intensity, which causes photooxidative stress, we treated the leaves with MV. In chloroplasts, MV takes electrons from PSI and transfers them to dioxygen, generating O2 radicals, effectively simulating the photooxidative stress. When leaves of different age were treated with 0.5 mM MV and then exposed to light with a moderate intensity at
Fig. 2. Steady-state levels of H2O2 in leaves of different ages. L2, L3, L4, L7 and L8 were the second, third, fourth, seventh, and eighth leaves from the top in 8-week-old tobacco plants. Leaves at mid-daylight were extracted with perchloric acid and the H2O2 levels were quantified by the homovanillic acid method. Data are means S.D. from three replicate experiments. Different letters indicate significant differences (P < 0.05).
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150 mmol photons m2 s1, the older leaves blanched, losing 68% of their chlorophyll during 24 h of the treatment, whereas the younger leaves lost only 21% chlorophyll (Fig. 3). Furthermore, the older leaves sustained significantly higher cellular damage as a result of the MV treatment compared to the younger leaves, as evident from ion leakage (Fig. 3). 3.2. Differences in levels of antioxidants and antioxidant enzymes of leaves of different ages In order to understand the differences in the photosynthetic capacity and the tolerance to photooxidative stress with leaf age, we determined the levels of various antioxidants and antioxidant enzymes in tobacco leaves of
different ages. As shown in Fig. 4, the levels of almost all the analyzed antioxidant enzymes and the antioxidants AsA and GSH were significantly lower in the older leaves than those in the younger leaves. Especially, the activities of catalase, Cu/Zn-SOD, and cytosolic APX in the older leaves were 14, 25, and 54%, respectively, of those in the younger leaves. However, there was no significant difference in the activities of Fe- and Mn-SOD in younger versus older leaves. The total AsA and glutathione pool sizes in older leaves were 44 and 33%, respectively, of those in the younger leaves. Further, the proportions of DHA and GSSG in the total AsA and glutathione pools, respectively, were higher in the older leaves. The low levels of antioxidants and antioxidant enzymes in the older leaves may account for the higher steady-state levels of H2O2 in the older leaves (Fig. 2). In contrast to the significantly lower activities of catalase and Cu/Zn-SOD, chloroplastic APX activity in the older leaves was only 35% lower than that in the younger leaves (Fig. 4). However, we recently demonstrated that chloroplastic APX, especially the thylakoid membrane bound APX, is a limiting factor in tolerance to photooxidative stress [14]. 3.3. Effect of photooxidative stress on levels of antioxidants and antioxidant enzymes in leaves of different ages
Fig. 3. Photooxidative-stress tolerance of leaves of different ages. (A) Chlorosis under induced photooxidative stress. Leaf discs were floated on a 0.1% Tween-20 solution containing the indicated concentrations of MV and preincubated in the dark for 12 h. They were then exposed to light of 150 mmol photons m2 s1 for 24 h at 25 8C. (B) Residual chlorophyll. The 0.5 mM MV-treated samples were extracted with acetone and the chlorophyll contents were measured. The residual chlorophyll contents are expressed as percentages of those of samples that were not exposed to light. (C) Ion leakage. Leaf discs were treated as described above at 1.0 mM MV for 12 h. Conductivities of the flotation solutions after the treatment were measured and expressed as percentages of the conductivity values obtained with total cell lysis attained by autoclaving the samples. L3, L4, L7 and L8 were the third, fourth, seventh, and eighth leaves from the top in 8week-old tobacco plants.
To clarify the role of chloroplastic APX in older leaves, we analyzed the levels of AsA and chloroplastic APX under the photooxidative stress induced by the MV treatment (Fig. 5). During the course of the induced photooxidative stress, both the younger and older leaves increasingly lost their chloroplastic APX with the increase in MV concentration in a dose-dependent manner, but the destruction of chloroplastic APX in the older leaves was much more marked at higher MV concentration. At 2 mM MV, 75% of the chloroplastic APX in the older leaves was destroyed, whereas the extent of the destruction in the younger leaves was only 31%. Furthermore, the total AsA level in the older leaves markedly decreased to 42% of that before the MV treatment, while it decreased to only 59% in the younger leaves. On the other hand, the activities of cytosolic APX, MDAR, DHAR, and GR were scarcely changed in both younger and older leaves, suggesting that among the antioxidant enzymes, chloroplastic APX is particularly destroyed during photooxidative stress.
4. Discussion In order to clarify the mechanism of the oxidative stress tolerance of leaves during the course of senescence, in this work we studied the differences in the levels of various antioxidant enzymes and antioxidants in leaves of different ages in tobacco plants. Chloroplasts are one of the earliest sites of catabolism in the senescence process, while mitochondria remain intact until late in the process [9]. In
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Fig. 4. Levels of antioxidants and antioxidant enzymes in leaves of different ages. L2, L3, L4, L7, and L8 were the second, third, fourth, seventh, and eighth leaves from the top of 8-week-old tobacco plants. cAPX, cytosolic APX; chlAPX, chloroplastic APX; mAPX, microbody APX; GR, glutathione reductase; DHAR, dehydroascorbate reductase; MDAR, monodehydroascorbate reductase; AsA, ascorbate; DHA, dehydroascorbate; GSH, reduced glutathione; GSSG, oxidized glutathione.
8-week-old tobacco plants, the total protein content was already significantly decreased in the older leaves (Fig. 1). Leaf senescence involves the degradation of proteins [25], nucleic acids [2] and membranes [26], and in advanced stages, the loss of chlorophyll [9]. Even though the chlorophyll content had not changed in the leaves of 8week-old tobacco plants, the photosynthetic capacity decreased with increasing leaf age. During senescence,
the transcript levels of several photosynthesis-related genes such as rbcS (small subunit of Rubisco) and cab (chlorophyll a/b-binding protein) decline [27,9]. The older leaves had constitutively higher levels of H2O2 (Fig. 2). H2O2 is not toxic by itself, but can be detrimental to plant metabolism by forming the highly reactive hydroxyl radical through the metal-catalyzed Haber–Weiss reaction [28]. The high level of H2O2 in the older leaves was clearly due to low levels of
Fig. 5. Effect of photooxidative stress on the levels of antioxidants and antioxidant enzymes in leaves of different ages. Leaf discs were floated on a 0.1% Tween-20 solution containing the indicated concentrations of MV, preincubated in the dark for 12 h, and then exposed to light of 150 mmol photons m2 s1 for 1 h at 25 8C. After the treatment, the leaf tissues were extracted and assayed for the levels of the indicated antioxidants and antioxidant enzymes. ‘Younger leaves’ were the third and fourth leaves, and ‘older leaves’ were the seventh and eighth leaves from the top in 8-week-old tobacco plants. See the legend of Fig. 4 for abbreviations. Data are means S.D. from three replicate experiments.
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antioxidant enzymes, particularly catalase, Cu/Zn-SOD, and cytosolic APX, and low levels of the antioxidants AsA and GSH (Fig. 4). Pastori and del Rı´o [5] reported that during senescence in pea leaves, peroxisomes generate AOS due to elevated levels of xanthine oxidase, urate oxidase, and MnSOD activities coupled with reduced catalase activity. Arabidopsis leaves have been found to loose the stressinducibility of catalase expression with senescence [29]. Similarly, the SOD expression in mature barley leaves was less sensitive to oxidative stress than that in the young leaves [30]. Thus, it seems that the dynamics of AOS regulation during senescence include both the proactive generation of AOS by up-regulation of certain enzymes as well as finetuned attenuation of AOS-scavenging mechanisms [31]. Photooxidative stress contributes to senescence through even more complex mechanisms, because photosynthetic electron transport is the major source of AOS even under optimal conditions [7,8]. Under the induced photooxidative stress conditions, the older leaves lost more chlorophyll and sustained cellular damage (Fig. 3). AOS can cause lipid peroxidation and membrane permeability, which can lead to decreased photosynthetic capacity and increased cellular damage [7]. During the induced photooxidative stress, chloroplastic APX in the older leaves was lost to a much greater extent than that in the younger leaves (Fig. 5). Even though there was no significant difference in the loss of total GR activity in the older and younger leaves during the induced photooxidative stress (Fig. 5), Casano et al. [32] reported that in barley, chloroplastic GR was inactivated more in the mature leaves than in the younger leaves. Chloroplastic APX, the key enzyme responsible for scavenging H2O2 generated in chloroplasts, is paradoxically sensitive to H2O2, with a half-life of only 15–20 s under limiting AsA concentration [33]. Both the chloroplastic APX activity and AsA content in the older leaves were significantly decreased (Fig. 4). Of all the APX isoenzymes, chloroplastic stromal and thylakoid-membrane bound APX isoenzymes are the most labile to H2O2, and are primary targets under photooxidative stress [34]. Taken together, these data suggest that the combination of low levels of AsA, catalase, Cu/Zn-SOD, and cytosolic APX in the older leaves (Fig. 4) might lead to a catastrophic state resulting in rapid inactivation of the already lowered level of chloroplastic APX during photooxidative stress (Fig. 5), and a resultant loss of chlorophyll and increased cellular damage.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (no. 15380078 to S.S.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by the Japan Society for the Promotion of Science Research for the Future Program grant (no. JSPS-RFTF 00L01604 to S.S.). This work was also supported in part by ‘‘Academic Frontier’’ Project for Private Universities:
matching fund subsidy from MEXT, 2004–2008. R.M. gratefully acknowledges a postdoctoral fellowship (ID no. P03127) from the Japan Society for the Promotion of Science.
References [1] K. Krupinska, J. Falk, Jon, K. Humbeck, Genetic, metabolic and environmental factors associated with aging in plants, in: S.I.S. Rattan (Ed.), Biology of Aging and its Modulation, vol. 4, Kluwer Academic Publishers, Dordrecht, 2003, pp. 55–78. [2] V. Buchanan-Wollaston, The molecular biology of leaf senescence, J. Exp. Bot. 48 (1997) 181–199. [3] A.M. Jones, J.L. Dangl, Logjam at the Styx: programmed cell death in plants, Trends Plant Sci. 1 (1996) 114–119. [4] L.M. Weaver, E. Himelblau, R.M. Amasino, Leaf senescence: gene expression and regulation, in: J.K. Setlow (Ed.), Genetic Engineering, vol. 19, Plenum Press, New York, 1997, pp. 215–234. [5] G.M. Pastori, R.A. del Rı´o, Natural senescence of pea leaves: an activated oxygen-mediated function for peroxisomes, Plant Physiol. 113 (1997) 411–418. [6] A. Jime`nez, J.A. Herna`ndez, G. Pastori, R.A. del Rı`o, F. Sevilla, Role of the ascorbate–glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves, Plant Physiol. 118 (1998) 1327–1335. [7] K. Asada, The water–water cycle as alternative photon and electron sinks, Phil. Trans. Roy. Soc. Lond. B 355 (2000) 1419–1431. [8] K. Asada, The water–water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601–639. [9] C.M. Smart, Gene expression during leaf senescence, New Phytol. 126 (1994) 419–448. [10] T. Takeda, A. Yokota, S. Shigeoka, Resistance of photosynthesis to hydrogen peroxide in algae, Plant Cell Physiol. 36 (1995) 1089–1095. [11] W.M. Kaiser, The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts, Biochim. Biophys. Acta 440 (1976) 476–482. [12] S. Shigeoka, T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta, K. Yoshimura, Regulation and function of ascorbate peroxidase isoenzymes, J. Exp. Bot. 53 (2002) 305–1319. [13] Y. Miyagawa, M. Tamoi, S. Shigeoka, Evaluation of the defense system in chloroplasts to photooxidative stress caused by paraquat using transgenic tobacco plants expressing catalase from Escherichia coli, Plant Cell Physiol. 41 (2000) 311–320. [14] Y. Yabuta, T. Motoki, K. Yoshimura, T. Takeda, S.T. Ishikawa, S. Shigeoka, Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress, Plant J. 32 (2002) 915–925. [15] Y. Miyagawa, M. Tamoi, S. Shigeoka, Overexpression of cyanobacterial fructose-1,6-/sedoheptulose-1,7-biphosphatase in tobacco enhances photosynthesis and growth, Nature Biotechnol. 19 (2001) 965–969. [16] S. Shigeoka, R. Yasumoto, T. Onishi, Y. Nakano, S. Kitaoka, Properties of monodehydroascorbate reductase and dehydroascorbate reductase and their participation in the regeneration of ascorbate in Euglena gracilis, J. Gen. Microbiol. 133 (1987) 227–232. [17] S. Shigeoka, Y. Nakano, S. Kitaoka, Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase, Biochem. J. 186 (1980) 377–380. [18] K. Yoshimura, Y. Yabuta, T. Ishikawa, S. Shigeoka, Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses, Plant Physiol. 123 (2000) 223–233. [19] B. Halliwell, C.H. Foyer, Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography, Planta 139 (1978) 9–17.
M. Ohe et al. / Plant Science 168 (2005) 1487–1493 [20] J.M. McCord, I. Fridovich, Superoxide dismutase, an enzymatic function for erythrocuprein (hemocuprein), J. Biol. Chem. 244 (1969) 6049–6055. [21] K. Asada, M. Takahashi, M. Nagata, Assay and inhibitors of spinach superoxide dismutase, Agric. Biol. Chem. 38 (1974) 471–473. [22] T. Shikanai, T. Takeda, H. Yamauchi, S. Sano, K. Tomizawa, A. Yokota, S. Shigeoka, Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts, FEBS Lett. 428 (1998) 47–51. [23] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [24] D.I. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris, Plant Physiol. 24 (1949) 1–15. [25] S. Lutts, J.M. Kinet, J. Bouharmont, NaCl-induced senescence in leaves of rice (Oryza sativa L) cultivars differing in salinity resistance, Ann. Bot. 78 (1996) 389–398. [26] S. Trippi, K.V. Thimann, The exudation of solutes during senescence of oat leaves, Physiol. Plant. 58 (1983) 21–28. [27] N.J. Bate, S.J. Rothstein, J.E. Thompson, Expression of nuclear and chloroplast photosynthesis-specific genes during leaf senescence, J. Exp. Bot. 42 (1991) 801–811. [28] M.L. Salin, Toxic oxygen species and protective systems of chloroplast, Physiol. Plant. 72 (1988) 681–689.
1493
[29] G. Orendi, P. Zimmermann, C. Baar, U. Zentgraf, Loss of stressinduced expression of catalase 3 during leaf senescence in Arabidopsis thaliana is restricted to oxidative stress, Plant Sci. 161 (2001) 301– 314. [30] L.M. Casano, M. Martı´n, B. Sabater, Sensitivity of superoxide dismutase transcript levels and activities to oxidative stress is lower in mature-senescent than in young barley leaves, Plant Physiol. 106 (1994) 1033–1039. [31] M.A. Longa, L.A. del Rı´o, J.M. Palma, Superoxide dismutase of chestnut leaves Castanea sativa: characterization and study of their involvement in natural leaf senescence, Physiol. Plant. 92 (1994) 227– 232. [32] L.M. Casano, M. Martı´n, J.M. Zapata, B. Sabater, Leaf age- and paraquat concentration-dependent effects on the levels of enzymes protecting against photooxidative stress, Plant Sci. 149 (1999) 13–22. [33] C. Miyake, K. Asada, Inactivation mechanism of ascorbate peroxidase at low concentrations of ascorbate: hydrogen peroxide decomposes compound I of ascorbate peroxidase, Plant Cell Physiol. 37 (1996) 423–430. [34] J. Mano, C. Ohno, Y. Domae, K. Asada, Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR, Biochim. Biophys. Acta 1504 (2001) 275–287.