Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence

ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 1572—1582

www.elsevier.de/jplph

Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence Hilda Araceli Zavaleta-Manceraa,, Humberto Lo ´pez-Delgadob, Herminia Loza-Taverac, Martha Mora-Herrerab, Claudia Trevilla-Garcı´ad, Martı´n Vargas-Sua ´rezc, Helen Oughame a

Programa de Bota´nica, Colegio de Postgraduados, Km. 36.5 Carr. Me´xico-Texcoco, Montecillo, Edo. Mex. 56230, Me ´xico b Programa Nacional de Papa, Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias (INIFAP), Metepec, Edo. Mex. 52142, Me´xico c Departamento de Bioquı´mica y Biologı´a Molecular de Plantas, Facultad de Quı´mica, Universidad Nacional Auto ´noma de Me ´xico. D.F. 04510, Me´xico d Facultad de Ciencias, Universidad Auto ´noma del Estado de Me ´xico, Toluca, Edo. Mex. 50000, Me´xico e Plant Genetics and Breeding Department, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, Wales, UK Received 5 September 2006; accepted 26 February 2007

KEYWORDS Ascorbate peroxidase; Catalase; Chloroplast; Cytokinin; ROS

Summary Increased oxidative stress displayed during dark-senescence of wheat leaves (Triticum aestivum L.) is caused not only by the increased levels of radicals but also by a loss of antioxidant capacity. Mature leaves were incubated in 6benzylaminopurine (BAP 104 M) or water (control) during 6 d in the dark. The senescence-delaying effect of BAP was associated with the retention of the chloroplast structure, 60% of the initial content of chlorophyll (Chl) and 77% of the initial content of protein. BAP reduced the degradation of the light-harvesting chlorophyll a/b binding protein (LHCP-2), and the large (LSU) and small subunits (SSU) of Rubisco. Our results indicated that the presence of the NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1) was not promoted by the cytokinin, leading to the conclusion that BAP maintains the level of Chl, preventing its degradation, rather than inducing Chl biosynthesis. The internal structure of

Abbreviations: APX, ascorbate peroxidase; BAP, 6-benzylaminopurine; CAT, catalase; Chl, chlorophyll; LHCP-2, light-harvesting chlorophyll a/b binding protein of photosystem 2; LSU, large subunit of Rubisco; POR, NADPH:protochlorophyllide oxidoreductase; SSU, small subunit of Rubisco; ROS, reactive oxygen species Corresponding author. Tel.: +52 595 95 20200x1362; fax: +52 55 595 95 2 0247. E-mail address: [email protected] (H.A. Zavaleta-Mancera). 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2007.02.003

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chloroplasts was maintained in BAP-treated leaves for up to 6 d, with well-organized grana thylakoids and small plastoglobuli; in contrast, chloroplasts of control leaves deteriorated rapidly from day 4 with disorganized internal membranes, and more and larger plastoglobuli. BAP increased the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) and reduced the level of H2O2 in the delayed-senescence tissue. The present research indicates that BAP reduces levels of reactive oxygen species (ROS), and enhances the activity of antioxidant enzymes (CAT, APX). Our results suggest that BAP protects the cell membranes and the photosynthetic machinery from oxidative damage during delay of senescence in the dark. & 2007 Elsevier GmbH. All rights reserved.

Introduction Leaf senescence is normally the last stage of leaf development from the fully expanded leaf state until death. Senescence is a sequence of structural, biochemical and physiological events, under a well regulated genetic program (Buchanan-Wollaston et al., 2003). Chlorophyll (Chl) breakdown leads to the yellowing of the blade, the main symptom of senescence (Matile et al., 1996). The chloroplast is the first organelle in the green cell to shows signs of decay (Biswal and Biswal, 1988), characterized by the dissociation of grana, increase in size and number of plastoglobuli and disruption of the chloroplast envelope (ZavaletaMancera et al., 1999a; Biswal et al., 2003). It is recognized that exogenous application of cytokinin delays degradation of Chl and photosynthetic proteins (Gan and Amasino, 1996) and decreases CO2 assimilation (Rulcova ´ and Pospı´silova ´, 2001). There is evidence that natural endogenous cytokinin levels decrease during dark-induced senescence in Cucurbita cotyledons and natural senescence in maize leaves (He et al., 2005). Some evidence indicates that cytokinins regulate the accumulation and expression of several thylakoid and stroma proteins, in Lupinus luteus cotyledons (Kusnetsov et al., 1998), etiolated cotyledons of watermelon (Longo et al., 1990) and wheat under water deficiency (Monakhova and Chernyade `v, 2004). Plants transformed with the bacterial cytokinin biosynthesis gene isopentenyl transferase (IPT) exhibited delay of leaf senescence in tobacco, maize, Arabidopsis, broccoli and other species (Smart et al., 1995; Gan and Amasino, 1995, 1996; Li et al., 2004). The reversal of leaf senescence of Nicotiana rustica, promoted by external application of 6-benzylaminopurine (BAP) under dim light, is one of the most striking pieces of evidence that leaf senescence follows a genetic program that can be regulated by cytokinins (Zavaleta-Mancera et al., 1999a, b).

In most plants the rate of CO2 fixation in the chloroplast is not high enough to convert the light energy received, therefore alternative acceptors lead to the formation of reactive oxygen species (ROS), such as superoxide radicals (O 2 ), singlet oxygen (1O2), hydroxyl radical (OH) and hydrogen peroxide (H2O2) (Foyer and Noctor, 2005; Zimmermann and Zentgraf, 2005). In addition, H2O2 is formed by the mitochondrial electron transport chain and photorespiration in the peroxisomes (Zimmermann and Zentgraf, 2005). During growth and development ROS are produced and their concentration increases during senescence, when ROS oxidize proteins, unsaturated fatty acids and DNA, resulting in membrane leakiness, cellular damage and cell death (Lin and Kao, 1998; Neill et al., 2002). Plant cells need to have protective mechanisms by which they respond to oxidative stress: (a) non-enzymatic antioxidants such as ascorbate and glutathione and (b) enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) (Navabpour et al., 2003; Zimmermann and Zentgraf, 2005). However during senescence the general antioxidant status of the leaf is diminished; levels of ROS are enhanced (Srivalli and KhannaChopra, 2004) and many antioxidant enzymes reduce their activity (del Rı´o et al., 1998; Prochazkova et al., 2001). In stay green maize cv. P3845 the delay of senescence was associated with higher CAT and SOD levels (He et al., 2005), but in the delayed leaf senescence mutants of Arabidopsis ore1, ore3 and ore9, resistance to oxidative stress was not due to enhanced activities of antioxidant enzymes (Woo et al., 2004). If leaf senescence is associated with the production of ROS, and cytokinins delay this process, then BAP might regulate the oxidative status of the tissue. In the present research wheat is studied as a model to explore the effect of BAP on the levels of H2O2, and the activity of the antioxidant enzymes CAT and APX, during delay of leaf senescence in the dark.

ARTICLE IN PRESS 1574 Chloroplast structure, levels of photosynthetic pigments (Chl a, Chl b and carotenoids), levels of chloroplast proteins (light-harvesting chlorophyll a/b binding protein of photosystem 2 (LHCP-2), NADPH:protochlorophyllide oxidoreductase (POR), large (LSU) and small subunits (SSU) of Rubisco) and electrolyte leakage percentages were studied to monitor the senescence-delaying effect of BAP. The possible role of cytokinins in the control of oxidative stress during leaf senescence is discussed.

Materials and methods Plant material Wheat (Triticum aestivum L.) seeds of the Mexican cv. Temporalera were provided by the National Institute of Forestry, Agricultural and Livestock Research (Instituto Nacional de Investigaciones Forestales Agrı´colas y Pecuarias, INIFAP) of the Mexican Ministry of Agriculture. Plants were grown in plastic trays (33  28  13 cm) containing a mixture of soil and peat-moss (Cosmocel) under greenhouse conditions, at 18–25 1C under a 16 h natural light period, over a period of 2172 d, until the second leaf was fully expanded and ligulated.

Delay of senescence Segments 5–6 cm long from the middle part of the second mature leaf were disinfected with a 0.15% (w/v) solution of sodium hypochloride and rinsed twice with distilled water. Delay of senescence was promoted by incubation of 4 segments per petri dish containing filter paper (Whatman No. 40) soaked with 5 mL of an aqueous cytokinin solution (BAP 10–4 M, Sigma) with 0.02% (v/v) Tween-20, in the dark at 20 1C for 2, 4 and 6 d. Control segments were incubated in the same way but without BAP in the solution.

Pigment measurements Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids including xanthophylls (x+c) were measured in 80% acetone extracts as described by Lichtenthaler and Wellburn (1983). Determinations were performed on twenty leaves per treatment (n ¼ 20).

Analysis of leaf proteins Three hundred milligrams of leaf tissue were ground with liquid N2, then 0.3 mL of extraction

H.A. Zavaleta-Mancera et al. buffer (0.1 M Tricine pH 8.1, 10 mM MgCl2, 10 mM NaHCO3, 5 mM EDTA, 10 mM dithiothreitol, 1.0 mM phenazine methosulphate, 2 mM benzamidine, and 0.01 mM leupeptin) were added and centrifuged for 10 min at 18 600g at 4 1C. Total protein was determined according to Bradford (1976). Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a Bio-Rad Mini Trans-Blot cell (Towbin et al., 1979). Immunological detection of proteins was carried out by standard procedures. The levels of the SSU, 12 kDa, LSU, 54 kDa of Rubisco, LHCP-2, 25 kDa and the POR, EC.1.6.99.1 of 36 kDa were investigated by immuno gel blot and densitometry. The antibodies against Festuca pratensis LHCP-2, Lolium perenne SSU and Lolium perenne LSU, and Arabidopsis POR were used at dilutions of 1:800; 1:2000, 1:5000; and 1:20 000, respectively. Peroxidase-conjugated swine anti-rabbit immunoglobulin (DAKO, UK) was used as secondary antibody at a 1:2000 dilution. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) following the kit manufacturer’s protocol (Amersham). Total protein determinations were performed on nine leaves per treatment (n ¼ 9), with two measurements per leaf.

Measurements of ion leakage The relative intactness of the plasma membrane was measured as the leakage percentage of electrolytes, as described by Jiang and Zhang (2001). Fresh leaves (0.1 g) were cut into pieces of 0.5 cm length, washed with distilled deionized water (DDW) and placed in glass vials containing 10 mL of DDW. The vials were incubated in a water bath at 30 1C for 2 h and the initial electrical conductivity (mV) in the medium was measured. The samples were boiled at 100 1C for 10 min to release all electrolytes, cooled and the final electrical conductivity was measured. Relative electrolyte leakage was calculated as percentage of electrolyte leakage after boiling. Three readings per leaf of each of five leaves per treatment were averaged (n ¼ 5).

Light and electron microscopy Tissue pieces (1–2 mm2), from the middle part of the leaf, were fixed in 5% (v/v) glutaraldehyde in 0.16 M phosphate-saline buffer (PBS) pH 7.2 for 2 h at room temperature. The tissue was postfixed in buffered 1% (w/v) OsO4 for 2 h at room temperature, and embedded in the epoxy resin Glycitether 100 (Merck). Semi-thin (2 mm) leaf cross-sections were stained with 0.5% toluidine blue. Silver/gold

ARTICLE IN PRESS Cytokinin promotes catalase and ascorbate peroxidase in dark-senescence (80–100 nm) sections were cut on an MT2 ultramicrotome (Sorvall), counter-stained with 5% (w/v) uranyl acetate for 15 min followed by a 3% (w/v) lead citrate solution for 3 min (Hayat, 2000), and examined with the electron microscope EM10 C (Zeiss), at 80 kV. Images were recorded on Kodak Electron Microscope Film.

Ascorbate peroxidase activity (APX EC 1.11.1.11) Leaf tissue (0.5 g) was ground with liquid N2. Soluble protein was extracted by homogenizing the powder in 2 mL of extraction buffer (100 mM potassium phosphate pH 7.1 mM dithiothreitol (DTT), 5 mM ascorbate). Insoluble materials were removed by centrifugation at 11 000g for 15 min at 4 1C. The activity of APX was determined according to Jime´nez et al. (1997), with some modification. The reaction mixture (2 mL) contained 50 mM potassium phosphate pH 7.0, 0.1 mM EDTA, 0.1 mM ascorbate, 2.7 mM H2O2 and 40 mL of crude enzyme extract. The reaction was initiated by the addition of H2O2. The decrease in absorbance due to the oxidation of ascorbate was determined at 290 nm every 30 s for 3 min at 26 1C.

Catalase activity (CAT, EC 1.11.1.6) The enzyme extraction was performed as described for APX but using 50 mM potassium phosphate buffer pH 7.2, containing 5 mM DTT, 1 mM EDTA and 1% (w/v) PVP (Anderson et al., 1995). The activity of CAT was determined according to the method of Aebi (1984). The total reaction mixture (3 mL) contained 50 mM potassium and sodium phosphate pH 7.0 and 20 mL of enzyme extract. The reaction was initiated by the addition of 30 mM H2O2. The decomposition was followed directly by the decrease in absorbance at 240 nm every 20 s for 3 min at 26 1C.

Determination of H2O2 content Tissue samples (0.2 g) were ground to a fine powder in liquid N2, homogenized in 1.2 mL icecold 5% (w/v) TCA, and centrifuged (20 min at 1400g). Samples (0.5 mL) of the supernatant fractions were eluted through a Dowex-1 resin (0.5 g, Fluka) column with 3.5 mL of 5% (w/v) TCA. H2O2 was measured in the eluates using the luminoldependent chemiluminescence method of Warm and Laties (1982). Samples (0.5 mL) of eluate were added to 0.5 mL of 0.5 mM luminol. The volume was adjusted to 4.5 mL with 0.2 M NH4OH pH 9.0.

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Aliquots (0.45 mL) of this mixture were analyzed in an Optocomp P luminometer (MGM Instruments, USA) for the chemiluminescence produced over 5 s following an injection (50 mL) of 0.5 mM potassium ferricyanide in 0.2 M of NH4OH pH 9.0. A parallel sample of each initial extract was processed after addition of a known concentration of H2O2 to provide a recovery correction factor (Warm and Laties, 1982). Determinations of APX, CAT and H2O2 were performed on the same samples. Data for those variables are expressed as means of three samples per treatment of three experiments (n ¼ 9), each measured in triplicate.

Statistical analysis Significance of data was tested by analysis of variance and Duncan’s Multiple Range Test (Duncan, 1955) at Pp0.05.

Results Pigments and photosynthetic proteins The retention of chlorophylls and proteins in the presence of cytokinins reflects the delay of senescence. Control leaves started to lose chlorophyll and proteins at day 4, earlier than the BAP treated leaves. At day 6, BAP treated leaves had retained 60% of Chl and 77% of leaf protein (Fig. 1A). The Chl a:b ratio of 3.14 in pre-senescent leaves (day 0) changed to 2.17 in 6 d BAP treated leaves. These results indicated that Chl b was the better retained pigment, with 78% in comparison to Chl a (54%) (Table 1). Leaves treated with BAP retained significantly more carotenoids (x+c: 0.2870.01 mg  g FW1) than control leaves (0.2370.02 mg  g FW1). However the initial x+c:total Chl ratio (0.18) in green leaves was maintained (0.20) in BAP treated leaves after 6 d (Table 1), data related to the retention of the green color during the delay of senescence. The POR protein was immunodetected most strongly in etiolated coleoptiles and foliage leaves of wheat (Fig. 2A) due to its abundance in the prolamellar body of etioplasts (Ougham et al., 2001), the level of this band was therefore set at 100% (Fig. 2A). In green leaves, little (13.7%) POR was found, as a normal characteristic of green mature tissues (Ougham et al., 2001) but this protein was not further detected during senescence (Fig. 2A). The BAP treated leaves retained 52.1% of LHCP-2, 54.7% of LSU, and 62.4% of SSU in

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Total chlorophyll (mg·g FW -1)

comparison with original levels after 6 d. In contrast, the amounts of these proteins dropped drastically from day 4 in senescent (control) tissue (Fig. 2B–D).

Control

2.0

BAP *

1.6

*

1.2 0.8 0.4 0.0

G-0

2

6

4

Chloroplast structure Electron microscopy showed that cytokinin treatment resulted in retention of chloroplast structure during senescence. Chloroplasts of pre-senescent leaves (day 0) showed a typical oval shape, with an intact double envelope, well-organized grana thylakoids and stroma membranes, high electron opacity of the stroma and small plastoglobuli (Fig. 3A). At day 2, the control and BAP treated leaves showed similar characteristics to green chloroplasts (Fig. 3B). At day 4 the control chloroplasts became smaller and rounder, with less electron opacity of the stroma, swollen and dispersed thylakoids and numerous and large plastoglobuli (Fig. 3G); in contrast BAP chloroplasts appeared intact (Fig. 3C). At day 6 no chloroplasts were observed in the control mesophyll cells (Fig. 3H); in contrast, 6 d BAP chloroplasts kept their shape and the internal membrane organization in grana and stroma thylakoids, with few plastoglobuli (Fig. 3D).

Days

-1

Total protein (mg·g FW )

9.0

control

8.0

*

7.0

Antioxidants and oxidative profiles

BAP *

6.0 5.0 4.0 3.0 2.0 1.0 0.0

G-0

2 Days

4

6

Figure 1. Effect of BAP on chlorophyll and total leaf protein, during 6 d of senescence in the dark: (A) total chlorophyll, data are means7SE (n ¼ 20); (B) total proteins, data are means7SE (n ¼ 9) with two replicates. G-0, green pre-senescent stage (0 d). (*) significantly different from the control (Pp0.05).

After 4 and 6 d of incubation, leaves in control solution showed significantly reduced CAT activity in comparison with the initial green stage and the 2 d control. However, BAP incubated leaves kept significantly higher CAT activity compared with the respective controls after 4 and 6 d; the activity after 6 d of senescence was unaffected, relative to that of the initial green stage (Fig. 4A). Leaves incubated in BAP showed significantly higher APX activity after 4 and 6 d of incubation relative to the respective controls, especially at day 4. The APX activity in BAP treated leaves was slightly but significantly higher at the end of the treatment compared with the initial green tissue (Fig. 4B). A significantly lower H2O2 content was measured in the BAP treated leaves relative to the respective controls. This difference was maintained from the beginning of the treatment up to day 6 (Fig. 4C).

Table 1. Changes in chlorophyll a (Chl a), chlorophyll b (Chl b), xanthophylls+carotenoids (x+c) and some ratios, from wheat leaves incubated in BAP 104 M or control solution (water) during 6 d of senescence in the dark. Days

0 2 4 6

Chl a (mg g FW1)

Chl b (mg g FW1)

x+c (mg g FW1)

Ratio Chl a: Chl b

Ratio x+c:total Chl

Control

BAP

Control

BAP

Control

BAP

Control

BAP

Control

BAP

1.5770.05 1.2970.07 0.8770.07 0.4870.06

1.5770.05 1.3570.06 1.1270.07 0.8570.05

0.5070.02 0.4370.02 0.3270.02 0.2170.02

0.5070.02 0.4670.01 0.4470.03 0.3970.02

0.3770.01 0.3270.01 0.2770.01 0.2370.02

0.3770.01 0.3370.02 0.2970.02 0.2870.01

3.14 3.0 2.71 2.31

3.14 2.93 2.54 2.17

0.17 0.19 0.22 0.33

0.17 0.18 0.18 0.22

Data are mean of 20 leaves7SE.  Indicates significant differences with respect to the control, Pp0.05.

ARTICLE IN PRESS Cytokinin promotes catalase and ascorbate peroxidase in dark-senescence E

G-0

C2

B2

C4

B4

C6

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A

B6

POR 100

13.7

---

---

---

---

---

---

(%)

B LHCP-2

100

53.2

49.5

13.8

19.4

0.8

12.4 (%)

C LSU

100

83.5

64.9

34.6

57.7

20.2

54.7

(%)

D SSU

100

91.4

78.9

69

69.3

20.8

62.4 (%)

Figure 2. Inmunoblot analysis of four chloroplast proteins from leaves treated with BAP 104 M during 6 d of senescence in the dark: (A) NADPH:protochlorophyllide oxidoreductase (POR 36 kDa); (B) light harvesting chlorophyll a/b binding protein of PS-2 (LHCP-2 of 25 kDa); (C) large subunit (LSU 54 kDa); and (D) small subunit (SSU 12 kDa) of Rubisco. G-0, green pre-senescent stage (d 0); C2, C4, C6 control leaves of 2, 4 and 6 d; B2, B4, B6, BAP treated leaves for 2, 4, and 6 d. Relative band intensities are indicated as % of the strongest band for each protein.

Membrane leakage The oxidation of lipids and proteins during membrane injury by ROS was evidenced by plasma membrane electrolyte leakage (Fig. 5). Senescing tissue (control) showed a marked increase of ion leakage, from 11.1% to 38.9%, indicating membrane damage. In contrast BAP treated leaves maintained low electrolyte leakage percentages (11.1%, 14.2%, 19.4% and 19.6%) at 0, 2, 4 and 6 d, respectively, evidence of the protective role of the cytokinin against membrane damage.

Discussion The current paper confirmed that BAP delays senescence in wheat (Xie et al., 2004) in the dark, as has been demonstrated also in broccoli florets (Costa et al., 2005), maize (He et al., 2005) rice (Ookawa et al., 2004). Sixty percent of initial levels of chlorophylls were retained, as was 77% of total leaf protein. In our study BAP promoted a preferential retention of Chl b, as in the ‘‘stay green’’ mutant cytG of soybean, where Chl b was more stable than Chl a (Guiamet et al., 1991). This contrasts with previous observations on the ‘‘stay green’’ Festuca/Lolium where Chl b was as labile as it was in the wild type (Thomas et al., 2002). Degradation of Chl a during senescence is well documented; chlorophyllase is the first enzyme to open the porphyrin macrocycle ring of Chl a;

however, it is assumed that Chl b is converted to Chl a before degradation (Matile et al., 1996). It has been demonstrated that cytokinin treatment reduced activity of chlorophyllase, Mg-dechelatase and peroxidase-linked chlorophyll bleaching in broccoli florets during postharvest (Costa et al., 2005). Therefore a similar mechanism is proposed in the present research for Chl retention but the high levels of Chl b may be related to the maintenance of the LHCP-2 protein, where Chl b is preferentially attached (Green, 1988). Analyses of the time-course of POR protein showed that BAP did not promote the accumulation of this chlorophyll biosynthesis enzyme in wheat (Fig. 2), in contrast to Nicotiana rustica, where regreening induced by BAP was mediated by the reappearance of POR and biosynthesis of new chlorophyll (Zavaleta-Mancera et al., 1999a). In the case of Triticum aestivum, BAP maintained levels of Chl, preventing its degradation rather than inducing Chl biosynthesis. The response of senescing tissue to cytokinins differs between gramineous and dicot species, this might be related to the reduced cell plasticity and totipotency observed in cell tissue culture of Gramineae species (Vasil and Vasil, 1992; Sharma et al., 2005). Gel protein blots indicate that the cytokinin BAP retained 52.1% of LHCP-2, 54.7% of LSU, and 62.4% of SSU, after 6 d of incubation. This observation agrees with previous reports in several species (Longo et al., 1990; Abdelgani et al., 1991; Chory et al., 1994; Kusnetsov et al., 1998). The retention

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Figure 3. Structure of plastids and mesophyll from BAP-treated (A–D) and control leaves (E–H), during delay of senescence. (A and E) Pre-senescent green leaf 0 d, (B) 2 d, (C) 4 d and (D) 6 d of BAP-treated leaves. (F) 2 d, (G) 4 d, and (H) 6 d of control leaves. g, grana; p, plastoglobuli; s, stroma; t, thylakoids; n, nucleus; vb, vascular bundle. Bars A–D and F–G ¼ 1 mm; Bars E and H ¼ 20 mm.

of the major thylakoid protein LHCP-2 conserved thylakoid membrane structure, whereas disappearance of this protein was accompanied by deterioration of internal chloroplast membranes. Control chloroplasts developed a typical gerontoplast appearance, characterized by disassembled grana thylakoids and numerous plastoglobuli (Biswal and Biswal, 1988).

Relatively few studies have addressed the relationship between oxidative stress and control of senescence by BAP. Increased proliferation of potentially toxic ROS is a common denominator of plant senescence, where chloroplasts are the main source of free radicals (Hodges and Forney, 2000). Also ROS have been found to increase the expression of senescence-enhanced genes

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Control

BAP

1000 *

800

Electrolyte leakage (%)

CAT activity (nmol·min·mgFW -1)

40 1200

*

600 400 200 0

G-0

2

4

APX activity (nmol·min·mgFW -1)

BAP

2

4

6

*

5

G-0

2

4

6

Days

H2O2 content (µmol·gFW -1)

0

Figure 5. Effect of BAP on membrane electrolyte leakage percentage during 6 d of leaf senescence in the dark. Three readings per leaf of each of five leaves per treatment were averaged (n ¼ 5). Leaves measured at day 0 were at the green pre-senescent stage.

*

10

Control

BAP

4 3

*

*

*

2 1 0

10

Days

15

5

20

0

20

0

Control

6

Days

Control

BAP

30

G-0

2 Days

4

6

Figure 4. (A) Catalase (CAT) activity, (B) ascorbate peroxidase (APX) activity and (C) hydrogen peroxide (H2O2) content in G-0, green pre-senescent stage; BAP and control (water) treated leaves at 2, 4 and 6 d. Data are means7SE bars of three samples per treatment of three experiments (n ¼ 9), each measured in triplicate. (*) significantly different from the control (Pp0.05).

(Navabpour et al., 2003). The increased levels of ROS displayed during senescence are caused not only by the elevated production of free radicals but also by a loss in antioxidant capacity (Srivalli and

Khanna-Chopra, 2004). In the present work, control wheat leaves showed a reduction in CAT activity, similar to the reduction observed in senescence of spinach leaves (Hodges and Forney, 2000) and early senescence of ginkgo and birch leaves (Kukavica and Jovanovic, 2004). The cytokinin BAP not only kept the chlorophyll levels significantly higher, but also resulted in higher activities of the antioxidant enzymes CAT and APX relative to the respective controls: CAT was kept at similar levels over the whole 6 d but APX increased dramatically at day 4, keeping this significant effect up to day 6. Similarly high levels of CAT were involved in the senescencedelaying process of muskmelon fruits (Lacan and Baccou, 1998). Our results suggest that BAP may be protecting the cell from oxidative damage, preventing peroxidation of fatty acids in the membrane, lipid that would otherwise lead to membrane leakage and cell death (Srivalli and Khanna-Chopra, 2004; Scandalios, 2005). ROS have been implicated in the direct or indirect degradation or bleaching of Chl (del Rı´o et al., 1998; Toivonen and Sweeney, 1998); thus it should not be surprising that the retention of chlorophyll and low content levels of H2O2 coincide in the delayed senescence of wheat leaves treated with BAP. In contrast leaves not treated with BAP showed higher levels of H2O2 and degradation of chlorophyll, proteins and chloroplast membranes. These observations are consistent with reports in which high levels of H2O2 and free radicals are associated with promotion of senescence where lipid peroxidation contributes to membrane

ARTICLE IN PRESS 1580 degradation (Lin and Kao, 1998; Hodges and Forney, 2000; Navabpour et al., 2003). Furthermore H2O2 accumulation has been considered necessary for senescence of rice leaves (Hung and Kao, 2004). There is some evidence that chloroplast proteins become non-functional due to interactions with oxygen species and degradation by proteolysis (Lindahl et al., 2000). Previous experiments, performed in Spirodela oligorrhiza, Chlamydomonas, T. aestivum and cucumber leaves, led to reports that Rubisco activity and stability in vivo were very susceptible to oxidative damage, resulting in: (a) an intermolecular crosslinking of LSU by bisulfide bonds within the holoenzyme, (b) rapid and specific translocation of the soluble enzyme complex to the chloroplast membranes, and finally (c) protein degradation (Mehta et al., 1992; Ishida et al., 1998; Nakano et al., 2006). Since APX is expressed in chloroplasts and is the primary H2O2 scavenging enzyme in this organelle (Asada, 1992), we suggest that the effect of BAP in preventing degradation of LHCP-2, LSU and SSU during senescence of wheat might be due to the accumulation of APX in the chloroplasts, which would protect against protein degradation. Both control and BAP treated leaves showed the highest values of APX activity on day 4; this was particularly so in the case of the BAP treated leaves, in which the chloroplast proteins, pigments and ultrastructure were well preserved. Lesser ion leakage in the BAP treated leaves, and lower H2O2 levels, suggest maintenance of membrane integrity in contrast to controls. It has been demonstrated recently that electrolyte leakage measurements may be correlated with antioxidative enzyme synthesis and membrane acyl lipid concentrations (Liu and Huang, 2000). It is well known that an increase in the endogenous H2O2 levels promotes senescence (Hung and Kao, 2004; Hossain et al., 2006; Vanacker et al., 2006). Reduction of H2O2 levels is one of the effects of BAP that leads to delayed senescence. Furthermore, BAP enhanced significantly the APX which has been reported as lowered during senescence (Hossain et al., 2006). The BAP could take part in the delay of the oxidative damage not only by inducing the activity of some antioxidative enzymes, but also by increasing the content of xanthophylls (Table 1). An increase in the content of xanthophylls could delay the onset of oxidative damage in wheat, and suppress the overexcitation of the photosynthetic apparatus (Vlcˇkova ´ et al., 2006). Panchuk et al. (2005) found that, while mRNA levels for some APX isoforms decline during leaf senescence in Arabidopsis thaliana, other isoforms, present in the

H.A. Zavaleta-Mancera et al. stroma of chloroplasts, seem to be unchanged or decline very late. The same authors concluded that expression of the individual APX genes is differentially regulated and coordinated during senescence, suggesting a possible functional specialization of the corresponding isoenzymes, in a complex regulatory network in different cell compartments. Similarly, it was suggested that a programmed down-regulation of APX enzyme activity may increase the endogenous H2O2 level, which seems to be the prerequisite for initiating senescence in gladiolus (Hossain et al., 2006). BAP increased activities of APX and CAT activities at the crucial 4-d stage when chloroplast senescence becomes obvious in control tissue. We conclude that the antioxidant system plays an important role in the protective effect of BAP against damage of cell membrane and the photosynthetic machinery during leaf senescence in the dark.

Acknowledgments This work was supported by the Mexican Council of Science and Technology, CONACYT (grant 43866/ A-1). IGER is grant-aided by BBSRC. We are grateful to Dr. Luis Felipe Jime´nez Garcı´a, Facultad de Ciencias UNAM, for electron microscopy facilities.

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