1、Resistance of Arabidopsis cpr5 mutants to photooxidation induced by H2O2HUANG Hong-Ying1，2 , SHU Zhan1, PENG Chang-Lian1* 1) Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, College of Life Sciences, South China Normal University, Guangzhou 510631,China; 2) Departme
2、nt of Chemistry and Life science, Xiangnan College,Chenzhou,Hunan, 423000,China Abstract: Chlorophyll fluorescence imaging and antioxidative capability in detached leaf discs of Arabidopsis cpr5 mutant and its wild type (Col) were investigated under photooxidation induced by exogenous H2O2. In compa
3、rison with wild type (WT) plant, photooxidation resulted in smaller decreases in fluorescence parameters (Fv/Fm， PS ，qP and NPQ) and the activities of SOD and APX, and an increase in cell membrane leakage rate in leaves of cpr5 mutant. After treatment for 240 min, PS in leaves of cpr5 mutant remaine
4、d 0.13, but nearly zero in WT. The sequence of sensitivity to photooxidation in leaves of two phenotypes were cpr5WT. The results indicated that cpr5 mutant exhibited higher antioxidative capability and more stable PSII than that in WT under photooxidative stress induced by exogenous H2O2. Key words
5、: Arabidopsis; cpr5 mutant; photooxidation; chlorophyll fluorescence imaging; antioxidative capacity.CPR5 gene is involved in several processes including signal transduction, cell proliferation , cell death and pathogendefence responses. CPR5 gene plays a repressive role in early processes for the i
6、nduction of general pathogendefence responses (Yoshida et al., 2002). CPR5 gene encodes a novel putative transmembrane protein containing five (pathogenesis-related genes) putative transmembrane helices at the C terminus. CPR5 is predicted to be a Type IIIa membrane protein, with the N-terminus bein
7、g cytoplasmatic (PSORT). In addition, a nuclear localization signal (NLS) is found at the N-terminus at position 40-56 (PSORT) (Kirik et al., 2001). Mutations in cpr5 have pleiotropic effects on the regulations of cell death, cell elongation, and trichome development (Kirik et al., 2001; Yoshida et
8、al., 2002).The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). SAR is a plant defense response that occurs after infection by an avirulent or other necrotizing pathogen and results in a long-lasting, nonspecific, systemic resistance to subs
9、equent pathogen infection (Ross, 1961; Kuc, 1982). The cpr5 rnutation is significantly smaller than the wild type. The cpr5 rnutation plants leaves were also found to the formation of spontaneous chlorotic lesions and a reduction in both trichome number and development, whereas wild-type leaves show
10、 no significant change(Bowling ,et al.1997). It is also found that a cpr5 mutant has spontaneous pathogendefence responses and constitutively expresses SAR, such as accumulation of reactive oxygen species, expression of PR-1 genes (pathogenesis-related gene 1) and elevated levels of SA, which might
11、be the potential inducers of senescence. (Boch et al., 1998; Bowling et al., 1997). Although leaf senescence and pathogen-defence responses are functionally mutually unrelated, both processes include common physiological events, such as increased levels of ethylene and salicylic acid (SA) (John et a
12、l., 1995; Morris et al., 2000; Ryals et al., 1996); accumulation of H2O2 (Levine et al., 1994; Pastori and Ro, 1997); and accumulation of transcripts from pathogenesis-related (PR) genes (Butt et al., 1998; Hanfrey et al., 1996; Pontier et al., 1999; Quirino et al., 1999; Quirino et al., 2000; Yoshi
13、da et al., 2001). Therefore, a cross-talk might exist between signaling pathways of leaf senescence and pathogen-defence responses.The influence of salicylic acid (SA) on plant growth, short-term acclimation to high light (HL), and on the redox homeostasis of Arabidopsis thaliana leaves have been re
14、ported recently (Mateo,et al.2006). However, little is known about the response of cpr5 mutant and its wild type to photooxidation induced by exogenous H2O2. In this study, effects of photooxidation induced by exogenous H2O2 on leaves of cpr5 mutant and its wild type (WT) were investigated. The meas
15、urement of corresponding fluorescence parameters was carried out with an IMAGING-PAM chlorophyll fluorometer (M- series, Walz, Germany). Seeds of Arabidopsis thaliana, ecotype Columbia (Col) and cpr5 mutant were obtained from the Arabidopsis Biological Resource Center in America. The detached leaf d
16、iscs (diameter: 5mm) were soaked in a solution of 100 mmol L-1 H2O2 (with 0.01 TritonX-100). The treatment was conducted in the growth chamber with the temperature of 25 , a light intensity of 90 molm-2s-1，and a relative humidity of 80 %. Chlorophyll fluorescence parameters (Fv/Fm， PS ，qP and NPQ) w
17、ere measured and their images were obtained simultaneously. Fv/Fm in leaves of Arabidopsis declined persistently during photooxidative treatment (Fig 1A). Within 0360 min of treatment, Fv/Fm in leaves of WT decreased at a rate of 0.1006/min, but only 0.046/min in cpr5 mutant. Clearly, the relatively
18、 rapid decrease of photochemical efficiency of PSII in WT revealed that cpr5 mutant had higher tolerance to oxidative stress than the WT plant. In addition, images of Fv/Fm in leaves of WT and cpr5 mutant exhibited heterogeneity when they were exposed to photooxidation(Fig 1C). During 360-min treatm
19、ent of photooxidation, images of Fv/Fm in WT faded gradually from blue (0.8) to red with partial orange (0.10.2), which indicated that the quantum efficiency of light energy transfer in PSII was close to 0.1. However, images of Fv/Fm in leaves of cpr5 mutant still retained blue color with partial gr
20、een, revealing PSII in leaves of cpr5 mutant was more stable; PS can reflect the actual efficiency of light energy capture under the close of partial reaction centers in PSII (Krall and Edward,1992). The changing tendency of PS under photooxidation induced by H2O2 in leaves of two phenotypes of arab
21、idopsis was different. PS in leaves of WT declined persistently during 360 min of treatment. However, PS in leaves of cpr5 mutant increased slowly in the early 30 min of treatment, then decreased sharply. Correspondingly imaging color of PS also faded from green (0.35) to red (0.1) at 180 min in WT
22、and at 360 min in cpr5 (Fig. 1B). The rapid decease in PS indcated that PSII was damaged severely by oxidative stress. Changes in these two chlorophyll fluorescence parameters (Fv/Fm and PS ) and their images(Fig. 1C,D) revealed that WT was damaged more severely than cpr5 mutant. The latter still re
23、tained higher inversion efficiency of light energy and PSII activity at the end of photoxidation treatment.qP (coefficient of photochemical quenching) is indicative of the proportion of open reaction centers in PSII (Genty et al., 1989.). The changing tendency of qP was compatible with that of PS (F
24、ig 1B). As shown in Fig 2A, qP in leaves of WT decreased continuously under photooxidation, indicating that the proportions of open reaction centers in PSII and electrons involved in CO2 fixation were decreased. However, qP in leaves of cpr5 mutant was increased slightly in the frist 30-min treatmen
25、t, then decreased persistently. During 360 min of photooxidative treatment, the capacities of photochemical quenching of PSII in leaves of Arabidopsis exhibited the sequence cpr5WT （P0.01). Change of imaging color was consistant with the numerical change of qP (Fig 2A). NPQ (non-photochemical quench
26、ing) is an effective index to reflect the dissipation capability of heat energy in plants (Hartel and Lokstein, 1995). A interested phenomenon was observed during 360 min of photooxidative treatment. The fluorescence imaging of NPQ appeared with three phases : rapid fall (060 min) slight fluctuation
27、 (6090 min) slow fall (90360 min) (Fig 2B). The drastic decrease in NPQ indicated the lose of capability to dissipate heat energy and the photo-protective potentials of both phenotypes induced by H2O2 in the light. As outline in qP and NPQ, a conclusion was obtained that the sensitivity of PSII to p
28、hotooxidation was present by wt cpr5 （qP: P0.01；NPQ: P0.05 Membrane permeability is a relevant index that reflects the degree of impaired membrane function. The higher the membrane permeability rate is, the more severe the cell membrane is damaged. Fig 3A showed changes in cell membrane permeability
29、 rates in leaves of Arabidopsis under phtotooxidation. After exogenous H2O2-induced photooxidative treatment for 6h, membrane permeability rates were increased noticeably, which suggested that their plasma membranes had been damaged. Increasing magnitude in cpr5 mutant was significantly different fr
30、om that of WT（P0.01. By 360-min treatment, plasma membrane permeability rates of leaf cells in two phenotypes of arabidopsis were increased to 2.85 times（ WT）and 2.03 times (cpr5) of that before treatment, respectively. Then activities of two antioxidative enzymes, SOD（Giannopolitis andRies , 1977）a
31、nd APX（Shen et al., 1996）were determined in leaves of two phenotypes. Before photooxidation treatment, the activity of SOD exhibited little difference between cpr5 and WT, whereas activity of APX is higher in cpr5 than in WT. However, the activities of SOD and APX decreased more significantly in WT
32、than that in crp after 360 min photooxidation treatment5 (Fig 3B and 3C). It is thus evident that cpr5 mutant possessed higher antioxidative ability and more stable cell membrane system than the WT under oxidative states condition. This is in accordance with the observation of fluorescence parameter
33、s.Production of excessive reactive oxygen species (ROS) causes severe oxidative stress in chloroplasts and leaf cells, finally induces photooxidative injury. In the present study, photooxidation induced by exogenous H2O2 resulted in the increase of electrolytes leakage rate and the decrease of chlor
34、ophyll fluorescence parameter (Fv/Fm, PS , qP, and NPQ) in leaves of cpr5 mutant and WT, which suggested that photosynthetic apparatus had been obviously damaged. Salicylic acid (SA) was accumulated in cpr5 mutant (John et al., 1995; Morris et al., 2000; Ryals et al., 1996). The activities of antiox
35、dative enzymes could be enhanced by SA treatment. Rao et al (1997) reported that SA treatments increased activity of Cu, Zn-SOD but inactivated catalase (CAT) and APX. The activities of both SOD and APX increased whereas CAT activity decreased under SA treatment in grape seedling (Wang et al 2003).
36、Our results showed that the activity of APX significantly increased in cpr5 and there is no difference in activity of SOD between WT and cpr5 (Fig 3). We proposed that the accumulation of SA possibly induced the increment of APX activity in cpr5. APX is the main enzyme located at chloroplast to degr
37、ade H2O2. This is a possible reason that cpr5 exhibited higher tolerance to photooxidation induced by H2O2 than WT. On the other hand, SA treatment could induce the resistance of plants to environmental stresses by increment the content of antioxidative substance such as GSH (Mateo et al 2006), AsA
38、(Shi et al 2004), and polyphenolic compound (Cevahir et al 2005). So we presumed that the content of antioxidative substance possibly increased in cpr5 mutant due to its accumulation of SA. These antioxidative substance can eliminate ROS to protect PSII against photooxidation induced by H2O2. This m
39、ay be another reason for cpr5 mutant in possession of higher tolerance to photooxidation. In a word, it was very clear that in comparison with WT, PSII in leaves of cpr5 mutant was more stable under photooxidative stress induced by exogenous H2O2, and cpr5 mutant exhibited higher antioxidative capab
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