Enhanced sensitivity of Arabidopsis anthocyanin ... - CSIRO Publishing

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Functional Plant Biology, 2008, 35, 714724

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Enhanced sensitivity of Arabidopsis anthocyanin mutants to photooxidation: a study with fluorescence imaging Ling Shao A,B,E, Zhan Shu A,E, Chang-Lian Peng A,F, Zhi-Fang Lin C, Cheng-Wei Yang A and Qun Gu D A

College of Life Science, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, South China Normal University, Guangzhou 510631, China. B College of Life Science, Zhao Qing University, Zhaoqing 526061, China. C South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. D Zealquest Laboratory for Ecological Research, Zealquest Scientific Technology Co. Ltd, Shanghai 200333, China. E These authors contributed equally to this work. F Corresponding author. Email: [email protected]

Abstract. Chlorophyll fluorescence imaging and antioxidative capability in detached leaves of the wild-type Arabidopsis thaliana ecotype Landsberg erecta (Ler) and in three mutants deficient in anthocyanin biosynthesis (tt3, tt4 and tt3tt4) were investigated under photooxidation stress induced by methyl viologen (5 mM) in the light. In comparison with the wild-type (WT) plant, photooxidation resulted in significant decreases in the contents of total phenolics and flavonoid, total antioxidative capability and chlorophyll fluorescence parameters (Fv/Fm, qP, FPSII, NPQ and ETR) as determined by chlorophyll fluorescence imaging, and in an increase in cell-membrane leakiness in the three anthocyanin mutants. The sequence of sensitivity to photooxidation in the leaves of the four phenotypes were tt3tt4 (deficient in both chalcone synthase locus (CHS) and dihydroflavonol 4-reductase locus (DFR)) > tt4 (deficient in CHS) > tt3 (deficient in DFR) > WT. The results demonstrate that anthocyanins might, along with other antioxidants, protect the photosynthetic apparatus against photooxidative damage. An interesting phenomenon was observed over the 270 min of the photooxidative treatment, that is, fluorescence imaging revealed that qP, FPSII and ETR appeared in three phases (fall ! partial recovery ! rapid fall). This was considered to be a modulation of reversible deactivation in PSII to cope with the moderate oxidative stress in the first two stages of short-term treatment ( tt4 > tt3tt4 during 60120 min of photooxidative treatment (P < 0.01). As shown in Fig. 7, over 0165 min of photooxidative treatment, FPSII and NPQ at first had a tendency to change in opposite directions, but then showed a similar decrease. The changing tendency of FPSII was compatible with that of qP (Fig. 5b). After treatment for 45 min, FPSII sharply decreased to a minimum (Fig. 7a). Correspondingly, the fluorescence imaging colours of FPSII changed to red (FPSII = 0.1) from the original green (FPSII = 0.5) (Fig. 8). Simultaneously, NPQ increased to the highest point after 45 min of treatment, which indicated that Arabidopsis had the strongest capability to regulate

excessive energy dissipation at this time. The rapid downregulation of FPSII revealed that it was very sensitive to photooxidation stress. Nevertheless, under continuous photooxidative treatment from 45 to 165 min, FPSII gradually increased, and WT and tt3 even showed double wave crests, which could be observed from the recovery imaging colour (Fig. 8). Meanwhile, NPQ showed a corresponding decrease (Fig. 7b). Therefore, the dynamic changes in FPSII and NPQ with photooxidation seem to involve three stages of alteration: first, FPSII decreased, but NPQ increased; second, FPSII increased and NPQ decreased; and third, both parameters decreased. The former two stages demonstrated the regulative response to moderate oxidative stress, and the later stage was inreversible damage. Among the four phenotypes it was evident that the sensitivity of PSII to MV-induced photooxidation in Arabidopsis displayed the sequence tt3tt4 > tt4 > tt3 > WT (FPSII, P < 0.01; NPQ, P < 0.05).

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Treatment time (min) Fig. 7. Effects of photooxidation on (a) effective PSII quantum yield (FPSII) and (b) non-photochemical quenching (NPQ) in the leaves of four Arabidopsis phenotypes. Photooxidative treatment was conducted with the whole leaves exposed to a solution of 5 mM methyl viologen for 270 min (WT, closed square; tt3, open triangle; tt4, open circle; tt3tt4, closed triangle). Data are the mean standard error (n = 5).

Non-cyclic ETR in PSII indicates the activity of PSII reaction centres (Genty et al. 1989). Curves of ETR had a similar changing tendency to that of FPSII under photooxidative treatment (Fig. 9). Deletion of anthocyanin biosynthesis obviously resulted in a greater susceptibility of ETR to photooxidative stress, and a smaller transient rise in ETR was observed. After treatment for 210 min, the ETR in the leaves of tt3tt4 was almost zero; in contrast, the ETR in the leaves of WT, tt3 and tt4 was 13.03 0.56, 8.67 0.42 and 5.07 0.95 mmol m2 s1, respectively. Fig. 10 further showed the ETR-light response curves in the leaves of WT and in the three mutants before photooxidative treatment. The patterns of ETR curves in the mutants were consistent with that of the WT plant, and ETR reached a maximum when the light intensity was ~800 mmol m2 s1 (Fig. 10a). Photooxidative stress aggravated the decline of ETR in different phenotypes of Arabidopsis and markedly reduced the saturating irradiance of ETR. After treatment for 15 min, the ETR in the leaves of WT, tt3 and tt4 reached a saturation value when the irradiance was ~500600 mmol m2 s1 (Fig. 10b). After 90 min, the saturating irradiance of ETR in the leaves of the mutants decreased to ~200 mmol m2 s1 (Fig. 10c). This saturating irradiance was kept until treatment for 180 min (Fig. 10d). The light saturation point of ETR in the leaves of WT was a little higher than that recorded in the mutants. In addition, the decreasing degrees of ETR in the leaves of the four phenotypes under photooxidation suggested that WT was the most-tolerant strain, and that tt3 and tt4 were intermediate, with similar tolerance; and tt3tt4 was the least-tolerant strain. Discussion Anthocyanin pigments, which are located outside the chloroplasts in the vacuoles of the epithelial cells, can absorb visible radiation

between 400 and 600 nm. Different levels of anthcoyanin might lead to different light absorption. Anthocyanin in the epidermal vacuoles of Zea mays L. leaves can absorb a lot of the incident light (~43%) (Pietrini and Massacci 1998). In the present study, the oxidative activity of MV is light dependent, so we estimated the effects of anthocyanin on leaf light absorption. The results indicated that the leaf absorption spectra displayed almost no difference between the WT plant and mutants deficient in anthocyanin after normalisation to the maximal absorption peak (Fig. 2b), and anthocyanin in the leaves of Arabidopsis had little effect on light absorption. Anthocyanin level in the leaves of the WT plant is as low as 0.75 0.01 g1 FW (Fig. 1b); moreover, the leafy colour of the WT plant, which is the same as the other three mutants deficient in anthocyanin, is green without appearing red. Given that such a low light intensity was used for treatment with MV (30 mmol m2 s1), in our opinion, the low anthocyanin level recorded in the WT plant could hardly contribute significantly to leaf absorptance. The leaf blade is the main organ that is rich in antioxidative enzymes and low-molecular-weight antioxidants. Ascorbic acid, tocopherol and carotenoids are recognised antioxidants. In addition, phenolics that are extensively present in cells, such as flavonoid, anthocyanins and phenolic acid, are highly active antioxidants (Larson 1988). Purified solutions of anthocyanins rate highly in the league of phenolic antioxidants and their capacities for absorbing oxygen free radicals are up to fourfold greater than those of ascorbic acid and vitamin E. Anthocyanin can directly scavenge ROS, such as H2O2, 1O2 and O2 (Gould et al. 2002). Neill et al. (2002) pointed out that anthocyanin contributed ~70% to the total antioxidant pool in methanol extracts from red leaves of Elatostema rugosum, more than all the other components, including phenolics, flavonols and flavones. Chalcone synthase and DFR are two key enzymes in the biosynthetic pathway of anthocyanin (Shirley et al. 1995). In the present study, the contents of anthocyanin, total phenolics and flavonoid were determined in the leaves of the four phenotypes of Arabidopsis (WT, tt3, tt4 and tt3tt4). The anthocyanin level in the WT plant was ~sevenfold as high as that in the three mutants (Fig. 1b). Moreover, our data revealed that total phenolics and flavonoid in the leaves of the mutants were significantly lower than the levels recorded in the WT plant, which indicates that lesions at CHS and DFR in the anthocyanin biosynthetic pathway might also influence the levels of phenolics in Arabidopsis, as well as the anthocyanin content. Although the contents of anthocyanin, flavonoid and phenolics in the leaves of the mutants were markedly lower than the levels in the WT plant, anthocyanin-deficient mutants and WT had a similar scavenging capability to DPPH before the photooxidative treatment. It is possible that under non-stressful conditions, the total antioxidative potential in the anthocyanin-deficient mutants might be compensated for by the other antioxidants. However, under continuous photooxidative treatment for 12 h, a considerably greater decrease in the total antioxidative capacity associated with the reduction of phenolics and flavonoid contents in the mutants was found. Total phenolics and flavonoid might be important antioxidants in the protection against photooxidation induced by MV in the three mutants, whereas anthocyanin together with other antioxidants in the WT

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Fig. 8. Changes in the fluorescence images of effective PSII quantum yield (FPSII) in the leaves of four Arabidopsis phenotypes. The whole leaves were exposed to a solution of 5 mM methyl viologen for 270 min. Fluorescence images are indicated by the false colour code at the bottom. The code ranges from black via red, orange, yellow, green, blue and violet to purple, and these colours code for numbers between 0 and 1.

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Treatment time (min) Fig. 9. Effects of photooxidation on the electron transport rate (ETR) in the leaves of four Arabidopsis phenotypes. Photooxidative treatment was conducted with the whole leaves exposed to a solution of 5 mM methyl viologen for 270 min (WT, closed square; tt3, open triangle; tt4, open circle; tt3tt4, closed triangle). Data are the mean standard error (n = 5).

plant exhibited efficiency in detoxification against a superoxide free radical (produced during MV photoreduction and subsequent oxidation) and its derivatives. In other words, anthocyanin, along with other antioxidants, protected the leaves of Arabidopsis from oxidative damage. This is consistent with the antiphotooxidation of anthocyanin-rich leaves of a purple rice cultivar (Peng et al. 2006). The membrane systems and photosynthetic pigments are two important targets of ROS. Under MV-induced photooxidative stress, the leaf cells in Arabidopsis were damaged to different degrees. The leakage of electrolytes intensified. The membrane leakage rate in leaves of the WT plant was significantly different from that recorded in the mutants. The WT plant exhibited greater tolerance than the mutants. The results from determinations of contents of antioxidants (anthocyanin, flavonoid and total phenolics), total antioxidant capacity and the retention of electrolytes in membranes in the leaves of the four phenotypes of Arabidopsis indicated that the capacity for mitigation against photooxidation occurred in the order of WT > tt3 > tt4 > tt3tt4.

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Fig. 10. Effects of photooxidative treatment periods on the electron transport rate (ETR)-light response curves in the leaves of four Arabidopsis phenotypes. Photooxidative treatment was conducted with the whole leaves exposed to a solution of 5 mM methyl viologen (MV) (WT, closed square; tt3, open triangle; tt4, open circle; tt3tt4, closed triangle). Exposures (20 s) to stepwise increased photon irradiance (0, 1, 21, 56, 111, 186, 281, 336, 461, 531, 611, 701, 801, 926, 1076 and 1251 mmol photons m2 s1) were provided. Data are the mean standard error (n = 5). (a) Untreated by MV (0 min), (b) treated for 15 min with MV, (c) treated for 90 min with MV and (d) treated for 180 min with MV.

An analysis of PSII functional activity in the leaves of Arabidopsis using a fluorescence imaging system yielded five chlorophyll parameters (Fv/Fm, qP, FPSII, NPQ and ETR) in the leaves of the four phenotypes of Arabidopsis. WT and the three mutants deficient at different sites in anthocyanin synthesis exhibited certain differences in response to photooxidative stress. In accordance with the determinations of antioxidants and total antioxidant capability, decreases in the magnitudes of Fv/Fm, qP, KPSII and ETR in the leaves of Arabidopsis occurred in the following sequence: tt3tt4 > tt4 > tt3 > WT, which suggested that deficiency in anthocyanin synthesis genes increased the sensitivity of Arabidopsis to photooxidative stress and lowered the stability and the capability of PSII to respond to photooxidative stress in leaves. Obviously, different sensitivities to oxidative stress in the leaves of the three mutants primarily resulted from their gene depletion sites. tt3, mutated in the DFR gene, is deficient in anthocyanins, but accumulates excess amounts of the antioxidants quercetin and kaempferol, and this has been confirmed experimentally (Peer et al. 2001; Heo and Lee 2004; Park et al. 2006). tt4, although containing the minor flavonoid, is deficient in the CHS gene, which is located upstream in anthocyanin biosynthesis. The double mutant tt3tt4 is deficient in both the CHS and DFR

genes, so its antioxidative capability was markedly decreased. Our results clearly demonstrate that anthocyanin in Arabidopsis is an effective antioxidant. Despite the fact that tt4 and tt3tt4 are both deficient upstream in anthocyanin biosynthesis, tt4 exhibited higher antiphotooxidative capability and higher contents of total phenolics and flavonoid than tt3tt4. Thus, we propose that tt4, which is not deficient in the DFR gene, might allow synthesis of some phenolics and flavonoid through other pathways. The toxic effects of MV on chloroplasts result from the acceptance of electrons from the ironsulfur cluster FeSA/ FeSB of PSI by MV, which results in depletion of NADPH and inhibition of CO2 fixation, and in the production of ROS, such as superoxide, H2O2 and the hydroxyl radical. ROS that are not scavenged promptly will rapidly destroy the chloroplast membranes (Chia et al. 1981; Babbs et al. 1989). In contrast, ROS are generally considered to serve as a signal in plant defence responses (Vranova et al. 2002). In the present study, more than 150 min of photooxidative treatment resulted in damage to PSII activity and to the cell membranes as expected. Interestingly, the patterns of fluorescence imaging and the relevant curves of qP, FPSII and ETR all showed an early decrease, followed by a partial increase that was accompanied by a contrary trend of NPQ after short photooxidative treatment. This phenomenon provided

Sensitivity of anthocyanin to photooxidation

useful evidence for reversible deactivation of PSII after short photooxidative treatment. Within the first 45 min of photooxidative treatment, qP, FPSII and ETR decreased sharply, while NPQ increased. It is thought that the presence of MV enhanced the Mehler reaction, with O2 acting as an electron acceptor (photoreduction pathway of O2). The ROS produced in this case induced PSII reaction centres to close, which then resulted in a decrease in ETR and FPSII. Simultaneously, the photosynthetic apparatus rapidly adjusted its capability to distribute and utilise excitation energy. Heat dissipation was used and excess excitation energy was dissipated through PSII antennae by means of heat. Soon after, the partial recovery of qP, FPSII and ETR with a decreasing NPQ was presumably mediated by accumulated ROS acting as a signal molecule in the induction of the antioxidant system in the chloroplasts. Hence, the transient rapid down-regulation and partial recovery of PSII activity reflected the fact that the photosynthetic apparatus could respond to moderate environments through reversible deactivation. The chlorophyll fluorescence parameters Fv/Fm and FPSII have been shown to be deactivated reversibly in the leaves of plants such as soybean, peanut and sugarcane under a diurnal variation process; and reversible deactivation of PSII is generally considered to be a physiological function for photosynthetic apparatus in plants that are able to adapt to complex survivable environmental changes under the long-term evolution process (Xu and Wu 1996; Lin et al. 1999). However, to the best of our knowledge, we report for the first time that PSII was transiently reversibly deactivated, based on a dynamic analysis of chlorophyll fluorescence imaging under MV-induced photooxidative treatment. Further investigations, however, are still required to understand this process more precisely and to explain it rationally. Acknowledgements We thank Professor Fred Chow (Research School of Biological Sciences, the Australian National University) and Professor Lars Olof Bjorn (Department of Cell and Organism Biology, Lund University) for critical reading of the manuscript and helpful suggestions. This research was supported by the National Natural Science Foundation of China (Grant No. 30770173) and the Zealquest Scientific Foundation.

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Manuscript received 9 March 2008, accepted 17 July 2008

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