Photosynthesis Research (2005) 84: 35–41
Springer 2005
Regular paper
Photoinactivation of Photosystem II in leaves Wah Soon Chow1,*, Hae-Youn Lee1,2, Jie He1,3, Luke Hendrickson1,4, Young-Nam Hong2 & Shizue Matsubara1,5 1
Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia; 2School of Biological Sciences, Seoul National University, 151-742 Seoul, South Korea; 3 Natural Sciences Academic Group, Nanyang Technological University, 1 Nanyang Walk, Singapore 637-616; 4 Umea˚ Plant Science Center, Department of Plant Physiology, Umea˚ University, 901 87 Umea˚, Sweden; 5 Institut fu¨r Phytospha¨re, ICG-III, Forschungszentrum Ju¨lich, 52425 Ju¨lich, Germany; *Author for correspondence (e-mail:
[email protected]; fax: +61-2-6125-8056) Received 11 September 2004; accepted in revised form 10 January 2005
Key words: law of reciprocity, photoinactivation of Photosystem II, quantum yield of photoinactivation, quenching of excitation energy
Abstract Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p=kDx, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N=Noexp()kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N=Noexp()kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N=Noexp()kx), the quantum yield of photoinactivation of PS II can be defined as )dN/dx=kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1 lmol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 107 photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely photoinactivated PS II complexes which are able to dissipate excitation energy efficiently and to protect the remaining active neighbours against damage by light.
Abbreviations: f – functional fraction of PS II; kr, ki – rate coefficient of repair and photoinactivation of PS II, respectively; N – number of functional PS II complexes; PS II – Photosystem II; QYI – quantum yield of photoinactivation; x – photon exposure
36 Introduction ‘Photosynthetic oxygenic evolution is intrinsically suicidal’ (van Gorkom and Schelvis 1993). This statement emphasizes the inevitability of lightinduced loss of Photosystem (PS) II activity, i.e. photoinactivation of PS II. Yet it is commonly thought that photoinactivation of PS II only occurs when light absorption exceeds the capacity of chloroplasts to utilize the light energy. Such a belief, however, conceals the fact that photoinactivation of PS II occurs even in low light, though on-going repair ensures little net loss of PS II activity. Thus, when repair is prevented, even very low continuous light (Keren et al. 1995) or single turnover flashes spaced many seconds apart (Keren et al. 1995, 2000) will cause net photoinactivation of PS II. Over a sunny day, the total number of photons (30 mol m)2) received by a leaf may be large enough to photoinactivate practically the entire population of PS II in a leaf (1 lmol m)2). Here we will examine the hypothesis that photoinactivation of PS II depends on light dosage. Comparing expectations from this simple hypothesis against experiments, we will further address some complexities in vivo that are not explained by this simple hypothesis.
The dependence of PS II photoinactivation on photon exposure Combinations of irradiance (lmol photons m)2 s)1) and duration of illumination that result in the same number of incident photons m)2 (‘‘photon exposure’’; Bell and Rose 1981) should give the same effect if the law of reciprocity is obeyed. According to this law, the important factor determining an effect of light is the total number of photons, rather than their rate of arrival per se. The photoinactivation of PS II in a number of photosynthetic systems has been reported to be consistent with this law (Jones and Kok 1966; Nagy et al. 1995; Park et al. 1995, 1996; Lee et al. 1999, 2001). For example, the loss of functional PS II centres showed a single dependence on photon exposure, whether the capsicum leaf discs were illuminated at 460, 900 or 1800 lmol photons m)2 s)1 (Figure 1a). In this experiment, capsicum leaf discs had been
treated with lincomycin to inhibit repair that requires chloroplast-encoded protein synthesis, thereby simplifying the system’s response to light. Similarly, isolated envelope-free chloroplasts that are devoid of any ability to repair photodamage obey the law of reciprocity (Jones and Kok 1966). In the presence of repair, however, capsicum leaf discs did not seem to obey the law of reciprocity of irradiance and duration; instead, the decrease in the functional fraction of PS II followed three different curves when three irradiances were used to vary the photon exposure (Figure 1b). In a leaf disc, there are irradiancedependent processes that interfere with the law of reciprocity. An obvious example is the rate of repair after photoinactivation, given by the product of the rate coefficient of repair (kr) and the concentration of photoinactivated PS II complexes (Kok 1956). Figure 1c depicts the complex dependence of kr on irradiance, corrected for the irradiance-dependent rate coefficient of gross photoinactivation of PS II (Figure 1d; see also He and Chow 2003). In darkness, kr was very small, but non-zero. It rose rapidly with small increases of irradiance, consistent with the requirement of low light for recovery from photoinactivation. The value of kr was maximal at moderate irradiances, but decreasing at higher irradiances. Similarly, Allakhverdiev and Murata (2004) reported that the rate of repair in Synechocystis was high under weak light, reaching a maximum under moderate light and decreased at a given irradiance by salt stress, oxidative stress or chilling stress. Based on their results, our observation of a decrease of kr at high irradiance (Figure 1c) could be due to oxidative stress. A complex dependence of kr on irradiance is probably the main factor that leads to violation of the law of reciprocity (Figure 1b).
The probability of photoinactivation Since the law of reciprocity of irradiance and duration of illumination is applicable at least to some simple systems (e.g. in the absence of repair), let us postulate for such a system that the probability of photoinactivation of a PS II complex is directly proportional to an increment in photon exposure Dx (Lee et al. 1999):
37
Figure 1. (a) and (b). The decrease in the functional fraction of PS II (f) with increase in photon exposure given to capsicum leaf discs floating on water. The value of f was determined from a chlorophyll fluorescence parameter that was directly proportional to the oxygen evolved per flash during repetitive, single-turnover flash illumination (Lee et al. 2001). Capsicum plants were grown in high light (500 lmol photons m)2 s)1) in a growth chamber. Light treatment was given at three irradiances, 460, 900 or 1800 lmol photons m)2 s)1 for various durations to vary the photon exposure. In (a), leaf discs were illuminated in the presence of lincomycin which inhibits repair of PS II after photoinactivation. In (b), leaf discs were illuminated in the absence of lincomycin. Replotted from Lee et al. (2001). The irradiance-dependence of the rate coefficient of repair kr (c) and of photoinactivation ki (d) of PS II in capsicum leaf discs, replotted from He and Chow (2003). Capsicum plants were grown in moderate light (200 lmol photons m)2 s)1) in a growth chamber. The rate coefficient of photoinactivation ki was determined from the time course of light treatment at each irradiance in the presence of lincomycin which inhibits repair; the rate coefficient of repair kr was obtained during recovery at each irradiance in the absence of lincomycin, corrected for the gross photoinactivation at each irradiance.
p ¼ kDx where k is the probability per unit photon exposure, a characteristic of PS II under a given set of experimental conditions. The probability of a PS II surviving an increment of photon exposure is 1)kDx. Let the probability of surviving a photon exposure x be F(x). Then F(x+Dx) is the probability of surviving a photon exposure x+Dx. That is
Fðx þ DxÞ ¼ ðprobability of surviving xÞ ðprobability of surviving DxÞ ¼ FðxÞ ð1 kDxÞ Rearranging, letting Dx fi 0, and integrating: FðxÞ ¼ expðkxÞ If, after photon exposure x, N functional PS II complexes remain of the initial No complexes,
38 N=No ¼ expðkxÞ That is, the fraction of functional PS II complexes (f=N/No) should decrease mono-exponentially with x. Indeed, the decrease in f in Figure 1a can be well described by a single exponential down to f=0.3. Thereafter, f decreases much more slowly (see below).
The rate coefficient of photoinactivation of PS II, ki Light treatments are usually conducted at constant irradiance I for various durations t. The decrease in f can then be plotted as a function of time. Since x=It, Equation (1) becomes: N=No ¼ exp½ðkIÞt The rate coefficient of the exponential time course is the product kI (=ki), which is directly proportional to I. The rate coefficient of photoinactivation of PS II has been shown to be directly proportional to irradiance under conditions where repair is inhibited (Tyystja¨rvi and Aro 1996; Lee et al. 2001; He and Chow 2003; Kato et al. 2003; Allakhverdiev and Murata 2004). Figure 1d illustrates the linear dependence of ki on I for capsicum leaf discs in the absence of repair. The rate of photoinactivation of PS II can be calculated as the product of ki and the concentration of remaining functional PS II complexes. Obviously, as the concentration of functional PS II complexes
decreases, the rate of photoinactivation decreases because fewer functional complexes are available for photoinactivation.
The quantum yield of photoinactivation From Equation (1), )dN/dx=kN can be taken as the quantum yield of photoinactivation of PS II, which varies with N. Its magnitude is maximum when N is maximum (i.e., when x=0). Table 1 shows some values of the quantum yield of photoinactivation of PS II. The highest values were obtained in vitro when photoprotective mechanisms could not operate. A typical value of 0.1 lmol PS II (mol photons))1 means that when 107 photons are received by leaf tissue, one PS II is photoinactivated. The lowest quantum yield of photoinactivation of PS II was observed in grapevine leaves, with a value of 0.02 lmol PS II (mol photons))1, corresponding to 5 · 107 photons per PS II. The low quantum yield of photoinactivation of PS II in grapevine leaves was observed in spite of the fact that the light treatments were conducted at 9 C and in the presence of lincomycin. Grapevine leaves are particularly well adapted to cope with high light stress (Hendrickson et al. 2004).
Severe photoinactivation of PS II Figure 1a shows that a residual population (ca. 15%) of PS II complexes in capsicum leaf discs
Table 1. Quantum yield of photoinactivation of PS II (QYI) in leaves, chloroplasts and chloroplast fragments Materiala 1
QYI [lmol PS II (mol photons))1] Reference
Grapevine leaves acclimated to glasshouse, illuminated at 9 C 0.02 Pumpkin leaves acclimated to very high light 0.06 3 Capsicum leaves 0.1 4 Pea leaves 0.3 5 Spinach membrane fragments 0.2–0.3 6 Alocasia and Atriplex thylakoids 0.3 2
Hendrickson et al. (2004) Tyystja¨rvi and Aro (1996) Lee et al. (1999) Park et al. (1995) Eckert et al. (1991) Bjo¨rkman et al. (1972)
a Light treatments were given to the materials at room temperature, except for grapevine leaves (9 C). QYI is defined as )dN/dx=kN, where N is the number of functional PS IIs; x, the photon exposure and k, the probability per unit photon exposure. QYI is highest when N is maximum in a fully active sample; the values for 2 pumpkin leaves; 3 capsicum leaves; 5 spinach membrane fragments and 6 Alocasia and Atriplex thylakoids were obtained this way. In the case of 6Alocasia and Atriplex thylakoids, k [=ki/I=( ln 2)/t1/2I, see text] was estimated from the half time t1/2 of loss of PS II activity, assuming [active PS II] to be 2.5 mmol (mol Chl))1 initially and that 70% of the incident light was absorbed by the thylakoid suspension. The values for 1 grapevine leaves and 4 pea leaves were average values obtained by dividing the loss of about half of the active PS II by the total photon exposure.
39 survived large photon exposures even when lincomycin was present to inhibit repair. A similar finding was reported for Arabidopsis plants grown hydroponically and illuminated at 1000 lmol photons m)2 s)1 with the inclusion of 2 mM lincomycin in the culture medium; about 15% of oxygen evolution capacity remained even after 24 h illumination (Nore´n et al. 1999). Clearly, photoinactivation of PS II was independent of photon exposure under prolonged light stress. It is interesting to consider why a residual population of functional PS II complexes was sustained. One plausible mechanism is that after a long illumination period, the photoinactivated PS II complexes might have undergone extensive modification and acquired a quenching mechanism for energy dissipation. This quenching mechanism in inactive PS II complexes might be so effective as to dissipate excitation energy received by remaining functional PS II complexes that are excitoni-
cally connected to the quenching centres (O¨quist et al. 1992; Chow et al. 2002; Ivanov et al. 2002). If photoinactivated PS II complexes act as quenching centres, they will cause a shortening of the lifetime of the excited state of chlorophyll, i.e., a shortening of the lifetime of chlorophyll fluorescence. We observed that the fluorescence lifetime of control capsicum leaves had a main fluorescence lifetime component centred at 2.22 ns (Figure 2a). Upon mild photoinactivation for 1 h (with 84% of functional PS II remaining under conditions which allowed repair), a new component with a lifetime of 1.25 ns appeared at the expense of the 2.22-ns component. On further photoinactivation beyond 1 h, yet another component appeared at 0.58 ns. Beyond 1 h illumination, the 1.25-ns component increased only slowly, while the 2.22-ns component was further decreased. At the same time, the 0.58-ns component increased considerably after the first hour of
Figure 2. (a). Fractional intensities of the three principal components of chlorophyll fluorescence decay with lifetimes centred at 2.22, 1.25 or 0.58 ns. Capsicum plants were grown at 250 lmol photons m)2 s)1. Leaf discs were floated on water at 25 C, and illuminated at 900 lmol photons m)2 s)1 for up to 5 h. After light treatment, samples were kept in the dark for 30 min during which leaf discs were infiltrated with 30 lM DCMU to block linear electron flow during subsequent measurement of fluorescence lifetime with excitation light at 140 lmol photons m)2 s)1 and )2 C. Chlorophyll fluorescence lifetimes were measured at the maximum-fluorescence state (Fm) using a multi-frequency phase fluorimeter (K2-004, ISS Instruments, Urbana, USA) equipped with a laser diode and a redsensitive micro channel plate detector. Excitation was modulated at 12 logarithmically spaced frequencies ranging from 25 to 200 MHz. The excitation wavelength was 635 nm, and the emission was detected at 689 nm. Data obtained by Matsubara and Chow (2004) were globally analysed by fitting to a continuous Lorentzian distribution model (Alcala et al. 1987). (b). A diagram showing three types of PS II complexes connected via their antennae (dashed envelope). An active PS II centre (non-hatched circle), presumed to give chlorophyll fluorescence emission at 2.22 ns, passes its excitation energy to, and is protected by, strongly quenching, photoinactivated PS II centres (hatched with solid lines) that are presumed to give rise to fluorescence lifetime at 0.58 ns. An intermediate type of PS II (hatched with dashed lines) is presumed to be weakly quenching and responsible for the lifetime component at 1.25 ns. Under the conditions of the measurement (low light and presence of DCMU), thermal dissipation in the antenna is expected to be limited, so it is not shown. Re-drawn from Matsubara and Chow (2004).
40 illumination, reaching a maximum at about 5 h illumination when about 45% of functional PS II remained. There appears to be two populations of photoinactivated PS II complexes in capsicum leaves, a weakly-quenching population with a fluorescence lifetime centred at 1.25 ns, and a strongly quenching one at 0.58 ns (Matsubara and Chow 2004). Conceivably, the strongly quenching centres helped to dissipate excitation energy in remaining functional PS II complexes, thereby helping to protect them against photoinactivation. The weakly quenching centres, though of limited help in protecting their neighbours, may nevertheless be able to avoid further damage to themselves (Krause 1988). Figure 2b depicts the dissipation of excitation energy in a mixed population of functional and photoinactivated PS II complexes. Where the three types of PS II are excitonically connected, excitation energy is shared, but the strongly quenching centres act as the predominant energy sinks, thereby ensuring that photoinactivation of the remaining PS II is independent of photon exposure. In turn, the residual population of functional PS II may be critical for recovery of the photoinactivated PS II complexes. In summary, the photoinactivation of PS II depends on photon exposure per se during mild photoinactivation and in the absence of repair. However, both the complex dependence of repair on irradiance and the generation of strongly quenching PS II centres after severe photoinactivation of PS II bring about a departure from this simple model.
Acknowledgements All our experimental work reported here was conducted at the Australian National University. WSC was supported by ARC Discovery Project Grant DP0343160. We are grateful to Professors Jan Anderson and Barry Osmond for helpful comments on the manuscript.
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