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AUSTRALIAN JOURNAL OF PLANT PHYSIOLOGY Volume 25, 1998 © CSIRO 1998
An international journal of plant function
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Aust. J. Plant Physiol., 1998, 25, 775–784 © CSIRO 1998
The electrochromic signal, redox reactions in the cytochrome bf complex and photosystem functionality in photoinhibited tobacco leaf segments W. S. ChowA and A. B. HopeB Photobioenergetics Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, A.C.T. 2601, Australia. A Author for correspondence; email:
[email protected] B School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, S.A. 5001, Australia.
Abstract. Photosynthetic electron transport in vivo was investigated in tobacco leaves pre-illuminated with strong light under conditions where Photosystem (PS) II repair was inhibited by lincomycin. Flashinduced redox changes of cytochrome b563, cytochrome f and plastocyanin, and the electrochromic (EC) signal (caused by a carotenoid band-shift due to charge separation across thylakoid membranes) from leaf segments were measured by deconvoluting absorbance changes at 520, 554, 564 and 575 nm. The EC signal was composed of easily separable fast and slow components. The fast EC signal decreased linearly with the loss of functional PS II centres, but there was a residual fast EC phase which was attributable to PS I centres alone. Inactivation of PS II centres by photoinhibitory light was also well-correlated with the quenching of variable fluorescence measured as the ratio of variable to maximum fluorescence, Fv /Fm. On complete photoinactivation of PS II centres, the slow rise of the flash-induced EC signal became more prominent, suggesting enhanced electrogenic charge transfer across the cytochrome bf complex as part of a path of electron flow involving PS I. Thus, both PS I and the cytochrome bf complex appeared to be fully functional after treatment of tobacco leaves with photoinhibitory light at room temperature. In totally photoinhibited leaf segments, the rate coefficients of cyt f III re-reduction increased from 59 s–1 (+ lincomycin, no photoinhibitory light) to 130 s–1, and that of cytochrome b 563 reduction also increased, from 270 s–1 to 500 s–1, suggesting that the prevailing plastoquinol concentration was higher after photoinhibitory light treatment. The source of the electrons entering the pool under these conditions was probably a high concentration of NADPH and reduced ferredoxin. Keywords: cyclic electron flow, cytochrome bf kinetics, electrochromic signal, photoinhibition, Photosystem I, Photosystem II, tobacco.
With regard to photoinhibition, defined as the loss of photosynthetic efficiency that occurs when the absorbed light exceeds that which can be utilized by photosynthetic reactions, Photosystem (PS) II is established as a prime site of damage (see Chow 1994; Osmond 1994; Andersson and Barber 1996). However, questions remain about the process — is PS II the only site of damage, or are PS I and the bf complex also affected? It has been established that at least at chilling temperatures, when presumably the enzymic scavenging of oxygen radicals is slow, PS I may be particularly susceptible to photodamage (see Sonoike 1996). Further, since singlet oxygen (1O2) may also be involved (Barber 1995), there is the possibility of its generation and local action in PS II as well as in neighbouring PS I units. Products and intermediates arising from superoxide formation, possibly on the acceptor side of
Introduction Despite initial studies of electron transfer reactions in intact leaves by kinetic optical spectroscopy (Klughammer et al. 1990; Kramer and Crofts 1990; Kramer and Crofts 1996), there are only sparse data on the kinetics of reactions of components of the electron transport chain between the photosystems in intact leaves, with the exception of the EC* signal, which was extensively studied by Kramer and Crofts (1989) using cucumber leaves. By contrast, information obtained using isolated chloroplasts is relatively comprehensive (reviewed by Hope 1993), and the bf complex itself has attracted several further reviews of its molecular organization and acclimation to the light environment (Anderson 1992) and on its structure and function (Hauska et al. 1994; Cramer et al. 1996).
*Abbreviations used: AMA, antimycin A; Chl, chlorophyll; cyt, cytochrome; DBMIB, 2-5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU, 3-(3,4dichloro-phenyl)-1,1-dimethyl urea; DCPIP, dichlorophenol indophenol; EC, electrochromic (signal at 515–520 nm); Fd, ferredoxin; FQR, ferredoxin/plastoquinone oxidoreductase; Fv, Fm, variable and maximum chlorophyll fluorescence yield, respectively; IR, infrared; NDH, NAD(P)H dehydogenase; PC(II), plastocyanin (oxidised); PI, photoinhibitory light; PQ, plastoquinone; PQH2, plastoquinol; PS I, PS II, photosystem I, II; SEM, standard error of mean; A, change in absorbance — absorbancy ‘units’ in figures and text are 104 3 A.
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10.1071/PP97160
0310-7841/98/070775
776
W. S. Chow and A. B. Hope
PS II, could also potentiate damage (Osmond and Grace 1995). Also, little is known about the effects of photoinhibition on the EC signal. With regard to PS I, Critchley (1981) found that in chloroplasts from partly-photoinhibited cucumber leaves, PS I activity (measured as O2 consumption by methyl viologen with ascorbate/DCPIP as the electron donor) was undiminished compared with controls. This report forms part of an ongoing study of aspects of photosynthetic electron transfer in chloroplasts in vivo, using leaves with different origins and subjected to certain stress treatments. Some results have already been presented in connection with antisense mutants of tobacco with reduced content of the Rieske centre and cytochrome bf complex (Anderson et al. 1997). The EC signal, measured as an absorbance change at 515–520 nm, is the response of pigments such as carotenoids and chlorophyll b to changes in the electric field in their environment (e.g. see Witt 1975). The fast phase (
