Photoinhibition of photosynthesis represents a ... - Springer Link

14 downloads 1 Views 1MB Size Report
King's Lynn, Norfolk, UK) according to Delieu and Walker (1983). The measurements ..... efficiency of PSII photochemistry in concert with the demand for ATPĀ ...
Planta

Planta (1992)186:450-460

(@ Springer-Verlag1992

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II Gunnar Oquist 1., W.S. Chow, and Jan M. Anderson Division of Plant Industry, CSIRO, GPO Box 1600, Canberra, ACT 2601, Australia Received 24 June; accepted 10 October 1991

Abstract. The obligate shade plant, Tradescantia albiflora Kunth grown at 50 ~tmol photons 9 m -2 s -1 and P i s u m sativum L. acclimated to two photon fluence rates, 50 and 300 p m o l . m - 2 . s -1, were exposed to photoinhibitory light conditions o f 1700 pmol 9 m -2 9 s- 1 for 4 h at 22 ~ C. Photosynthesis was assayed by measurement of CO2saturated Oz evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 ~tmol- m -2- s -~ was most resistant, with pea grown at 50 ~tmol 9m - 2 . s-1 showing an intermediate sensitivity. A very good correlation was found between the decrease o f functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (qp) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F'F~, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81-92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F'vF~, since qp at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield o f PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoini Permanent address: Department of Plant Physiology, University of UmeA, S-901 87 UmeA, Sweden * To whom correspondence should be addressed Abbreviations: F o and F~,= minimal fluorescence when all PSII reac-

tion centres are open in darkness and steady-state light, respectively; Fm and F~ = maximal fluorescence when all PSII reaction centres are closed in dark- and light-acclimated leaves, respectively; F~, = variable fluorescence (F--F~) under steady-state light conditions; Fs = steady-state fluorescence in light; QA= the primary, stable quinone acceptor of PSII; qNe= non-photochemical quenching of fluorescence due to high energy state (ApH); qNi=non photochemical quenching of fluorescence due to photoinhibition; qp = photochemical quenching of fluorescence

hibition o f the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid ApH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to qp, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority o f their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.

Key words: Light acclimation (photoinhibition) Photoinhibition of photosynthesis - Photosynthesis (photoinhibition)- Photosystem II (regulation) Pisum (photoinhibition) Tradescantia

Introduction Photoinhibition of photosynthesis results from excessive excitation of the photosynthetic apparatus (Osmond 1981). The functional consequences of photoinhibition of photosynthesis are inhibition of the maximum quantum yield (see Powles 1984 for a review), a decline in the convexity of the light-response curve (Leverenz et al. 1990), and a decreased rate of light-saturated photosynthesis. However, quantum yield and convexity are decreased well before effects are observed in light-saturated photosynthesis (Oquist and Malmberg 1989; Leverenz et al. 1990). At the chloroplast level, photoinhibition is manifested as a decrease of the quantum yield of PSII photochemistry (Powles 1984). The mechanism of photoinhibition is not yet fully understood (Critchley 1988; Krause 1988), but it typically involves inhibition of PSII at the reaction-centre level (Kyle 1987), coupled to in-

G. Oquist et al. : Photoinhibition and regulation of PSII

creased thermal de-excitation of excited chlorophyll (Ogren and Oquist 1984). Recovery from photoinhibition usually requires protein synthesis since it involves replacement of photodamaged reaction-centre components, particularly the D1 protein (Kyle 1987). Alternatively, another type of photoinhibition has been proposed to result from the formation of a quencher in the antenna of PSII (Demmig and Bj6rkman 1987). The quencher is suggested to be a carotenoid, zeaxanthin (Demmig-Adams 1990), which lowers the yield of photochemistry by competing with the PSII reaction centres for excitation energy. Recovery from this latter type of photoinhibition is not likely to require protein synthesis and is relatively fast. Since protection of PSII from photodamage always appears to be of high priority, PSII is characterized by its dynamic properties (Krause 1988; Horton 1989). In the short-term, the quantum yield of photochemistry of PSII is regulated within seconds to minutes by both non-photochemical quenching of excitation energy as induced by a high transthylakoid ApH (Oxborough and Horton 1988) and State 1/State 2 transitions, which adjust the distribution of excitation energy between the two photosystems (Horton and Hague 1988). It has recently been suggested that this short-term "photosynthetic control" serves to adjust the syntheses of ATP and NADPH to the rate by which these metabolites are required for carbon metabolism (see Foyer et al. 1990). According to this view, State 1/State 2 transitions would optimize the quantum yield of photosynthesis at low irradiance by balancing the energy distribution to the two photosystems (Horton 1985), while under high irradiance, the build-up of ApH would serve to avoid overreduction of PSII by triggering non-radiative dissipation of excitation energy (Weis and Berry 1987; Horton and Hague 1988; Krause et al. 1988). The aim of our study was to test the hypothesis that photoinhibition of PSII reaction centres from a functional viewpoint is a down-regulation of PSII that is "lockedin" under prevailing high-light conditions, and represents a stable, long-term down-regulation of this vulnerable photosystem in the time-scale of hours or longer. Our methodological approach has been to study the function of photosynthesis and PSII at limiting and at saturating light in relation to the level of photoinhibition displayed by three differently light-acclimated plants, before and after exposure to a high irradiance. We measured (i) modulated fluorescence to study the function of PSII, (ii) flash yield to determine the amount of functional reaction centres of PSII, and (iii) polarographic determination of CO2-saturated O2 evolution to assay leaf photosynthesis. The obligate shade plant Tradescantia albiflora was chosen as a species with high sensitivity to photoinhibition and unable to acclimate its light-harvesting components to high light (Chow et al. 1991a), whereas Pisum sativum was chosen to represent species with the ability to acclimate photosynthetically to both low and high irradiances (Chow and Anderson 1987), thereby implying a variable sensitivity to photoinhibition depending on growth light conditions. We present in this paper evidence that photoinhibi-

451

tion of photosynthesis represents a long-term, downregulation of the yield of PSII photochemistry, which under light-saturating conditions is indistinguisable from a short-term, down-regulation of PSII as exerted by a high transthylakoid ApH gradient. Photoinhibiton is related to the loss of functional PSII reaction centres. Furthermore, we provide evidence that, irrespective of light acclimation, plants are prone to photoinhibition when the majority of PSII reaction centres are still in an open state. Materials and methods Plant material. Pea (Pisum sativum L. cv. Greenfeast (local dealer)) and Tradescantia albiflora K u n t h (Chow et al. 1991a) were grown from seeds and cuttings, respectively, in a compost/Perlite mixture (1: 1, w/w) watered daily with 1/4-strength Hoagland's (No. 2) solution (Hewitt 1966). The plants were grown in growth cabinets (12-h light/22 ~ C; 12-h dark/18 ~ C; TLD 55W/86 fluorescent tubes; Philips; Roosendaal, The Netherlands). Tradescantia was grown at a photon fluence rate of 50 lamol 9 m - 2 . s-1, whereas pea was grown at 50 or 300 l a m o l ' m 2. s-1. Tradescantia represents an obligate shade plant unable to acclimate to high irradiance (Chow et al. 1991a), whereas pea represents a plant with ability to adequately acclimate to both low and high irradiences (Chow and Anderson 1987). The leaves used for experiments were the last fully-developed leaf pair of a shoot. When used for experiments, the pea seedlings were three to six weeks old and the Tradescantia cuttings were four to eight weeks old. Additional species were used for Fig. 6 (see figure legend). Photoinhibition. Leaf discs (diameter 15 or 19 mm) were floated on water in a Petri dish with their upper surface exposed to air. Unless otherwise specified, photoinhibition was imposed on floating leaf discs by exposing them for 4 h to 1700 lamol - m -2 9 s -1 using two slide projectors. To avoid effects of heat radiation, the photoinhibitory light was filtered through 6 cm water, and the Petri dish with floating leaf discs was thermostatted to 22 ~ C. Fluorescence measurements. Photoinhibition was routinely assayed at room temperature by measuring time-resolved fluorescence induction kinetics with an instrument described previously (Strid et al. 1990), which was connected to a Gould 2 M H z (Type 1421) storage oscilloscope (Hainault, UK). Prior to measurements, the leaf discs were dark-acclimated at room temperature for at least 30min. The p h o t o n fluence rate of the actinic light was 600 lamol 9 m - 2. s - 1, which was sufficient to reach Fm (maximal fluorescence) at the P-peak in dark-acclimated leaves. The minimal fluorescence (Fo) was measured after 1.2 ms, which was equal to the opening time of the shutter. The ratio of variable to maximal fluorescence (Fv/F~) was calculated and used as a measure of the maximal photochemical efficiency of PSII (Butler 1978). Modulated fluorescence was measured using a P A M 101 Chlorophyll Fluorometer equipped with accessories: P A M 103, a Schott lamp KL1500 FL103 and a polyfurcated fibre-optic system (H. Walz, Effeltrich, Germany) . The experimental protocol of Genty et al. (1989) was followed. Photosynthetic light was provided by a slide projector and different photon fluence rates (0 to 2830 ~tmol - m - 2 . s - 1) were obtained by the use of neutral-density filters. The values of Fm (determined after at least 30 min of dark acclimation) and F~ (in the presence of photosynthetic light) were obtained by imposing 1-s flashes using the Schott lamp, and applying the extrapolation method of Markgraf and Berry (1990). Three flashes of decreasing p h o t o n fluence rates were applied ( 2100, 1040 and 725 l a m o l - m - 2 - S-1) allowing the fluorescence to relax completely down to the steady-state fluorescence level (Fs) between each flash. The minimal fluorescence level (Fo) was sensitized by a p h o t o n fluence rate of 0.01 lamol" m -2" s -1 (modulation at 1600 Hz). To

452

G. Oquist et al.: Photoinhibition and regulation of PSII

ensure complete oxidation of QA (the primary , stable quinone acceptor of PSII), F o and F o (minimal fluorescence in darkness and steady-state light, respectively) were always determined with a farred background light (3 W . m-2; Balzers 710 nm (Geisenheim/ Rhein, Germany) plus Schott RG 695 ( Schott Glaswerke, Mainz, Germany)) peaking at 713 nm and with a half-bandwidth of 5 nm. The F o level was determined immediately after turning off the photosynthetic light. Photochemical (qp) and nonphotochemical quenching (qN) were calculated according to van Kooten and Snel (1990), and F o quenching was accounted for in the calculations according to Bilger and Schreiber (1986). The terminology suggested by van Kooten and Snel (1990) and Walters and Horton (1991) has been used. Modulated fluorescence and photosynthetic 02 evolution were measured simultaneously on leaf discs (diameter 15 mm) enclosed in a leaf-disc electrode (see below).

Measurement of photosynthetic

0 2

evolution. Photosynthetic

0 2

evolution was measured in a leaf-disc oxygen electrode (Hansatech, King's Lynn, Norfolk, UK) according to Delieu and Walker (1983). The measurements were done at 25 ~ C in air containing 21% 02 and 1% CO/(from a 1 M carbonate/bicarbonate buffer solution at pH 9). The photosynthetic light was provided by the same slide projector as used for the measurements of modulated fluorescence. The top of the leaf-disc electrode was for this purpose modified to fit the fibre-optic light guide. The quantum yield determinations of O2 evolution, as calculated by regression analyses, were based on absorbed photons as given by the leaf absorptance. Leaf absorptance (A) was estimated from the emperical relationship A = 0.96 x~ (x+0.047) established for a large number of species (J.R. Evans, RSBS, Australian National University, Canberra, Australia; personal communication). In this equation, x is the chlorophyll content of leaf discs (mmol " m-2), determined after extraction with buffered 80% acetone (25mM 4-2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), pH 7.5) using the extinction coefficients and wavelengths suggested by Porra et al. (1989).

Determination of functional PSH reaction centres. The method developed for leaf discs by Chow et al. (1989a, 1991b) was applied. The leaf-disc-electrode system described above was used for leaf discs with a diameter of 19 mm. Following an initial dark equilibration of the leaf discs of about 20 min, repetitive xenon flashes (15 Hz; type FX200; EG & G Electro Optics, Salem, Mass., USA) of a certain intensity were applied for 4 min, followed by 4 min of darkness, before the procedure was repeated with another flash intensity. Different flash intensities as set by neutral-density filters were used to ensure that the calculations of functional PSII centres were based on the 02 yield of saturating flashes. The amount of functional PSII was expressed as mmol PSII per mol of chlorophyll.

Results Nature o f photoinhibition. F i g u r e 1 c o n f i r m s the n o w w e l l - e s t a b l i s h e d a p p r o x i m a t e l y l i n e a r c o r r e l a t i o n between the fluorescence r a t i o o f Fv/Fm, w h i c h is a m e a s u r e o f the m a x i m u m q u a n t u m efficiency o f P S I I , a n d the m a x i m u m q u a n t u m yield o f p h o t o s y n t h e s i s in differently p h o t o i n h i b i t e d p l a n t s ( D e m m i g a n d B j 6 r k m a n 1987). Clearly, the f a c u l a t i t v e s h a d e p l a n t Tradescantia g r o w n a t 50 ~tmol 9 m - 2 . S - 1 was m u c h m o r e sensitive to p h o t o i n h i b i t i o n w h e n e x p o s e d to 1700 Ixmol 9 m -2 9 s - 1 for 4 h a t 22 ~ C t h a n w a s p e a g r o w n at 50 ~tmol 9 m - 2 . S - 1 . P e a g r o w n at 300 ~tmol- m - 2 - s - 1 s h o w e d the highest resistance to p h o t o i n h i b i t i o n . This is c o n s i s t e n t with s h a d e - o r l o w - l i g h t - a c c l i m a t e d p l a n t s b e i n g m o r e susceptible to p h o t o i n h i b i t i o n t h a n sun- o r high-light-acclim a t e d p l a n t s ( A n d e r s o n a n d O s m o n d 1987). T h e finding t h a t Tradescantia a n d differently l i g h t - a c c l i m a t e d p e a s

1.0

i

a

i

i

0.8 0.6

~

0.4

~

J J J

0.2

/

/

J

/ I" 0.0

,

0.00

i

0.02

,

I

0.04

,

i

0.06

,

I

,

0.08

0.10

mol O 2-mol - lphotons absorbed Fig. 1. The relationship between the quantum yield of photosynthetic 02 evolution and the PSII fluorescence ratio, Fv/Fm of noninhibited and photoinhibited leaves of Tradescantia ([]) grown at 50 lamol 9m -2 S-1 and pea grown at 50 (*) or 300 ktmol" m - 2 " s - 1(11). Oxygen evolution and fluorescence were measured on leaf discs (diameter 15 mm) in a leaf-disc electrode containing an atmosphere of 21% 02 and 1% CO2. The temperature was 25 ~ C. Fv/Fm was measured using modulated fluorescence and saturating flashes. Points are means 4- SD for each treatment with n ranging from 5 to 10 independent determinations. Line drawn by linear regression of mean values fits the equation Y = 7.94X + 0.034, r = 0.992 "

fall o n the s a m e line o f the r e l a t i o n s h i p b e t w e e n F v / F m a n d the q u a n t u m yield o f p h o t o s y n t h e s i s (Fig. 1)justifies the use o f the e a s i l y - d e t e r m i n e d Fv/Fm r a t i o as a reliable m e a s u r e o f the efficiency o f p h o t o s y n t h e s i s in o u r c o m parative study of photoinhibition. It has b e e n discussed w h e t h e r p h o t o i n h i b i t i o n results f r o m direct i n h i b i t i o n o f P S I I r e a c t i o n centres a n d - o r f r o m i n d u c t i o n o f i n c r e a s e d a n t e n n a q u e n c h i n g o f excitation e n e r g y ( B j 6 r k m a n 1987). Therefore, we c a n n o t exclude the p o s s i b i l i t y t h a t the different levels o f p h o t o i n h i b i t i o n s h o w n in Fig. 1 result f r o m different m e c h a nisms. T o e v a l u a t e w h e t h e r the c r e a t i o n o f a n a l t e r n a t i v e energy q u e n c h e r in the p i g m e n t a n t e n n a was involved, we used flash-yield m e a s u r e m e n t s o f 0 2 e v o l u t i o n to d e t e r m i n e the a m o u n t o f f u n c t i o n a l P S I I r e a c t i o n centres in b o t h p h o t o i n h i b i t e d a n d n o n i n h i b i t e d p l a n t s (Fig. 2). By v a r y i n g the flash intensity we e n s u r e d t h a t the calculat i o n o f f u n c t i o n a l P S I I r e a c t i o n centres w a s b a s e d o n the yield o f s a t u r a t i n g flashes. T h e c o n t e n t o f f u n c t i o n a l P S I I r e a c t i o n centres ( m m o l - m o l - 1 , c h l o r o p h y l l ) in n o n i n h i b i t e d p l a n t s w a s : 2.36 4-0.16 for Tradeseantia g r o w n at 50 g m o l . m - 2 - s -1, a n d 2.41 + 0 . 0 5 a n d 2.664-0.09 for p e a g r o w n at 50 o r 300 lamol" m - 2 " s -1, respectively (Fig. 2 A - C ) . This o b s e r v a t i o n is in a c c o r d a n c e w i t h s h a d e p l a n t s u s u a l l y possessing a lower a m o u n t o f P S I I p e r unit c h l o r o p h y l l t h a n sun p l a n t s ( C h o w et al. 1990). A f t e r p h o t o i n h i b i t i o n , the n u m b e r s o f f u n c t i o n a l P S I I r e a c t i o n centres were r e d u c e d to 1.23 + 0.18 for Tradescantia g r o w n at 50 ~tmol - m -2 - s -1, a n d 1.86_+0.28 a n d 2.32_+0.16 for p e a g r o w n at 50 o r 300 g m o l 9 m -2 9 s 1, respectively. O u r e x p e r i m e n t a l a p p r o a c h o f v a r y i n g the flash intensity also allowed us to d i s c r i m i n a t e between p h o t o i n h i b i -

G. Oquist et al.: Photoinhibition and regulation of PSII i

i

|

i

453

'/" ' U

3.0

i

A

2.5

A

2.0

2

1.5 1.0

J

_o /

0.5 ,/

0

,

I

i

I

,

I

,

I

,

0.0 0.0

I

O

-~

3

I

I

I

|

I

2

2.0 1

O

1.5

r O e-

1.0

f f

0

=

3

,

I

,

|

I

,

I

I

,

i

I

i

i

i

i

1.0

i

S

f

I

0.5

J J f

i

0.0 0.00

I

0.02

i

I

0.04

,

I

0.06

i

I

0.08

i

0.10

tool O2.mo1-1 photons absorbed

e~