Photosynthesis Research 35: 305-310, 1993. (~) 1993 KluwerAcademic Publishers. Printed in the Netherlands. Regular paper

Chlorophyll fluorescence transients in a barley mutant lacking Photosystem I Ren6 K. Juhler 1, Mette Miller 1, David Simpson 2 & Raymond P. Cox 1.

llnstitute of Biochemistry, Odense University, Campusvej 55, DK 5230 Odense M, Denmark; 2Department of Physiology, Carlsberg Laboratory, Gamle Carlsbergvej 10, DK 2500 Copenhagen Valby, Denmark; *Author for correspondence Received 17 June 1992; accepted in revised form 4 November 1992

Key words:

gas phase, modulated fluorescence, pigment composition

Abstract

We have compared the properties of a mutant of barley lacking Photosystem I (viridis-zb 63) with the corresponding wild type using modulated fluorescence measurements. The mutant showed two unexpected characteristics. Firstly, there was a slow decline in the fluorescence signal in the light which was dependent on the presence of 0 2 at concentrations similar to that in air; 2% 0 2 in N 2 had no effect. The observed decline was mainly due to an increase in the non-photochemical quenching. Secondly, in the absence of 02, saturating light pulses caused a pronounced transient decrease in the fluorescence signal; a similar effect could also be observed in wild type plants when neither CO 2 nor 0 2 was present.

Abbreviations: PPFD-photosynthetic photon flux density; qN-non-photochemical quenching of chlorophyll fluorescence; q p - photochemical quenching of chlorophyll fluorescence

Introduction

Our current picture of the organisation of the photosynthetic apparatus is based on the idea of two types of reaction centre operating in parallel, as suggested by Robert Hill and Fay Bendall in 1960 (Hill and Bendall 1960). Since changes in the proportion of excitation energy re-emitted as fluorescence at ambient temperatures depend on the state of the Photosystem II but not directly on the state of Photosystem I, measurements of fluorescence intensity are a powerful tool for the study of the interactions between the two photosystems, with the advantage that intact plants can be studied as well as subcellular preparations. The introduction of the modulated fluorescence technique (Quick and Horton 1984, Ogren

and Baker 1985, Schreiber 1986) allowed new types of measurement by using periodic pulses of measuring light, with negligible integrated actinic effect, to detect the level of fluorescence against a background of actinic light. Measurements of the effect of short saturating light pulses during induction revealed that the quenching of fluorescence could be separated into a component which was abolished by the light pulse (photochemical quenching, qp), and a component which could not be abolished by illumination (non-photochemical quenching, qN)" The use of the modulated fluorescence technique has been reviewed by B61har-Nordenkampf et al. (1989), Horton and Bowyer (1990) and Krause and Weis (1991). The changes in fluorescence yield, qp and qN, with time are complex are not yet fully under-

306 stood. One approach to a better understanding is the use of simplified systems such as mutants, and we report here measurements with a mutant of barley (viridis-zb 63) which contains normal levels of Photosystem II but lacks Photosystem I (Hiller et al. 1980) and the corresponding wildtype.

Materials and methods

Plant material Barley plants (wild type Hordeum vulgare cv. Sval6vs Bonus and the mutant viridis-zb 63) were grown in aluminium trays containing vermiculite moistened with tap water. Measurements were made with material that had been grown at 20 °C for 7 days in a growth cabinet under a 16h l i g h t - 8 h dark cycle. Plants were illuminated with fluorescent tubes and the photosynthetic p h o t o n flux density at the level of the vermiculite was 30/xmol m -2 s -1. The pale green mutants were selected by visual inspection.

Modulated fluorescence measurements A PAM 101/103 (Heinz Walz Mess- und Regeltechnik, Effeltrich, G e r m a n y ) was used to measure fluorescence transients. All measurements were conducted on the leaf section between 1.5 cm and 2 . 5 c m from their leaf tip. Leaves were harvested in the light and the section excised and dark-adapted in a thermostatted measuring chamber for 15 min at 20°C before being subjected to a saturating light pulse (1 s, P P F D 11 0 0 0 ~ m o l m -2 s -1) using a Schott K L 1500 flash lamp for the determination of F m. The leaf section was then left in dark for a further 4 min before measurement of the fluorescence transients. F 0 was determined for a period of 3 s using red light modulated at 1 . 6 k H z (PPFD 0.05/~mol m - 2 s - 1 ) . The actinic light source (Walz 102-L, PPFD 50/~mol m -2 s -1) was then applied and the frequency of the measuring light was increased to 100 kHz. A train of saturating pulses (800 ms, PPFD of 11 000/zmol m -2 s -1) was started 14 s after onset of actinic illumination and continued at 20 s intervals. The apparatus was connected to a microcomputer for control of

the measurements and collection of data using a specially written program. Each trace presented is representative of at least 3 independent measurements with leaf segments from different plants. Fluorescence nomenclature is according to van K o o t e n and Snel (1990). During dark adaptation and measurements of fluorescence, the atmosphere of the leaf chamber was flushed with water-saturated air or gas mixtures produced using flow meters and a computer-controlled digital gas mixer equipped with a three-way magnetic valve. The composition of the gas mixtures was determined using a quadrupole mass spectrometer (Dataquad, Spectramass, Congleton, UK).

Pigment content The pigment composition of the pooled 1-cm leaf segments which had been used for the fluorescence measurements, and of whole shoots, was determined by HPLC. The plant material (about 0.3 g) was frozen in liquid N 2 and pulverized in the presence of an equimolar mixture of solid K H 2 P O 4 and K2HPO 4. The material was successively extracted three times with 2.0 ml acetone, twice with 2.0 ml 80% acetone and twice with 2.0 ml methanol, the residue being separated by centrifugation after each extraction. Pigments were analysed on a Kontron H P L C system using a 120 x 4 m m I.D. column packed with octadecylsilica (Shandon Hypersil 5-~m spherical particles) with detection at 450 nm as described previously (Juhler and Cox 1990).

Results

The absence of Photosystem I in the viridis-zb 63 mutant (Hiller et al. 1980) was confirmed by E P R measurements of P700 in shoot segments which had previously been used for fluorescence measurements. The segments were illuminated with far-red light and the size of the reversible light-induced signal was determined. There was no detectable signal in mutant shoot segments; any residual signal was less than 3% of that in the corresponding segments from wild type plants (results not shown). Figure 1A shows the results of fluorescence

307

WT

Air

(A)

u_* 14.

I

I

I

'

I

zb 63' Air (BI

It.

1

I

I

I

I

Extract

(Cl

\ 0

I

I

L

L

50

100

150

200

c o m p o n e n t s , one abolished by intense illumination (qp) and one representing a loss o f the potential fluorescence signal which c a n n o t be reversed by illumination (qN). Figure 1B shows the results of a similar experim e n t with the barley m u t a n t viridis-zb 63 which lacks P h o t o s y s t e m I. T h e decline in the fluorescence yield is m u c h slower than in the wild-type, and the initial series of transients is abolished. E a c h saturating pulse caused a small decline in the m e a s u r e d fluorescence signal. This is apparently an artefact of the m e a s u r i n g system, as r e p o r t e d by Ting and O w e n s (1992); we observed similar d o w n w a r d s transients with a solution o f chlorophyll in e t h a n o l (Fig. 1C). It thus seems that in this case qp is z e r o and the o b s e r v e d q u e n c h i n g is entirely c o m p o s e d of qN. T h e changes in q u e n c h i n g in the m u t a n t cannot be linked to n o r m a l p h o t o s y n t h e t i c processes, but might be the result of reactions involving 0 2 . In o r d e r to study the changes w i t h o u t interference f r o m the artefacts caused by the saturating pulses, we r e p e a t e d the m e a s u r e m e n t s w i t h o u t the pulse train and investigated the effects of various gas p h a s e compositions. T h e results are shown in Fig. 2. A n appreciable decrease in fluorescence (increase in qN) was o b s e r v e d in air, and also u n d e r an a t m o s p h e r e o f 1.1

Time (s)

\

1.0

N2 2~ 0 2

Fig. 1. Fluorescence induction kinetics under a gas phase of

air. (A) leaf segment from wild type barley; (B) leaf segment from the barley mutant viridis-zb63; (C) ethanol extract of pigments from a barley leaf. The origin of the time scale is the start of the actinic illumination. The signal intensity has been normalised so that F 0 has a value of 1.0. The inserts show a 3 second segment of the trace during a light pulse on an expanded scale. Further details are as given in the Methods section.

E

.~ 0.9 LI-

21~ Air

0.8

0.7 0

m e a s u r e m e n t s in wild type barley leaf segments in a n o r m a l air a t m o s p h e r e , showing the characteristic transients for leaves d a r k - a d a p t e d for a few m i n u t e s ( O g r e n and B a k e r 1985, Schreiber 1986) with a decline in fluorescence yield to a stable value within the first minute. T h e use of a train o f saturating 4 0 0 m s pulses allows the q u e n c h i n g to be s e p a r a t e d into the e x p e c t e d two

02

50

100

150

200

Time Is) Fig. 2. Effect of gas phase on the decay of the fluorescence

signal from leaf segments of the barley mutant viridis-zb 63. The percentage 02 values given correspond to the partial pressure in the gas phase, with the remainder being N 2. Times are from the beginning of the actinic illumination; the initial phase of the fluorescence transient is not shown. The fluorescence signal is normalised to Fm. Further details are as given in the Methods section.

308 21% 0 2 and 79% N 2. However, in the presence of pure N 2 o r an atmosphere containing 2% 0 2 in N 2 the fluorescence yield remained at a high level. The intensity of the actinic illumination used in the fluorescence measurements was low, and similar to those used during cultivation, so the effects were not caused by exposure to light intensities greater than those to which the plants were adapted. When the mutant was subjected to a train of saturating pulses in an atmosphere of N 2 , the pulses caused a reversible decline in the fluorescence yield which was greater than could be accounted for by the artefact associated with the pulse (Fig. 3A). The fluorescence signal returned to the level measured before the light pulse with complex kinetics which had a half-time of about a second (not shown). Similar results were obtained with N 2 containing 0.5% CO 2 (Fig. 3B) but with 21% O 2 and 79% N 2 the effects of the 1

I

i

i

pulse train were no larger than those obtained in air and thus attributable to the artefact (results not shown). A similar decrease in fluorescence yield caused by saturating light pulses could also be observed in wild type barley under an atmosphere of N z (Fig. 3C). When the wild type was exposed to 0.5% CO 2 in N 2, the observed fluorescence changes (Fig. 3D) were similar but not identical to those observed in normal air (see Fig. 1A). One possible explanation for the observed differences between the mutant and wild-type barley could be the absence of the xanthophylls involved in photoprotective processes (DemmigAdams 1990). Table 1 shows the content of photosynthetic pigments in whole shoots and in the leaf segments previously used for fluorescence measurements in both the wild type and the v i r i d i s - z b 63 mutant. In both cases the pigment content of the segments used for measure-

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zb 63 I

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[C)

150

2(~0

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0

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[D)

150

2()0

0

Time (s)

Fig. 3. Fluorescence induction kinetics from leaf segments of the barley m u t a n t viridis-zb 63 and wild type barley, u n d e r different atmospheres. (A) viridis-zb 63 under N2; (B) viridis-zb 63 under 0.5% C O 2 in N2; (C) wild type u n d e r N2; (D) wild type u n d e r

0.5% C O 2 in N 2. T h e origin of the time scale is the start of the actinic illumination. The fluorescence intensities are normalised so that F 0 has a value of 1.0. Further details are as given in the M e t h o d s section.

309 T a b l e 1.

C o n t e n t of photosynthetic pigments in the barley m u t a n t viridis-zb 63 and the corresponding wild-type

Material

Pigment content ( m g / g chlorophyll a) chl b

neox

viol

anth

zeax

lutein

~-car

wild type s e g m e n t viridis-zb 63 s e g m e n t

390 300

wild type shoot viridis-zb 63 shoot

420 290

v+ a+ z

29 18

28 25

ND 4

ND ND

116 172

102 87

28 29

40 36

41 107

ND 25

ND 39

128 212

109 75

41 172

M e a s u r e m e n t s were m a d e on whole shoots, harvested at the level of the vermiculite, and on the pooled leaf s e g m e n t s previously used for the fluorescence m e a s u r e m e n t s . Wild type shoots contained 1.13 m g chl per g fresh weight; the corresponding value for t h e shoots of the viridis-zb 63 m u t a n t was 0.44. A b b r e v i a t i o n s : chl b, chlorophyll b; neox, neoxanthin; viol, violaxanthin; anth, antheraxanthin; zeax, zeaxanthin; /3-car, /3-carotene; v + a + z, violaxanthin plus antheraxanthin plus zeaxanthin. ND, not detectable.

ments differs from the average for the whole shoot. The shoots of the mutant are greatly enriched in the xanthophylls involved in the photoprotective cycle (violaxanthin, antheraxanthin and zeaxanthin), but these seem to be concentrated away from the leaf segment used for the fluorescence measurements. Thus, there is no evidence to suggest that the observed difference in fluorescence is due to qualitative changes in the amounts of photosynthetic pigments.

Discussion The viridis-zb 63 mutant is reported to be devoid of Photosystem I reaction centres and to be depleted in the cytochrome b6- f complex, but to contain normal Photosystem II and a normal arrangement of the thylakoid membranes into stacked grana and exposed stroma membranes (Hiller et al. 1980, Simpson 1983). In the absence of Photosystem I to reoxidise plastoquinol, one might expect that the pool would become fully reduced and Photosystem II photochemistry would be blocked, leading to a high and constant fluorescence yield. In earlier studies with the viridis-zb 63 mutant, Simpson and von Wettstein (1980) observed a high F 0 in measurements of fluorescence induction curves in continuous light; however, the rapid rise to a maximum value was followed by a slow decline. We observed a similar effect in the presence of atmospheric concentrations of O 2 but not in its absence, demonstrating the involvement of oxygen in the process; in addition the use of modu-

lated fluorescence allowed the decline to be attributed to an increase in qr~- It is known that the plastoquinol pool can be oxidised by oxygen (McCauley and Melis 1986) and lower potential reductants would be expected to react readily with oxygen if the normal reoxidation pathways are blocked. However, it is not clear why such processes should lead to a continual decline in fluorescence during a period of illumination. The trivial explanation of irreversible destruction of the components involved can be excluded because the conditions of measurement are similar to those which the plants were exposed to during growth. A second noteworthy phenomenon is the transient decrease in fluorescence caused by a saturating light pulse in leaves of the mutant incubated under N 2 and in the wild type in the absence of either 0 2 or CO 2. These transients are clearly greater than the small instrumental artefacts which can be observed with chlorophyll in solution and probably result from the diversion of energy to processes other than normal electron transport, since the phenomenon is observed under conditions where reoxidation of the plastoquinone pool is prevented. High intensity illumination under these conditions will generate relatively high steady-state concentrations of low potential reductants in Photosystem II. This can lead to back reactions or cyclic electron transport pathways (Rees and Horton 1990), or to the reduction of other electron acceptors within the chloroplast. Other possible dissipative processes include the formation of the triplet states of chlorophyll and carotenoids (Siefermann-Harms 1987). Under natural condi-

310 t i o n s , c h l o r o p l a s t s in p h o t o s y n t h e t i s i n g leaves will always b e e x p o s e d to o x y g e n c o n c e n t r a t i o n s close to a t m o s p h e r i c so processes o c c u r i n g u n d e r a n o x i c c o n d i t i o n s m a y have n o physiological significance. T h e use of m o d u l a t e d fluorescence has b e c o m e a p o p u l a r t e c h n i q u e in b o t h basic a n d a p p l i e d p l a n t biology, b e c a u s e of the ease with w h i c h results c a n b e o b t a i n e d a n d the n o n - p e r t u r b i n g n a t u r e of the a p p r o a c h . H o w e v e r , m u c h r e m a i n s to b e u n d e r s t o o d a b o u t the biophysical a n d b i o c h e m i c a l processes causing the o b s e r v e d p h e n o m e n a . T h e use of m u t a n t s deficient in o n e of the two types of r e a c t i o n c e n t e r m a y be of v a l u e in p r o v i d i n g e x p e r i m e n t a l m a t e r i a l with a simplified c o m p o s i t i o n of the p h o t o s y n t h e t i c apparatus.

Acknowledgements T h i s r e s e a r c h was s u p p o r t e d by the Carlsberg F o u n d a t i o n a n d the D a n i s h N a t u r a l Science Research Council.

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