Photosynthesis Research 16:187-202 (1988) 'C Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

Regular paper

Interaction of electron acceptors with thylakoids from halophytic and non-halophytic species

CHRISTOPHER PRESTON l & CHRISTA CRITCHLEY 2 Botany Department, The Faculties, Australian National University, GPO Box 4, Canberra, ACT 2601, Australia, (present address." Photoconversion Research Branch, Solar Energy Research Institute, 1617 Cole Blvd., Golden, CO, USA); 2author for correspondence at present address. Botany Department, Universi O, of Queensland, St Lucia, QLD 4067, Australia

Received 30 June 1987; accepted in revised form 26 November 1987

Key words: : electron transport, halophyte, mangrove, photosystem II, spinach, thytakoid

Abstract. Thylakoid membranes isolated from halophytic species showed differences in their interactions with ionic and lipophilic electron acceptors when compared to thylakoids from non-halophytes. FeCN was considerably less efficient as electron acceptor with halophyte thylakoids, supporting much lower rates of 02 evolution and having a lower affinity. FeCN accepted electrons at a different, DBMIB insensitive, site with these thylakoids. 1,4-Benzo- quinones with less positive midpoint potentials were less effective in accepting electrons from halophyte thylakoids compared to nonhalophyte thylakoids, also reflected in lower rates of O, evolution and lower affinity. Considering the lipolphilic nature and the fact that there was no apparent change in the site donating electrons to the quinones, an alteration in the midpoint potential of this site by about + 100 mV is postulated for the halophyte thylakoids.

Abbreviations: AMPD - 2-amino-2-methyl- 1,3-propanediol, Cyt b 6 / f - cytochrome b 6/f com- plex, DBMIB - 2,5-dibromo-6-isopropyl-3-methyl-l,4-benzoquinone, DCBQ - 2,6-dichloro- 1,4-benzoquinone, DCIP - 2,6-dichlorophenol-indolphenol, DMBQ - 2,5-dimethyl-l,4- benzoquinone, Era7 - midpoint redox potential at pH 7.0, FeCN-K 3 Fe(CN)6 , HNQ - 5-hy- droxy- 1,4-naphthoquinone, MV - methylviologen, NQ - 1,4-naphthoq uinone, PBQ - phenyl- 1,4-benzoquinone, PC - plastocyanin, PQ - plastoquinone

Introduction

Photosynthetic electron transport occurs through a series of redox active molecules, with stepped redox potentials, from water to the terminal electron acceptor NADP ÷ . The energy to drive this electron transport is supplied by light absorption and charge separation in the two photosystems. This electron transport chain can be studied in sections by the use of artificial electron donors, acceptors and inhibitors. The commonly used electron acceptors have quite different properties and sites of actions (Izawa

188

1980). These electron acceptors have been divided into several classes de- pending on their nature and whether they support photophosphorylation. There are i) ionic compounds such as Fe(CB)~- (FeCN) which are lipid insoluble, ii) weak acid dyes such as DCIP which are lipid soluble in the protonated form only, and iii) lipid soluble compounds such as 1,4-benzo- quinones and oxidized p-phenylenediamines (Saha et al. 1971). The latter group accepts electrons only from PSII, methyl viologen (MV) only from PSI (Izawa 1980), whereas FeCN and DCIP can accept electrons from both PSI and PSII (Bohme et al. 1971, Nolan and Bishop 1975).

Although the oxidized p-phenylenediamines are excellent PSII electron acceptors (Saha et al. 1971) they are rarely used as they are chemically unstable (Izawa 1980). Instead FeCN and the benzoquinones seem to be preferred. The ability of these molecules to be effective electron acceptors is dependent both on their lipophilicity (Oettmeier et al. 1978), and their redox potential (Izawa 1980). Several substituted benzoquinones such as DBMIB are potent inhibitors of FeCN and MV supported electron transport (Bohme et al 1971, Oettmeier et al. 1978), as well as being able to accept electrons (Gould and Izawa 1973). This behaviour is concentration depen- dent where at low concentrations DBMIB aqcts as a platoquinone antagon- ist; at higher concentrations it can accept electrons in its own right (Gould and Izawa 1973).

With the nature of thylakoid membranes from halophytic species being somewhat different to those from non-halophytic species (Preston et al. 1987 a,b), it was considered likely and indeed observed that the interaction of electron acceptors with the membranes may also be different. The effec- tiveness of the charged molecule FeCN as an electron acceptor was tested for a variety of species. The use of several substituted benzoquinones allowed differences in the apparent midpoint potential of the quinone accep- tor site between non-halophytic and halophytic thylakoids to be observed.

Materials and methods

Isolated thylakoids from Pisum sativum, Spinacia oleracea, Beta vulgaris, Sarcocornia quinqueflora, Selliera radicans and A vicennia marina were prepared as described by Robinson and Yocum (1980) or Preston and Critchley (1986). Thylakoids were assayed for 02 evolution activity accord- ing to Critchley (1983) with buffer compositions as listed in Table 1.

K3 Fe(CN)6 was dissolved in H20. Five substituted benzoquinones whose structures and midpoint potentials (calculated from Clark 1960) are shown in Fig. 1, were dissolved in ethanol. The concentration of ethanol in the assay buffer did not exceed 1%.

Table 1

.Buffer compositions for O

Zevolution assays of halophytic and non-halophytic species used in t

his

stud

y.

All

conc

entr

atio

ns i

n mM

.

S. oleracea

P.

sati

vum

B. vulgaris

A.

mari

naS.

quin

quef

lora

S. radicans

HEPE

S-AM

PD p

H 7

.825

2525

2525

25(N

H4)

2S0

42

22

22

2NaC1

5050

5025

2525

HEPE

S-AM

PD p

H 7

.025

2525

250

250

250

(NH

4)2 S0 4

22

22

22

NaCl

5030

3015

0150

150

MES-

AMPD

pH

6.5

2525

2525

2525

(NH

4)2S0 4

22

22

22

NaCl

3030

3010

0100

100

190

O li

OH O HNQ 5-Hydroxy- t , 4-naphthoqulnone

Em7 = +33mY

O / ~ CH3

CH; O

DMBQ 2,5-Dlmethyl- 1,4-benzoqtutnone

Era7 = +180mV

0

il

II 0

NO I, 4-Haphthoqulnone

Em7 - +143mV

0

PBO PhenTl - ! , 4-beo.zoqLd.uone

~7 = +277=V

O C l ~ C l

H 0

DCBQ 2,6-Dlchloro-l,4-benzoqu£none

Em7 - +315mV

Fig. 1. Structures and midpoint redox potentials of the substituted 1,4-benzoquinone electron acceptors.

191

For all experiments an activity versus electron acceptor concentration curve was plotted and from these c u r v e s Wma x and K1/z were determined. The K~/2was the concentration of electron acceptor required for 50% of maxim- um activity and was calculated directly from the curves.

Results

FeCN was able to accept electrons from thylakoids of all species examined, but the efficiency varied considerably. Typical activity versus concentration data are shown in Fig. 2. Increasing FeCN concentration led to increasing activity with maxima at 100#m FeCN for S. oleracea thylakoids and at 750ym for S. quinqueflora thylakoids. The rate of FeCN supported 02 evolution by S. oleracea thylakoids was approximately 3.5 times greater than that by S. quinqueflora thylakoids. Addition of a quinone as well as

A

I j =

~'~ 400 o

E

@

o E 300

g ',7, 2 o 20C

UJ

"a

100 g~

i I I

0 0.5 1.0 FeCN Concentrat ion ( m M )

Fig. 2.02 evolution by S. oleracea and S. quinqueflora thylakoids as a function of the electron acceptor concentration assayed at pH 7.8. S. oleracea thylakoids; FeCN (O); FeCN with 500/~M DMBQ (o). S. quinqueflora thylakoids; FeCN only (a); FeCN with 250#M DCBQ (z~).

192

FeCN increased activity, but to a much larger extent for S. quinqueflora thylakoids than for S. oleracea thylakoids.

The Ki/2 for FeCN supported 02 evolution was several times higher for the halophytic species compared to the non-halophytic species both at pH 7.8 and pH 6.5 (Figs. 3 and 4). At pH 7.8 the non-halophytic species also had a higher g m a x for FeCN supported 02 evolution that did the halophytes (Fig. 3). However at pH 6.5 the Vm,x for all species had decreased, more so for the non-halophytic than for the halophytic species, such that the Vmax was similar for all species (Fig. 4). The C1 concentration present in the assay had no effect on the K1/z for FeCN for either S. oleracea or A. marina thylakoids (Table 2), but had an effect on V . . . . probably due to inhibition of 02 evolution at C1- concentrations away from the optimum. For this reason assays were performed at C1 concentrations close to the optimum.

The plastoquinone antagonist DMBIB inhibits 02 evolution when added at low concentrations (Lozier and Butler 1972, Trebst 1980). 2.5/~m DBMIB inhibited thylakoids from the non-halophytic species by 80%. With thylakoids from A. marina or S. quinqueflora, however, activity was only

1.2

1.0

' ~ 0.8 m,.

0.6 x co 0.4

E > 0.2

0

100

" " " 80 IE

80

~4o ~ 2 O

T T

O O

-.-- ~ 0 :) :3 I0 iJ E O" 0 > ~. o "r_ =

"~ {3 " - o 3 G, E ~- o

1-

T

I Fig. 3. The affinity (K~,2) and effectiveness (V,,,x) of FeCN as an electron acceptor for several species at pH 7.8. The Vm, ~ is measured relative to the O 2 evolution activity supported by 250#M DCBQ ( = 1) for each species. The K,.2 is the concentration of FeCN at which half the Vmax had been attained. Data are means _+ s.e.m. (n = 4).

I.-i,q. 4. The affinity and effectiveness of FeCN as an electron acceptor for several species assayed ; j t pH 6.5. For an explanation of V,,,,, and K , see Fig. 3. Data are means s.e.m. ( n = 4).

inhibited 20% by DBMIB, and there was a 10% increase in activity of S. rudicuns thylakoids in the presence of DBMIB (Fig. 5). These results suggest that in halophytes FeCN accepts electrons from a site situated close to the Q, binding site. DBMIB is not a perfect inhibitor and at high concentra- tions (20-30pm) it is able to accept electrons (Gould and Izawa 1973), however at the low concentrations used here it is unlikely to contribute much to electron transport. The residual activity remaining (20%) for the non-

Trrhir~ 2. The egect of NaCl on the affinity and effectiveness of FeCN as an electron acceptor for S. alr~raccw and A . niurinu thylakoids at pH 6.5. V,,,,, was measured relative to the activity with 250pM DCBQ as an electron acceptor.

+ 2 . 5 p M DBMIB

Fig. 5. FeCN supported O2 evolution in the presence of 2.5pM DBMIB for thylakoids from several species. All assays were performed at pH 7.0 with 500pM FeCN as an electron acceptor. 100% activity is the activity in the absence of DBMIB. Data are means s.e.m. (n = 4).

halophyte thylakoids is typical for this electron acceptor/inhibitor system (Trebst et al. 1970). The differences observed with DBMIB inhibition were not due to DBMIB being ineffective as an inhibitor for halophyte thyla- koids, as MV supported electron transport of both S. oleracea and S. quinquejlora thylakoids was inhibited by DBMIB (Table 3).

Lipophilic electron acceptors such as 1 ,Cbenzoquinones are able to byp- ass the site of DBMIB inhibition (Saha et al. 197 I , Izawa et al. 1973, Sarojini and Daniell, 1981) and accept electrons from PSI1 only. Several substituted 1,4-benzoquinones with midpoint redox potentials (E,,) varying from + 33 to + 315 mV were tested as electron acceptors. Typical responses of 0, evolution to quinone concentration are shown in Fig. 6. These data are for P. sativum thylakoids and the K,, has been indicated by a cross. HNQ was unable to support 0, evolution at all. NQ was able to support some 0, evolution and required 5OOpM for maximum activity. PBQ was the next most effective quinone and required 60 pM for maximum activity. DCBQ

Table 3. DBMIB inhibition of electron transport from H,O to MV by thylakoids from S. oleracea and S. quinquejora. Thylakoids were assayed in 25mM HEPES-AMPD pH 7.0, 2 m M (NH4),S04, 30pM MV, with 30mM (S. oleracea), or 150mM (S. quinqueflora) NaCI.

Activities are given in @moles O2 taken up. mg Chl- ' .h-l . - DBMIB + 2.5 pM DBMIB

S. oleracea 250 0 S. quinqueflora 94 0

195

I . c 0

E e0 ®

o

D

0 >

UJ

I I

e¢-

,°° t

300

20G

100

DMBQ

+

/

HNQ v • • I

O.S 1.0 Quinone Concentration ( m M )

Fig. 6. O~ evolution activity of P. sativum thylakoids as a function of quinone concentration assayed at pH 7.0. HNQ (v); NQ (0) ; DMBQ (11); PBQ (A): and DCBQ (o). The crosses indicate the position of K, ~ for each quinone.

and DMBQ were equally effective, but DMBQ required 5 times as much (500/~M) quinone for maximum activity. All the quinones when added at super-saturating concentrations became inhibitory.

The effectiveness of the quinones as electron acceptors can be plotted as a function of the midpoint potential of the quinone (Fig. 7). HNQ could not support 02 evolution of any thylakoid preparation tested. The other quin- ones showed varying effectiveness with the different species. In general activity increased with increasing Em7. DMBQ at t80mV and DCBQ at 315 mV provided the greatest activity for all three species of non-halophytes. PBQ at 277 mV occupied an anomalous position with activity lower than for DCBQ or DMBQ. The pattern was a little different for the halophytic species where PBQ and DCBQ (A. marina), or just DCBQ (S. quinqueflora and S. radicans) provided the greatest activity. The Kl/2 for these quinones showed the opposite trend (Fig. 8) where the quinones of high midpoint potential had the highest affinity. The K1/2 for PBQ and DCBQ did not vary with species, however the K1/2 for DMBQ for the halophytic species was about twice that for the non-halophytic species. The K~/2 for NQ also varied with species being much larger for S. quinqueflora and S. radicans thyla-

196

0 ii

E >

0 x

>E

1.5 t P.sativum ~ 1

1"5 ~ 1.0 7 1.5 ~ 1.0 0"5 0

1

0

4 S.qulnqueflora ~T~ ~T~

2 0 100 200 300

Em 7 (mV)

Fig. 7. The effectiveness of quinones as electron acceptors for thylakoids from several species as determined by the midpoint redox potential of the quinone. The effectiveness is measured as the maximum rate of O, evolution supported by the quinone, V ..... (Q), divided by the maximum rate of O, evolution supported by FeCN, V,,,x (FeCN), for that species. Assays were performed at pH 7.0. Data are means ± s.e.m. (n = 4).

koids. The K]/2 is p r o b a b l y no t influenced as m u c h by the midpo in t potent ia l

as the Vm~x is, and is p r o b a b l y also m o r e dependen t on the l ipophilicity o f

the qu inone (Oet tmeier et al. 1978).

The possibil i ty tha t the differences observed between the ha lophy t i c and non -ha lophy t i c species could be due to qu inones accept ing electrons at different sites was tested by using the inhibi tors D C M U and D B M I B .

197

g

v"

25

,

7Os025 I ~ [~1 B.vulgaris

0 i

100150 [-~ A.marina

0 , , ~ I , F ' 7

150 -= S.quinquef o r a

100

50

0

150

100

50

0

-i- S . r a d i c a n s

l | ~ Jr-~7 100 200 300

Em 7 (mV)

1"7,,4. <~'. The affinity of quinones for thylakoids from several species as determined by the midpoint redox potential of the quinone. The K~, is the concentration of quinone at which half the maximum activity tor that quinone and for that species had been attained. Assays were perlk~rmed at pH 7.0. Data are means + s.e.m. ( n - 4 ) .

DCMU inhibited electron transport from H~0 to all the quinones in all thylakoid preparations tested (Table 4). DBMIB inhibited quinone suppor- ted O~ evolution for both species by between 13-53%. There were some differences in DBMIB inhibition between species for some of the quinones, but these did not indicate a change in the acceptor site. However, there does appear to be a change in the nature of this site for halophytic species.

198

Table 4. DCMU and DBMIB inhibition of substituted 1,4-benzoquinone supported 02 evolution by S. oleracea and S. quinqueflora thylakoids at pH 7.0.

Quinone

Assay conditions are as described in materials and methods. % Activity remaining

S. oleracea S. quinqueflora

+ 1/~M DCMU + 2.5 #M DBMIB + 1 #M DCMU + 2.5/~M DBMIB

NQ 5 76 0 87 DMBQ 0 47 9 69 PBQ 4 61 0 59 DCBQ 0 64 0 58

Discussion

The interaction between artifical electron acceptors and thylakoid mem- brane components appears to be different for halophytic species compared to non-halophytes. The differences in sites of interaction are shown in Fig. 9, which is a schematic representation of the electron transport chain in the membrane showing the sites of action of several acceptors and inhibitors. FeCN accepts electrons from non-halophytic thylakoids at PSI (Bohme et al. 1973) leading to high rates of FeCN supported O2 evolution which is DBMIB sensitive (Fig. 9A). In contrast, FeCN supported 02 evolution by halophyte thylakoids occurs at low rates and is DBMIB insensitive (fig. 9B). There are three possible explanations for this behaviour, all of which may be involved: 1) FeCN, which is a charged molecule, has more difficulty in interacting with the thylakoid membranes from halophytes because of a higher surface electrical charge (Preston et al. 1987 a,b); 2) the site of FeCN interaction has a different redox potential; or 3) FeCN accepts from a different site. The third possibility is perhaps the most likely because of the DBMIB insensitivity, and the change in K1/2 also points to a different site. It is unlikely that this new site is close to PSI as whole chain electron transport is blocked in at least one (A. marina) of these species (Critchley 1982). McCauley et al. (1984) found that at low concentrations (< 200 pM) FeCN accepts solely from PSI whereas at higher concentrations it can also accept from PSII, pointing to the PSII site having a higher KI/2. The halophyte site also has a higher KI/2 suggesting it may be a PSII site. The response to FeCN by halophyte thylakoids is similar to that reported by Aoki et al. (1986) for thylakoids from Dunaliella tertiolecta, a halophytic green alga.

The ability of a 1,4-benzoquinone to support 02 evolution is highly dependent on its midpoint potential, with quinones of too low a midpoint potential being unable to support 02 evolution. Trebst et al. (1963) reported a sharp decrease in O2 evolution with quinones of Em7 less than 100mV

A

DCMU t

Q A - ' ~ B i

t

Pheo

Z,-,,~P680

O~

PsE

B Q ~ --~FeCN

/---DBMIB

,0

DBMIB

Cyt bl/f

199

FeCN NADP+ NADPH

, J ..--~Fd--~F N R

i

~PC

ps T_

DCMU t

Q A"~ ~ 8 i

I

Pheo

1 Z - - ~ - P 6 8 0

H20 O

PSiT

B Q ~ _..,~FeCN

FeCN ~ - / - - - D B M I B

"~.-.-,.,,~ p Q

DBMIB PC

FeCN MV J NADP + NADPH

,,,~.-~ ~ .._-.~ Fd -.-*-- F N R

Cyt b6/f PS I--

Fig. 9. A model showing electron transport pathways of thylakoids with the sites of electron acceptor and electron transport inhibitor action shown. Non-halophytic thylakoids (A); and halophytic thylakoids (B). The three intrinsic membrane complexes; PSII, Cyt b6/f and PSI are separated by the mobile carriers PQ and PC. BQ is any of the substituted 1,4-benzoquin- ones. The sites of inhibition by DCMU and DBM-[B are shown by dotted lines. In the halophytic thylakoids (B) FeCN is shown to accept electrons from the PQ pool prior to the sites of DBMIB inhibition.

200

presumably for S. oleracea thylakoids. This has also been observed here where for thylakoids from three species, S. oleracea, P. sativum and B. vulgaris, NQ at 143 mV gave slightly less activity, and HNQ at 33 mV gave no activity. Thylakoids from the halophytic species show this effect of Era7 to a greater extent, where DMBQ at 180 mV and sometimes PBQ at 277 mV gave lower activity. This suggests a change in the midpoint potential of the site of interaction with quinone electron acceptors. The relative location of the site was not altered as 02 evolution by S. oh, racea and S. quinqueftora was equally inhibited by DCMU or DBMIB.

Quinones are presumed to accept electrons from the PQ pool (Izawa et al. 1973, Sarojini and Daniell 1981, Cohen and Barton 1983), although it is more likely that they accept electrons (as well as protons) close to the QB site and PSI1 electron transport should be DBMIB insensitive. Quinone suppor- ted O, evolution proved to be inhibited by DBMIB by 20-30% depending on the quinone. This inhibition is probably due to competition by DBMIB for the quinone binding site. DBMIB at higher concentrations (20-30 #M) is able to accept electrons but does not do so as effectively as other quinones (Gould and Izawa 1973), and may have two sites of interaction with different affinities in much the same way as 5-n-undecyl-6-hydroxy-4,7-dioxoben- zothiazole, another PQ antagonist (Oettmeier et al. 1981). There are several possible sites for quinones to accept electrons within the PQ pool. In addition to the DCMU and DBMIB sites at the reducing and oxidizing sides of the PQ pool, respectively (see Fig. 9), there is a third site which can be inhibited by halogenated naphthoquinones (Pfister et al. 1981). It is also possible that the exact site of electron acceptance may vary between species.

Figure 9 shows the proposed sites of interaction of electron acceptors in thylakoids from non-halophytes (Fig, 9A) and halophytes (Fig. 9B). The major difference is in the site of interaction of FeCN which is the PQ pool in halophytic thylakoids. The other important difference is an apparent increase in the midpoint potential of the PQ pool of about 100 inV.

Whether these alterations are related to pronounced changes in stacking properties of halophytic thylakoids (Preston et al. 1987 a,b) based on modi- fications of proteins and lipid-protein interactions remains to be elucidated.

Acknowledgements

CP was supported by a Commonwealth Postgraduate Scholarship and CC by the National Research Fellowship Scheme and a CSIRO Extra Mural Grant. Both authors are most grateful to Ms Joanne Perks for expert handling of the manuscript.

201

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