See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/284293643

Electron Transport, Photophosphorylation and Thylakoid Stacking

Chapter · January 1984

DOI: 10.1007/978-94-017-4973-2_19

CITATIONS READS

19 1,196

1 author:

Wah Soon Chow

Australian National University
256 PUBLICATIONS   13,292 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Measuring Canopy Photosynthesis View project

All content following this page was uploaded by Wah Soon Chow on 17 April 2016.

The user has requested enhancement of the downloaded file.

 111.1. 83

ELECTRON TRANSPORT, PHOTOPHOSPHORYLATION AND THYLAKOID STACKING

w. s. CHOW/Glasshouse Crops Research Institute

Lettuce chloroplasts in the stacked or unstacked state have been compared

with respect to cyclic photophosphorylation mediated by phenazine metho-
sulphate, and non-cyclic photophosphorylation and uncoupled electron trans-
port mediated by methyl viologen. The same ionic medium was used to assay
chloroplasts in either structural state, in order to equalize effects of
ionic composition which may influence reaction rates. Over a wide range
of irradiance (400-700 nm, 20°C), chloroplasts in the stacked state ex-
hibited higher rates of non-cyclic photophosphorylation, but lower rates
of cyclic photophosphorylation compared with unstacked thylakoids.
Unstacked membranes gave higher rates of uncoupled non-cyclic electron
flow at relatively high irradiance, but not at low irradiance. The results
are discussed in terms of the lateral heterogeneous distribution of
membrane components of chloroplasts in the stacked state.

1. INTRODUCTION

An outstanding structural feature of chloroplasts of mo~t higher

plants and some green algae is the formation of grana! stacks consisting
of appressed thylakoids connected by non-appressed membranes. Associated
with grana! formation is a lateral heterogeneous distribution of certain
components of the thylakoid .membranes, e.g. the coupling factor (Miller,
Staehelin, 1976), Ferredoxin-NADP reductase (Jennings et al. 1979) and
the two photosystems (Andersson, Anderson, 1980).

Isolated envelope-free chloroplasts can be induced to lose their

grana! structure by changing the ionic composition of the suspension
medium. Upon unstacking, there is a random distribution of thylakoid
membrane components. In this work, chloroplasts with or without a grana!
structure were prepared and assayed in the same medium in which the initial
structural state was largely maintained. Differences in activity may be
ascribed to the structural differences.

2. MATERIALS AND METHODS

Chloroplasts were isolated from Lactuca sativa L. cv. Celtuce grown

at 20°C~ 70% relative humidity and 12 h day-length, the irradiance being
80 W m- of cool white fluorescent light. The ice-cold grinding medium
consisted of 400 mM sorbitol, 5 mM MgC1 2 , 1 mM Mncl 2 , 2 mM EDTA, 10 mM
KCl, 20 mM Hepes {pH 7.6, KOH), 0.5% BSA, 0.5% polyvinylpyrrolidone and
5 mM sodium isoascorbate.

To prepare unstacked membranes, chloroplasts were osmotically shocked

in 20 mM KCl. An equal volume of a solution was added to give final con-
centrations of 100 mM sorbitol, 20 mM KCl and 0.1 mM Hepes (pH~ 7.4).
The suspension was kept at room temperature for 10 min. After centri-
fugation, the pellet was resuspended in a small volume of the supernatant.
Stacked chloroplasts were obtained similarly, except that 4 mM MgC1 2
replaced 20 mM KCl in the medium used for osmotic shock, giving 2 mM MgC1 2
in the final suspension. Both preparations containing about 2 mg Chl/ml
were kept on ice for about 1 h before use.
Sybesma, C. (ed.), Advances in Photosynthesis Research, Vol. III. ISBN 90-247-2944-0.
© 1984 Martinus Nijhoff/Dr W. Junk Publishers, The Hague/Boston/Lancaster.
111.1. 84

For measurements of electron transport and photophosphorylation,

chloroplasts were diluted into a basic medium containing 100 mM sorbitol,
3.5 mM MgC1 2 , 3 mM ADP, 2 mM K2HP0 4 , and 0.1 to 0.5 mM Tricine (pH 8.0)
to which was added either (i) 50 ~M phenazine methosulphate (PMS) or
(ii) 0.1 mM methyl viologen (MV) + 0.5 mM NaN 3 ± 10 ~M Gramicidin D. Each
sample containing 20 ~g Chl/ml was incubated in the dark for 2 min before
illumination. Steady-state rates of o2 uptake and photophosphorylation
were measured with an o2 electrode and a pH electrode, respectively, at 20°C.

3. RESULTS

In order to select an assay medium which maintained the greatest

difference between pre-stacked and pre-unstacked membranes, the MgC1 2 con-
centration was varied. Fig. 1 shows that the chlorophyll fluorescence
level showed the greatest difference at about 3.5 mM MgC1 2 , the background
concentrations of ADP and K2HP0 4 being fixed at 3 and 2 mM, respectively.
At this optimal concentration of MgC1 2 , the apparent absorbance spectra
were measured. Fig. 2 shows the spectral differences between pre-stacked
and pre-unstacked samples ascribable to different light-scattering properties.
§
/" \

~
! 4
1.:
.;.
.~.r'/ _____
~-. Ql
u
0:::
0.8
.,... -"" '
''
'
' \
~-~

' 'I
"'5 \\
i....
.0
•I
S / I VI 0.6
0 / I
.0 \
2 I
<{
'' '
~ ~·
u
Ql
J
I "1::
~ 0.4
'' '
-
~ "'
t
>
:;: u
~
0::

0 2 4 6 8 10
O.Z'------.J4s'-o---s-'s-o---6,-Ls-o---=7=so
IMgCl 2 1 (mM) Wavelength ( nm)

FIGURE 1. Room-temperature chlorophyll FIGURE 2. Apparent absorbance spectra

fluorescence (686 nm) from pre-stacked of pre-stacked and pre-unstacked
(S) and pre-unstacked (U) thylakoids thylakoids. Assay medium as in
suspended in the basic assay medium Figure 1, except with 2 ~M Gramicidin
supplemented with 0.1 mM MV, 0.5 mM D instead of DCMU. Chlorophyll con-
NaN 3 and 10 ~M DCMU, with varying centration was 6.7 ~g/ml. Slit
concentrations of MgC1 2 • Blue-green width 1 nm.
excitation light, about 5 W m2.

Fig. 3A illustrates the irradiance dependence of non-cyclic photo-

phosphorylation mediated by methyl viologen. Pre-stacked chloroplasts
gave higher rates than pre-unstacked membranes. The situation was reversed
with regard to cyclic photophosphorylation mediated by PMS (Fig. 3B).
Cyclic photophosphorylation also required much higher irradiance for
saturation. Fig. 3C illustrates the irradiance dependence of uncoupled,
non-cyclic electron transport. At high irradiance, pre-stacked membranes
gave lower rates of uncoupled electron flow than pre-unstacked membranes.
Uncoupled electron flow required higher irradiance for saturation than
did non-cyclic photophosphorylation.
 111.1. 85

FIGURE 3. Steady-state rates of ATP-

formation and uncoupled electron
transport for pre-stacked (S) and
2
&! 5 r-----~ -.------.--------------___ .,
.6
pre-unstacked (U) thylakoids.
Chlorophyll concentration was 20~g/ml
in all cases. Maximum irradiance ~ 0..
.--:.!;(
1 ~

2.8 mmol photons m-2s-1 (400-700 nm). ~ ....

'i3
Basic medium was supplemented by (A) (;-
I
0.1 mM MV + 0.5 mM NaN 3 , (B) 50 ~M ~
0•
PMS and (C) 0.1 mM MV + 0.5 mM NaN3
+ 10 ~M Gramicidin D. 2 min dark S2
pre-incubation in each case.
Rates in mol ATP or o 2
(mol Chl)- 1 s- 1 •

..
8
--------:--------------------- ---~

..
~
.>::
J!! 4
-w c.

.
:::>
ON

0
S2
0 20 40 60 80 100
Relative lrrndiance

4. DISCUSSION

When pre-stacked or pre-unstacked thylakoids were diluted into the

assay medium containing an optimal concentration of MgC1 2 , they main-
tained a high or low level of chlorophyll fluorescence (Fig. 1), and
greater or lesser light scattering, respectively (Fig. 2). As the level
of chlorophyll fluorescence (Barber, 1980) and light scattering (Wollman,
Diner, 1980) can sometimes be correlated with thylakoid stacking, it is
proposed that the chloroplasts largely maintained their initial structural
state when diluted into the assay medium, a similar case having been
reported for a colloid (Overbeek, 1977). Since the same medium was used for
comparing pre-stacked and pre-unstacked membranes, the observed differences
in their photosynthetic activities could not have arisen from the effects
of different ionic composition, but probably originated from different
degrees of thylakoid stacking being maintained in the assay medium.

As shown in Fig. 3A, pre-stacked membranes carried out non-cyclic

photophosphorylation at about twice the rate of pre-unstacked thylakoids.
This difference was not primarily due to the unstacked membranes being
leakier to H+, since the 'P/2e' ratio was only 20% higher in pre-stacked
membranes, and since pre-unstacked membranes in fact showed a higher rate
of cyclic ATP formation. Neither was the difference in non-cyclic photo-
phosphorylation rates due to differing capacities for electron transport,
since the uncoupled rate of non-cyclic electron flow was lower in pre-
stacked membranes at high irradiance (Fig. 3C).

It is proposed that the movement of H+ from the sites of production

(water splitting and oxidation of plastoquinol) to the coupling factor
(CF) may be a significant limiting step in non-cyclic photophosphorylation.
Haraux and de Kouchkovsky (1982) have suggested the existence of a
111.1. 86

significant lateral resistance to protons. Presumably pre-stacked membranes

gave higher rates of non-cyclic photophosphorylation because of an effective
and controlled proton movement from the sites of production to the site of
utilization (see also Albertsson, 1982; Anderson, Melis, 1983). Such con-
trolled movement may result from the proper positioning or 'anchoring' of
membrane components. In unstacked membranes, although the average distance
betweenPSIIand CF is smaller, the random motional fluctuations of the
membrane components may be so large as to hinder proton movement to the
coupling factor. It is noticeable that uncoupled, non-cyclic electron
transport required higher irradiance for saturation than non-cyclic photo-
phosphorylation, suggesting that a limiting step (most probably the H+
gradient) has been eliminated by the uncoupler. Further work on proton
movement using the present approach may give an insight into the mechanism
of conduction to the coupling factor.

REFERENCES
Albertsson PA (1982) FEBS Letters 149, 186-190.
Andersson B and Anderson JM (1980) Biochim. Biophys. Acta 593, 426-439.
Anderson JM and Melis A (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 745-749.
Barber J (1980) FEBS Letters 118, 1-10.
Haraux F and de Kouchkovsky Y (1982) Biochim. Biophys. Acta 679, 235-247.
Jennings RC, Garlaschi FM, Gerola PD and Forti G (1979) Biochim. Biophys.
Acta 546, 207-219.
Miller KR and Staehelin LA (1976) J. Cell Biol. 68, 30-47.
Overbeek J Th G (1977) J. Colloid Interface Sci. 58, 408-422.
Wollman FA and Diner BA (1980) Arch. Biochem. Biophys. 201, 646-659.

ACKNOWLEDGEMENTS
The author wishes to thank Mrs J M Le Fay for laboratory assistance.

Authors address: w. S. Chow, Glasshouse Crops Research Institute,

Worthing Road, Littlehampton, w. Sussex, BN16 3PU, U.K.

View publication stats