AbstractThe accumulation of the 33-, 23- and 16-kDa polypeptides of the oxygen-evolving complex has been compared with the development of oxygen evolution activity during greening. Both the 33- and 23-kDa proteins are present in etioplast membranes whereas the 16-kDa species is detectable only in trace amounts. The accumulation of the 3 polypeptides' greening is asynchronous. Only the 16-kDa polypeptide is formed in concert with the appearance of oxygen evolving activity.Thylakoid developmentPhotosynthetic oxygen evolution33-kDa protein23-kDa protein16-kDa proteinWestern blottin

Andersson, Bertil

Ryrie, Ivan J.

Young, Susan

Elsevier - Publisher Connector 

Volume 177. number 2 FEBS 1987 November 1984 
Development of the 33-, 23- and 16-kDa polypeptides of the 
photosynthetic oxygen-evolving system during greening 
Ivan J. Ryrie, Susan Young and Bertil Anderson* 
Department of Applied Mathematics, Research School of Physical Sciences and Department of Environmental Biology, 
Research School of Biological Sciences, Australian National University, Canberra, ACT 2601. Australia, and *Department 
of Biochemistry, University of Lund, PO Box 124, S-221 00 Lund, Sweden 
Received 13 August 1984 
The accumulation of the 33-, 23- and 16-kDa polypeptides of the oxygen-evolving complex has been com- 
pared with the development of oxygen evolution activity during greening. Both the 33- and 23-kDa proteins 
are present in etioplast membranes whereas the 16-kDa species is detectable only in trace amounts. The 
accumulation of the 3 polypeptides’ greening is asynchronous. Only the 16-kDa polypeptide is formed in 
concert with the appearance of oxygen evolving activity. 
Thylakoid development Photosynthetic oxygen evolution 33-kDa protein 
Western blotting 
23-kDa protein IGkDaprotein 
1. INTRODUCTION 
The structure of the PS II oxygen-evolving 
system in thylakoids consists of the reaction center 
and light-harvesting assemblies, intimately 
associated with the water-splitting complex which 
contains the 33-, 23- and 16-kDa polypeptides and 
probably also a so far unidentified Mn-carrying 
protein [l]. Studies of the oxygen evolution 
mechanism have so far been focussed on the 33-, 
23- and 16-kDa proteins since these are hydrophilic 
and are bound to the inner membrane surface, and 
are thus amenable to resolution-reconstitution ex- 
periments. Extraction of either inside-out 
thylakoids or oxygen-evolving particles with con- 
centrated NaCl releases the 23- and 16-kDa species 
and inhibits oxygen evolution [2,3]. Activity is 
reportedly restored either by rebinding the 23-kDa 
protein [2,3], by high chloride concentrations [4,5] 
or by 10 mM CaClz [6,19], suggesting that one 
function of the 23-kDa species is to increase the af- 
finity of the catalytic center for essential Ca and 
chloride. Additional release of the 33-kDa protein 
occurs upon sonication [7] or treatment with high 
Tris [8] or CaCl2 [9] concentrations. Provided Mn 
is retained during extraction, activity can ap- 
parently be restored by rebinding the 33-kDa [lo] 
or 23-kDa species [7] or simply by high chloride 
concentrations [l 11. Thus, a somewhat confused 
picture is emerging which nonetheless uggests that 
the 33- and 23-kDa proteins are non-catalytic but 
are in some way involved in the retention or bind- 
ing of Mn, Ca and chloride at the active center. 
The role of the 16-kDa protein is completely 
unknown and only one report has appeared show- 
ing any beneficial effect in the reconstitution of ox- 
ygen evolution activity [ 121. 
* To whom correspondence should be addressed 
Abbreviations: PS, photosystem; IML, intermittent il- 
lumination; LIDS-PAGE, lithium dodecyl sulphate- 
polyacrylamide gel electrophoresis 
One means of probing the essentialness or other- 
wise, and possible roles, of the 33-, 23- and 16-kDa 
proteins is to compare their accumulation during 
greening with the development of oxygen evolution 
activity. Here, this correlation has been studied 
during prothylakoid differentiation under either 
continuous or intermittent light conditions, using 
Published by Elsevier Science Publishers B. V. 
00145793/84/$3.00 0 1984 Federation of European Biochemical Societies 269 
Volume 177, number 2 FEBS LETTERS November 1984 
the sensitive immunological technique of Western 
blotting for unambiguous detection of the 
polypeptides. 
2. EXPERIMENTAL 
Barley seedlings (Hordeum vulgare cv. Clipper) 
were grown for 6-7 days in darkness then trans- 
ferred to an IML regime consisting of cycles of 2 h 
A B 
1 2 3 4 1 
ORIGIN- 
darkness interrupted by 2 min light of intensity 
90 ~E.m-2.s-1 [13]. Where indicated, the plants 
were further exposed to continuous illumination at 
170~E~m-2~s-‘. Intact chloroplasts [13] and 
etioplasts [14] were isolated as described then 
membrane fractions were prepared by osmotic 
lysis in 10 mM NaCl and 5 mM Tricine-NaOH, 
pH 7.5, for 15 min followed by centrifugation at 
10000 x g for 10 min. Control thylakoids were 
2345 6 78 
., ~- I.- .* 
-33 
-23 
-16 
Fig.1. Development of the 33-, 23- and 1dkDa polypeptides of PS II during intermittent illumination of dark-grown 
barley seedlings. (A) Specificity of the antisera. Samples (60 pg protein) of continuous-light thylakoids were 
electrophoresed by LiDS-PAGE then the lanes were processed individually by Western blotting using 1: 800 dilutions 
of the antisera against the 33 (lane l), 23 (lane 2) or 16 (lane 3) kDa proteins. Lane 4 was processed with a combination 
of the antisera. (B) Prothylakoids were prepared from IML plants and the 3 polypeptides were detected by LiDS-PAGE 
and Western blotting. Lanes l-4, 12, 24, 36 and 48 IML cycle prothylakoids, respectively; lane 5, control (continuous- 
light) thylakoids; lane 6, etioplast membranes; lanes 7-8, 120-pg samples of etioplasts and control thylakoid 
membranes, respectively. Both lanes were processed at 1:400 dilutions of the antisera. 
270 
Volume 177, number 2 FEBS LETTERS November 1984 
isolated from plants grown in the greenhouse using 
a 16 h per day photoperiod. 
Antisera against the 33-, 23- and 16-kDa pro- 
teins were raised in rabbits following injection of 
the purified proteins [4]. PS II oxygen evolution 
was assayed in 50 mM sorbitol, 10 mM NaCl, 
0.5 mM NH&l, 10 mM Tricine-NaOH, pH 7.4, 
using dimethylbenzoquinone as acceptor [ 151. Pro- 
cedures for LIDS-PAGE and Western blotting 
[ 131 are described elsewhere. 
3. RESULTS 
The specificities of the antisera raised against he 
33-, 23- and 16-kDa polypeptides were first ex- 
123456 
ORIGIN- rd 
-33 
-23 
-16 
FRONT- * KY 
Fig.2. Development of the 33-, 23- and I6-kDa 
polypeptides during continuous illumination of 12 IML 
cycle barley seedlings. Samples (6Opg protein) of 
prothylakoid membranes were electrophoresed by 
LiDS-PAGE then subjected to Western blot analysis. 
Lanes l-5, 30 min, 3, 6, 10 and 16 h light, respectively; 
lane 6, control (continuous-illumination) thylakoids. 
amined by Western blotting using thylakoids from 
control (continuous-light) plants as the antigen for 
electrophoresis. Each antiserum was highly 
specific and showed reactivity with only the single 
band amongst the thylakoid polypeptide profile 
(fig. 1 A, lanes l-3). Where the antisera were com- 
bined only the 3 sharp bands were seen on blots 
(lane 4); thus, the mixture of antisera enabled a 
simultaneous examination of the 3 polypeptides 
and was therefore employed routinely. 
The putative relationship between these 
polypeptides and the development of oxygen 
evolution activity was initially examined in plants 
greened under IML. Since the assembly of the 
PS II complex under these conditions is quite ab- 
normal [16,17] the IML regime represents an in- 
teresting contrast with the continuous-light system 
(see below). After only 12 IML cycles both the 33- 
and 23-kDa polypeptides were very prominent on 
blots (fig.lB, lane 1). Both proteins continued to 
accumulate during greening (lanes l-4) and after 
48 IML cycles were as abundant as in control 
thylakoids (lane 4 vs 5). In contrast, the 16-kDa 
protein, though detectable after 12 IML cycles, 
was low in concentration (lane 1) but increased 
quite markedly by 24 IML cycles (lane 2) to an 
amount approximating that in control membranes 
(lane 5). The accumulation of the 16-kDa protein 
is therefore asynchronous with the 33- and 23-kDa 
species but notably, quite closely correlates with 
the onset of oxygen evolution which also com- 
mences between 12 and 24 IML cycles (table 1). 
Given the prominence of the 33- and 23-kDa 
species after only 12 IML cycles, we questioned 
whether they might also be present in etioplasts 
and thus synthesized before exposure of the seed- 
lings to light. Indeed, both proteins were clearly 
present although the 16-kDa band could not be 
seen under these conditions of detection (lane 6). 
However, by increasing the sensitivity of detection 
(see fig.1 legend) the 16-kDa protein was also 
detectable but was clearly present only in trace 
amounts compared with control thylakoids (lane 7 
vs 8). The fact that identical blots were obtained 
whether intact or osmotically lysed etioplasts were 
used as antigen (unpublished results) strongly sug- 
gests that all 3 proteins are already associated with 
the prothylakoid membranes. 
The relationship between the accumulation of 
the 3 polypeptides and the onset of oxygen evolu- 
271 
Volume 177, number 2 FEBS LETTERS November 1984 
Table 1 
The development of photosynthetic oxygen evolution in 
prothylakoids from plants greened under intermittent or 
continuous illumination 
Illumination 
conditions 
PS II activity 
IML cycles 12 
IML cycles 24 
IML cycles 36 
IML cycles 48 
pm01 02. minm2 am01 02. min-’ 
. mg protein-’ .mg chl-’ 
0.01 0.09 
0.13 0.55 
0.26 0.65 
0.34 0.82 
12 IML cycles 
+ l/2 h light 0 0 
+ 3 h light 0 0 
+ 6 h light 0.23 0.25 
+ 10 h light 0.49 0.43 
+ 16 h light 0.39 0.31 
Control: 
continuous light 0.64 0.50 
Intact plastids were prepared from plants after various 
stages of illumination then prothylakoids were isolated 
from osmotic lysates and assayed for oxygen evolution 
activity 
tion during greening has been further examined 
where early stage (12 cycle) IML plants were expos- 
ed to continuous illumination. As shown in fig.2 
the 33- and 23-kDa proteins were again prominent 
in the early stages of greening and accumulated 
further under continuous light. By contrast, the 
amount of the 16-kDa protein was initially very 
low but increased most dramatically between 3 and 
6 h of illumination (lane 2 vs 3). Again, this in- 
crease coincided with the onset of oxygen evolu- 
tion activity, which also begins to develop between 
3 and 6 h of illumination (table 1). 
4. DISCUSSION 
Our findings demonstrate a close correlation 
between the accumulation of the 16-kDa protein 
during greening and the onset of PS II oxygen 
evolution. In contrast with the 33- and 23-kDa 
polypeptides, which are present at high levels in 
etioplasts, the 16-kDa species is initially present 
only in trace amounts, either under continuous or 
212 
intermittent light conditions, but accumulates 
quite strongly during development of the 
photosynthetic activity. These findings are thus 
strong evidence for an essential role of this protein 
in the oxygen-evolving complex. The nature of this 
role still remains unclear, however, and further 
progress will probably depend on clarification of 
the so far conflicting results from in vitro 
reconstitution experiments (see section 1). What is 
presently certain is that from the developmental 
standpoint, all 3 polypeptides are synthesized and 
inserted in barley prothylakoids before oxygen 
evolution activity becomes measurable. 
The detection of the 33-, 23- and 16-kDa 
polypeptides, and previous reports of the presence 
of cytochromes f, b-559Lp and b-563, and the 
CFr-ATPase complex [ 181, show clearly that some 
components of the thylakoid photochemical ap- 
paratus are present in etioplasts and thus do not re- 
quire light for their biogenesis. Alternatively, 
many polypeptides are completely missing at this 
stage and may eventually be assembled into the 
multi-subunit, supramolecular membrane com- 
plexes in an asynchronous fashion, as is the case 
for PS I [15]. From the present results, and other 
developmental studies [ 16,171, this also seems to be 
true of the PS II oxygen-evolving complex. As a 
means of studying the overall assembly of the 
PS II complex, it will clearly be interesting to cor- 
relate the present findings with similar studies on 
the formation of the reaction center core, and 
light-harvesting, polypeptides during greening. 
ACKNOWLEDGEMENT 
We thank Dr Christer Larsson for a generous 
gift of antibodies. 
REFERENCES 
[l] Haehnel, W. (1984) Annu. Rev. Plant Physiol. 35, 
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[4] Andersson, B., Critchley, C., Ryrie, I.J., Jansson, 
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273 


Development of the 33-, 23- and 16-kDa polypeptides of the photosynthetic oxygen-evolving system during greening 

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Development of the 33-, 23- and 16-kDa polypeptides of the photosynthetic oxygen-evolving system during greening

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