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Running Title: 1
State Transitions in chlorina and cpSRP Mutants 2
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Corresponding Author 4
Bernhard Grimm 5
Department of Plant Physiology, Humboldt University Berlin, 10115 Berlin, Germany 6
Phone: 0049-302093-6106 7
Fax: 0049-30-2093-6337 8
E-mail: [email protected]
Plant Physiology Preview. Published on September 23, 2016, as DOI:10.1104/pp.16.01009
Copyright 2016 by the American Society of Plant Biologists
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Chloroplast chlorina and cpSRP Mutants Implicate LHCI in LHCII-PSI Complex 10
Assembly 11
Peng Wang and Bernhard Grimm* 12
Department of Plant Physiology, Humboldt University Berlin, 10115 Berlin, Germany 13
*Address corresponding to [email protected] 14
One-sentence Summary: 15
Comparative analysis of chlorina and cpSRP mutants provides the novel genetic evidence for 16
the flexible organization of light-harvesting complexes, and their dynamic and reversible 17
allocation to the two photosystems. 18
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Footnotes 21
Author Contributions 22
P.W. and B.G. designed the research; P.W. performed the experiments; P.W. and B.G. 23 analyzed the data and wrote the article. 24
Financial Support 25
This work was supported by the Alexander von Humboldt Foundation (WP) and by the 26 Deutsche Forschungsgemeinschaft FOR2092 (Grant No. GR 936/18-1 to BG). 27
Corresponding Author 28
Bernhard Grimm 29
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ABSTRACT 34
State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of 35
light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) 36
in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates 37
with PSI, to favor absorption cross-section of PSI. Here, we used Arabidopsis mutants with 38
defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle 39
(cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we 40
found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized 41
LHCI rather than LHCII, while the contents of both LHCI and LHCII are equally depressed in 42
the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state 43
transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were 44
used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under 45
state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. 46
LHCII phosphorylation was drastically reduced in ch1-2 and the inactivation of STN7 47
correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was 48
observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII 49
in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence 50
for the flexible organization of LHCs, and enhances our understanding of the reversible 51
allocation of LHCII to the two photosystems. 52
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243 words 55
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INTRODUCTION 57
In oxygenic photosynthesis, photosystems II and I (PSII and PSI) function in series to 58
convert light energy into the chemical energy that fuels multiple metabolic processes. Most of 59
this light energy is captured by the chlorophyll and carotenoid pigments in the light-harvesting 60
antenna complexes (LHCs) that are peripherally associated with the core complexes of both 61
photosystems (Wobbe et al., 2016). However, since the two photosystems exhibit different 62
absorption spectra (Nelson and Yocum, 2006; Nield and Barber, 2006; Qin et al., 2015), PSI or 63
PSII is preferentially excited under naturally fluctuating light intensities and qualities. In order 64
to optimize photosynthetic electron transfer, the excitation state of the two photosystems must 65
be rebalanced in response to changes in lighting conditions. To achieve this, higher plants and 66
green algae require rapid and precise acclimatory mechanisms to adjust the relative 67
absorption cross-sections of the two photosystems. 68
To date, the phenomenon of state transitions is one of the well-documented short-term 69
acclimatory mechanisms. It allows a mobile portion of the light-harvesting antenna complexes 70
of PSII (LHCII) to be allocated to either photosystem, depending on the spectral composition 71
and intensity of the ambient light (Allen and Forsberg, 2001; Rochaix, 2011; 72
Goldschmidt-Clermont and Bassi, 2015; Gollan et al., 2015). State transitions are driven by the 73
redox state of the plastoquinone (PQ) pool (Vener et al., 1997; Zito et al., 1999). When PSI is 74
preferentially excited (by far-red light), the PQ pool is oxidized and all the LHCII is associated 75
with PSII. This allocation of antenna complexes is defined as the state I. When light conditions 76
(blue/red light or low light) favor exciton trapping of PSII, the transition from state I to state II 77
occurs. The over-reduced PQ pool triggers the activation of the membrane-localized 78
serine-threonine kinase STN7, which phosphorylates an N-terminal threonine on each of two 79
major LHCII proteins, LHCB1 and LHCB2 (Allen, 1992; Bellafiore et al., 2005; Shapiguzov et 80
al., 2016). Phosphorylation of LHCII results in the dissociation of LHCII from PSII and triggers 81
its reversible relocation to PSI (Allen, 1992; Rochaix, 2011). Conversely, when the PQ pool is 82
re-oxidized, STN7 is inactivated and the constitutively active, thylakoid-associated 83
phosphatase TAP38/PPH1 dephosphorylates LHCII, which then re-associates with PSII (Pribil 84
et al., 2010; Shapiguzov et al., 2010). The physiological significance of state transitions has 85
been demonstrated by the reduction in growth rate seen in the stn7 knock-out mutant under 86
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fluctuating light conditions (Bellafiore et al., 2005; Tikkanen et al., 2010). 87
The canonical state transitions model implies spatial and temporal regulation of the 88
allocation of LHC between the two spatially segregated photosystems (Dekker and Boekema, 89
2005). PSII-LHCII supercomplexes are organized in a tightly packed form in the stacked grana 90
regions of thylakoid membranes, while PSI-LHCI supercomplexes are mainly localized in the 91
non-stacked stromal lamellae and grana margin regions (Dekker and Boekema, 2005; 92
Haferkamp et al., 2010). It has been proposed that, in the grana margin regions, which harbor 93
LHCII and both photosystems, LHCII can migrate rapidly between them (Albertsson et al., 94
1990; Albertsson, 2001). This idea is supported by the recent discovery of mega complexes 95
containing both photosystems in the grana margin regions (Yokono et al., 2015). Furthermore, 96
phosphorylation of LHCII was found to increase not only the amount of PSI found in the grana 97
margin region of thylakoid membranes (Tikkanen et al., 2008), but also to modulate the pattern 98
of PSI-PSII megacomplexes under changing light conditions (Suorsa et al., 2015). 99
Nonetheless, open questions remain in relation to the physiological significance of the 100
detection of phosphorylated LHCII in all thylakoid regions, even under the constant light 101
conditions (Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013), although LHCII 102
phosphorylation has been shown to modify the stacking of thylakoid membranes (Chuartzman 103
et al., 2008; Pietrzykowska et al., 2014). 104
State I-to-state II transition is featured by the formation of LHCII-PSI-LHCI 105
supercomplexes, in which LHCII favors the light-harvesting capacity of PSI. Recently, 106
LHCII-PSI-LHCI supercomplexes have been successfully isolated and purified using various 107
detergents (Galka et al., 2012; Drop et al., 2014; Crepin and Caffarri, 2015) or a 108
styrene-maleic acid copolymer (Bell et al., 2015). These findings yielded further insights into 109
the re-organization of supercomplexes associated with state transitions, and it was suggested 110
that phosphorylation of LHCB2 rather than LHCB1 is the essential trigger for the formation of 111
state-transitions supercomplexes (Leoni et al., 2013; Pietrzykowska et al., 2014; Crepin and 112
Caffarri, 2015; Longoni et al., 2015). Furthermore, characterization of mutants deficient in 113
individual PSI core subunits indicates that PsaH, L, and I are required for docking of LHCII at 114
PSI (Lunde et al., 2000; Zhang and Scheller, 2004; Kouril et al., 2005; Plochinger et al., 2016). 115
Recently, the state transitions capacity has been characterized in the Arabidopsis mutants 116
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with missing LHCI components. Although the Arabidopsis knock-out mutants lacking one of 117
the four LHCI proteins (LHCA1-4) showed enhanced accumulation of LHCII-PSI complexes, 118
the absorption cross-section of PSI under state II conditions was still compromised in the 119
lhca1-4 mutants, and it is suggested that LHCI mediates the detergent-sensitive interaction 120
between ‘extra LHCII’ and PSI (Benson et al., 2015; Grieco et al., 2015). Furthermore, the 121
Arabidopsis mutant ΔLhca lacking all LHCA1-4 proteins was shown to be compensated for the 122
deficiency of LHCI by binding LHCII under state II conditions (Bressan et al., 2016). In spite of 123
this finding, the significant reduction in the absorption cross-section of PSI was still observed in 124
the ΔLhca mutant, suggesting a substantial role of LHCI in light absorption under canopy 125
conditions (Bressan et al., 2016). However, these findings emphasize the acclimatory function 126
of state transitions in balancing light absorption capacity between the two photosystems by 127
modifying their relative antenna size, and imply the dynamic and variable organization of 128
PS-LHC supercomplexes. 129
LHC proteins are encoded by the nuclear Lhc superfamily (Jansson, 1994). The 130
biogenesis of LHCs includes the cytoplasmic synthesis of the LHC precursor proteins, their 131
translocation into chloroplasts via the TOC/TIC complex, and their post-translational targeting 132
and integration into the thylakoid membranes by means of the chloroplast SRP (cpSRP) 133
machinery (Jarvis and Lopez-Juez, 2013). The post-translational cpSRP-dependent pathway 134
for the final translocation of LHC proteins into the thylakoid membrane includes interaction of 135
cpSRP43 with LHC apo-proteins and recruitment of cpSRP54 to form a transit complex. Then 136
binding of this tripartite cpSRP transit complex to the SRP receptor cpFtsY follows, which 137
supports docking of the transit complex to thylakoid membranes and its association with the 138
LHC translocase ALB3. Ultimately, ALB3 inserts LHC apo-proteins into the thylakoid 139
membrane (Richter et al., 2010). Importantly, stoichiometric amounts of newly synthesized 140
chlorophyll (Chl) a and Chl b as well as carotenoid are inserted into the LHC apo-proteins by 141
unknown mechanisms to form the functional LHCs that associate with the core complexes of 142
both photosystems in the thylakoid membranes (Dall'Osto et al., 2015; Wang and Grimm, 143
2015). 144
The first committed steps in Chl synthesis occur in the Mg branch of the tetrapyrrole 145
biosynthesis pathway. 5-Aminolevulinic acid synthesis provides the precursor for the formation 146
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of protoporphyrin IX, which is directed into the Mg branch (Tanaka and Tanaka, 2007; 147
Brzezowski et al., 2015). Chl synthesis ends with the conversion of Chl a to Chl b catalyzed by 148
Chl a oxygenase (CAO) (Tanaka et al., 1998; Tomitani et al., 1999). It has been hypothesized 149
that coordination between Chl synthesis and the post-translational cpSRP pathway is a 150
prerequisite for the efficient integration of Chls into LHC apo-proteins. 151
In this study, we intend to characterize the assembly of LHCs when the availability of Chl 152
molecules or the integration of LHC apo-proteins into thylakoid membranes are limiting. To this 153
end, we compared the assembly of LHCs and the organization of PS-LHC complexes in two 154
different sets of Arabidopsis mutants. Firstly, we used the chlorina1-2 (ch1-2) mutant, which is 155
defective in the CAO gene. The members of the second set of mutants carry knock-out 156
mutations in genes involved in the chloroplast SRP pathway (Richter et al., 2010). 157
Our studies revealed distinct accumulation of PS-LHC supercomplexes between the two 158
sets of mutant relative to wild-type plants. In spite of the defect in synthesis of Chl b, ch1-2 159
retains predominantly intact PSI-LHCI supercomplexes, but has strongly reduced amounts of 160
LHCII. In contrast, the chaos (cpsrp43) mutant exhibits synchronously reduced contents of 161
both LHCI and LHCII, which results in the accumulation of PS core complexes without 162
accompanying LHCs. Thus, the distribution of LHCs in the thylakoid membranes of the two 163
mutants, ch1-2 and chaos, were explored under varying light conditions with the aim of 164
elucidating the influence of modified LHCI/LHCII antenna size on state transitions. Our results 165
contribute to an expanding view on the variety of photosynthetic complexes, which can be 166
observed in Arabidopsis plants with specified mutations in LHC biogenesis. 167
168
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169
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RESULTS 170
Reduced Contents of LHCs in ch1-2 and cpsrp Mutants 171
To examine the prerequisites for the precise re-allocation of LHCII in response to an 172
imbalance in the distribution of absorbed light energy between PSII and PSI, we examined 173
mutants that are impaired in Chl b biosynthesis or in the cpSRP machinery. These mutants 174
enable comparative studies on LHC accumulation during state transitions when the availability 175
of either Chl b or LHC apo-proteins are limiting (Fig. 1A). 176
Three allelic Arabidopsis cao mutants have been reported and termed chlorina1-1, 1-2 and 177
1-3 (ch1-1, ch1-2, and ch1-3). They either accumulate reduced amounts of Chl b or fail to 178
synthesize it altogether, and in turn show significantly reduced levels of LHC proteins (Murray 179
and Kohorn, 1991; Espineda et al., 1999; Havaux et al., 2007; Kim et al., 2009; Takabayashi et 180
al., 2011). ch1-1 and ch1-3 entirely lack Chl b due to a CAO null mutation (Murray and Kohorn, 181
1991; Espineda et al., 1999; Havaux et al., 2007; Kim et al., 2009; Takabayashi et al., 2011). 182
In ch1-2, Chl b synthesis is compromised, and the CAO protein contains a V274E point 183
mutation within its Rieske-binding domain (Espineda et al., 1999). In agreement with previous 184
reports, the ch1-2 mutant accumulated only about 20% as much Chl b as the wild-type plants. 185
As a result, the Chl a/b ratio in ch1-2 rises to about 9.55 (Fig. 1B). 186
Plants bearing knockout mutations in the nuclear genes encoding cpSRP43 (chaos) (Amin 187
et al., 1999; Klimyuk et al., 1999), cpSRP54 (ffc) (Pilgrim et al., 1998; Amin et al., 1999), both 188
cpSRP43 and cpSRP54 (chaos/ffc) (Hutin et al., 2002) or cpFtsY (cpftsy) 189
(Tzvetkova-Chevolleau et al., 2007) exhibited always a pale-green leaf phenotype (Fig. 1A) 190
and contained reduced Chl levels (Fig. 1B). In contrast, the alb3 mutant, which lacks the LHC 191
translocase, shows an albino phenotype (Sundberg et al., 1997). Interestingly, in addition to 192
chaos and ffc mutants, an additive effect on delayed plant growth and reduced Chl contents 193
was found in chaos/ffc mutant (Figs. 1A and 1B), highlighting the role of cpSRP43-cpSRP54 194
heterodimer in targeting of LHC proteins to thylakoid membranes. Moreover, the strongest 195
pale-green phenotype and the most retarded plant growth were observed in the cpftsy mutants 196
among the cpsrp mutants analyzed here (Figs. 1A and 1B), indicating the indispensable 197
function of cpFtsY in the cpSRP pathway. 198
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In the mutants analyzed here, the LHC contents were examined by immunoblotting with 199
antibodies raised against LHCA1 and LHCB1, as representative subunits of LHCI and LHCII, 200
respectively. As shown before (Espineda et al., 1999), LHCB1 was strongly reduced in ch1-2 201
(Fig. 1C), while the LHCA1 content was unexpectedly slightly diminished (Fig. 1C). In contrast 202
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to ch1-2 mutant, the cpsrp mutants contained severely reduced contents of the LHCPs of both 203
photosystems. Combining previous detailed descriptions of the effects of cpsrp mutations on 204
levels of various LHCI and LHCII subunits (Pilgrim et al., 1998; Espineda et al., 1999; Hutin et 205
al., 2002; Tzvetkova-Chevolleau et al., 2007; Ouyang et al., 2011), we concluded that, in each 206
of the three cpsrp mutants studied here, steady-state amounts of LHCI and LHCII proteins are 207
equally affected (Fig. 1C). Interestingly, levels of LHC proteins were clearly higher in ffc than in 208
chaos, chaos/ffc, and cpftsy (Fig. 1C), indicating that cpSRP43 functions predominantly and 209
independently from cpSRP54 in targeting of LHC proteins to the thylakoid membranes 210
(Tzvetkova-Chevolleau et al., 2007; Liang et al., 2016). In summary, our initial results suggest 211
that malfunction of the cpSRP pathway depresses steady-state levels of both LHCI and LHCII, 212
while strongly reduced Chl b biosynthesis preferentially affects LHCII. 213
214
Accumulation of Photosynthetic Apparatus in ch1-2 and cpsrp Mutants 215
The diminished LHC contents observed in ch1-2 and cpsrp mutants enabled us to examine 216
the consequences of each mutation for the assembly of PS-LHC complexes in the thylakoid 217
membranes. For this purpose, the isolated thylakoid membranes were treated with the 218
non-ionic detergent n-dodecyl-β-D-maltoside (β-DM) to efficiently solubilize both grana and 219
non-stacked regions (Jarvi et al., 2011; Grieco et al., 2015). The thylakoid membranes were 220
then fractionated in a large-pore Blue-Native (lpBN)-PA gel (Jarvi et al., 2011), followed by 221
SDS-PAGE in the second dimension to determine the protein composition of each of the 222
various photosynthetic complexes. 223
In the thylakoid membranes, LHCII is peripherally associated with PSII to form a 224
PSII-LHCII supercomplex, which is mainly localized in the grana core regions (Dekker and 225
Boekema, 2005). Depending on the binding strength of LHCII to PSII, four variants of 226
PSII-LHCII supercomplexes (II) were observed on the lpBN-PA gel (Fig. 2A and 2B). Apart 227
from the PSII-LHCII supercomplexes, several PSII sub-complexes, including the PSII dimer 228
(III), PSII monomer (V), LHCII assembly complex (VI), trimeric and monomeric LHCII (VII and 229
VIII) could be detected (Figs. 2A and 2B), which is in consistency to previous reports (Jarvi et 230
al., 2011). The ch1-2, ffc and chaos mutants were characterized by reduced amounts of the 231
PSII-LHCII supercomplexes and LHCII trimers, which are in turn associated with elevated 232
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levels of the PSII monomer and LHCII assembly complex (Figs. 2A and 2B). The chaos/ffc and 233
cpftsy mutants showed a more severe reduction in PSII-LHCII supercomplexes and PSII 234
dimers (Figs. 2A and 2B), suggesting that simultaneous loss of cpSRP43 and cpSRP54 or 235
deficiency of the cpFtsY receptor not only affects the stability of antenna proteins, but also the 236
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assembly of the PSII core complex in the thylakoid membranes. This observation is supported 237
by the earlier finding that cpSRP54 and cpFtsY cooperate in the co-translational integration of 238
plastid-encoded PSII core subunits (Richter et al., 2010). 239
In contrast to the various PSII-LHCII supercomplexes seen in wild-type plants, only a single 240
PSI-LHCI supercomplex (III) was observed in control plants, which migrates close to PSII 241
dimers on lpBN-PA gels. In mutants defective in LHCI formation, only PSI core complexes are 242
observed (Havaux et al., 2007; Wientjes et al., 2009; Takabayashi et al., 2011; Benson et al., 243
2015). A dominant band of PSI core complexes was observed in the chaos mutant (Figs. 2A 244
and 2B), confirming reduced accumulation of LHCI subunits in chaos relative to ch1-2 (Figs. 245
1C). In addition to drastically disrupted assembly and/or reduced stability of PSII-LHCII 246
supercomplexes and PSII dimers in chaos/ffc and cpftsy mutants, accumulation of both 247
PSI-LHCI and PSI core complexes was strongly impaired (Figs. 2A and 2B). In contrast to 248
these observations, the slight reductions in LHCI proteins seen in ch1-2 and ffc are consistent 249
with a minor perturbation of PSI-LHCI supercomplex formation (Figs. 2A and 2B). 250
In summary, based on the accumulation of PS-LHC complexes in the thylakoid membranes, 251
ch1-2 and the different cpsrp mutants can be classified into three groups: (i) ch1-2 exhibited a 252
drastically reduced content of LHCII and only a slightly impaired LHCI content; (ii) chaos and 253
ffc were both characterized by impaired accumulation of both LHCI and LHCII, with levels of 254
both complexes being more severely affected in the chaos mutant than in ffc; (iii), the chaos/ffc 255
and cpftsy mutants showed the greatest reductions in LHC content, and accumulated 256
photosystem core complexes. 257
258
Impaired State Transitions in ch1-2 and cpsrp Mutants 259
Short-term state transitions enable the reversible allocation of LHCII to PSI when PSII 260
rather than PSI is preferentially activated (Allen and Forsberg, 2001; Rochaix, 2011; 261
Goldschmidt-Clermont and Bassi, 2015; Gollan et al., 2015). The observations that ch1-2 and 262
chaos mutants exhibited distinct accumulation of PSI-LHCI complexes (Figs. 1 and 2) led to 263
further exploration of the association of LHCII with PSI or PSII during state transitions. It was 264
recently shown that an intact LHCI complex is required for a complete state I-to-state II 265
transition (Benson et al., 2015). To explore these findings further, we compared state 266
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transitions in ch1-2 and chaos with control seedlings. We hypothesized that the defects in 267
formation of PSI-LHCI supercomplexes observed in chaos would lead to an aberrant transition 268
relative to ch1-2 and control plants under state II conditions (Figs. 1 and 2). As additional 269
controls, we examined the ffc mutant, in which levels of both LHCs were only slightly reduced, 270
and the stn7/8 double mutant. The latter mutant is unable to phosphorylate LHCII proteins and 271
PSII core subunits, and thus fails to undergo state transitions during changes in light quality. 272
Light-dependent state transitions were marked by the allocation of LHCII to PSII in state I 273
and its partial transfer to PSI in state II. Thus, modification of the antenna sizes (i.e., 274
absorption cross-sections) of the photosystems, as determined by 77K fluorescence emission, 275
reflected the capacity to undergo state transitions (Bellafiore et al., 2005; Tikkanen et al., 276
2008). In wild-type plants, the transition from state I (induced by exposure to far-red light) to 277
state II (upon exposure to red light) was accompanied by an obvious relative increase in PSI 278
fluorescence emission at 733 nm, indicating the redistribution of excitation energy from PSII to 279
PSI (Fig. 3). Although a slightly reduced content of LHCs was observed in the ffc mutant (Figs. 280
1 and 2), the PSI peak in ffc showed a greater enhancement under state II conditions than that 281
in wild-type plants (Fig. 3). In contrast, the PSI fluorescence of ch1-2, chaos and stn7/8 282
mutants showed no obvious increase under state II conditions (Fig. 3), implying that the state 283
I-to-state II transition is blocked not only in stn7/8, but also in ch1-2 and chaos. The spectral 284
response of the photosynthetic complexes in the thylakoids of chaos was consistent with a 285
previous report (Pesaresi et al., 2009). Furthermore, it is worth noting that the PSII and PSI 286
fluorescence peaks in ch1-2 differed by more than 2-fold (Fig. 3). This observation is explained 287
by strongly impaired assembly of LHCII relative to the LHCI content at PSI in ch1-2 as a result 288
of its deficiency in chlorophyll synthesis. Apparently, LHCII assembly is far more susceptible to 289
perturbation of Chl synthesis than formation of LHCI (Figs. 1 and 2). 290
Next, the formation of the LHCII-PSI-LHCI supercomplexes under state II conditions was 291
analyzed by 2D lpBN-SDS-PAGE. To keep the LHCII-PSI-LHCI supercomplexes intact, the 292
mild non-ionic detergent digitonin was used instead of β-DM to specifically solubilize the 293
non-appressed grana margins and stromal lamellae of thylakoid membranes (Jarvi et al., 2011; 294
Grieco et al., 2015). As expected, when plants were exposed to red light (state II), 295
LHCII-PSI-LHCI supercomplexes were formed (Figs. 4), which raised the photochemical work 296
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rate of PSI (Galka et al., 2012). In agreement with 77K fluorescence emission spectra analysis 297
(Fig. 3), LHCII-PSI-LHCI supercomplexes were only observed in wild-type plants and, to a 298
lesser degree, in ffc under state II conditions (Figs. 4A and 4B). In contrast, stn7/8, ch1-2, and 299
chaos lacked these supercomplexes (Figs. 4A and 4B). As shown in Fig. 2, a stable PSI-LHCI 300
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complex was observed in ch1-2, while chaos accumulated PSI core complexes lacking LHCI 301
(Figs. 4A and 4B). Both ch1-2 and chaos mutants exhibited diminished levels of the LHCII 302
trimer (Figs. 4A and 4B). Nevertheless, megacomplexes containing PSI-LHCI and/or 303
PSII-LHCII supercomplexes were still observed in all of the mutants analyzed under state II 304
light (Figs. 4A and 4B). Altogether, these results indicate ch1-2 and chaos mutants failed to 305
perform state transitions and did not form LHCII-PSI-LHCI supercomplexes under state II 306
conditions. 307
308
Phosphorylation of LHCII in ch1-2 and cpsrp Mutants 309
Phosphorylation of LHCII is reported to be a prerequisite for the state I-to-state II transition 310
(Allen, 1992; Rochaix, 2011). The phosphorylation state of LHCII (P-LHCII), as well as of PSII 311
core subunits (P-D1, P-D2 and P-CP43), was analyzed on a phospho-threonine immunoblot 312
(Anti-P-Thr), when plants were acclimated to state I or state II light conditions. Both wild-type 313
plants and the ffc mutant showed increased phosphorylation of LHCII and PSII core subunits 314
in state II conditions, while P-LHCII and phosphorylated PSII core subunits were absent in 315
both ch1-2 and stn7/8 mutants (Fig. 5A), implying that the kinases STN7 and STN8 are not 316
activated in the ch1-2 mutant. Thus, it is suggested that the lack of state transitions-dependent 317
excitation energy transfer from LHCII to PSI in ch1-2 (Figs. 3 and 4) correlates with the lack of 318
P-LHCII (Fig. 5A). It is worth mentioning that the content of phosphorylated PSII core subunits 319
in the ffc mutant in state II conditions was higher than in the wild-type plants, which indicates 320
that ffc is subjected to photoinhibition (Bonardi et al., 2005; Tikkanen et al., 2008). In contrast, 321
in the chaos mutant, P-LHCII was detected in state II conditions, but in lesser amounts than in 322
Ler-0 plants (Fig. 5A). Semi-quantitative analysis of the immunoblot in Fig. 5A suggested that 323
the LHCB1 level in chaos was highly correlated with the P-LHCII level (Fig. 5B). This finding 324
implies that STN7 in chaos was activated to phosphorylate LHCII under state II conditions. 325
However, no state transition was actually observed in the chaos mutant, which in this respect 326
behaves like ch1-2 and stn7/8. 327
328
Protein Composition of Photosynthetic Complexes in ch1-2 and cpsrp Mutants 329
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Although phosphorylation of LHCII occurred in the chaos mutant under state II conditions 330
(Fig. 5), LHCII-PSI-LHCI supercomplexes were not detectable by BN gel electrophoresis (Fig. 331
4). It has been suggested that PsaH and PsaL serve as docking site for P-LHCII in PSI (Lunde 332
et al., 2000; Zhang and Scheller, 2004; Kouril et al., 2005). Thus, we hypothesized that the 333
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failure of chaos to form LHCII-PSI complexes might be due to impaired docking of P-LHCII at 334
PSI. To test this possibility, we analyzed the accumulation of core subunits of four 335
photosynthetic complexes, including D1 and CP43 subunits of the PSII complex, cytochrome f 336
(Cyt f) of the Cyt b6f complex, PsaA, PsaH and PsaL of the PSI complex, and the β-subunit of 337
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20
the ATP synthase, in plants which were adapted to state II light conditions (Fig. 5C). We found 338
increased PsaH and PsaL protein contents in chaos in comparison to wild-type plants (ecotype 339
Ler-0), while the other proteins analyzed were not affected in chaos (Fig. 5C). These 340
observations do not only indicate that the docking site of P-LHCII at PSI is not affected in 341
chaos, but also prompted us to propose that cpSRP43 deficiency leads to a specific defect in 342
the LHC biogenesis (Klimyuk et al., 1999; Hutin et al., 2002). Furthermore, we found reduced 343
levels of plastid-encoded D1 and PsaA, and nuclear-encoded PsaH and PsaL in the ffc mutant 344
(Fig. 5C). This finding supports previous results (Pilgrim et al., 1998; Amin et al., 1999), 345
indicates the role of cpSRP54 in the biogenesis of plastid-encoded PS core subunits, such as 346
D1 (Richter et al., 2010) and suggests an instability of PS core complexes in the ffc mutant. 347
348
Distribution of Phosphorylated Proteins in the Thylakoid Membranes from ch1-2 and 349
cpsrp Mutants 350
To address the distribution of P-LHCII in the grana margin regions of thylakoid membranes, 351
thylakoid membranes adapted to state II conditions were isolated and solubilized with digitonin. 352
The dominant photosynthetic pigment-protein complexes obtained were analyzed on 2D 353
lpBN-SDS-PA gels. Phosphorylated LHCII and PSII core subunits were quantified by the 354
phospho-threonine immunoblot. In agreement with a recent report (Grieco et al., 2015) and in 355
contrast with stn7/8 (Fig. 6B), P-LHCII was not only found in LHCII-PSI-LHCI supercomplexes, 356
but also in the megacomplexes containing PSII-LHCII-PSI-LHCII and/or PSII-LHCII 357
supercomplexes, dimeric and monomeric PSII complexes, and in LHCII trimers in both 358
wild-type and ffc plants (Figs. 6A, 6D and 6F). In consistency with Fig. 5A, very low levels of 359
phosphorylated LHCII and PSII core subunits were detected in ch1-2 (Fig. 6C). Notably, since 360
we found that P-LHCII exhibited the same migration rate on the lpBN-PAGE as P-D1, P-D2, 361
and P-CP43 in the chaos mutant, we assume that P-LHCII is associated with PSII complexes 362
rather than with the remaining PSI-LHCI complexes or PSI core complexes (Fig. 6E). 363
364
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365
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DISCUSSION 366
367
Diverse Accumulation of LHCI and LHCII When the Availability of Chl b or the 368
Integration of LHC Apo-proteins into Thylakoid Membranes are Limiting 369
Integration of newly synthesized Chl a and Chl b into the LHC apo-proteins is essential for 370
the stability, folding and membrane insertion of functional LHCs, and is a prerequisite for the 371
association of LHCs with core complexes of two photosystems (Dall'Osto et al., 2015; Wang 372
and Grimm, 2015). Thus, impaired synthesis of Chl and LHC apo-proteins, as well as 373
dysfunctional post-translational translocation of LHC apo-proteins from the cytosol to the 374
chloroplasts by the TOC/TIC translocons and from the stroma to thylakoid membranes by the 375
cpSRP machinery, could disrupt the association and assembly of LHCs in the thylakoids. 376
For the first time, we have measured the accumulation of multiple PS-LHC 377
supercomplexes and the allocation of phosphorylated LHCII to the two photosystems during 378
state transitions in a Chl b-less mutant and in cpsrp mutants (Figs. 1 and 2). It was expected 379
that these mutants show the formation of different photosynthetic protein complexes with the 380
intention to balance the excitation status of PSI and PSII. Lack of one or two components of 381
cpSRP machinery, cpSRP43 and cpSRP54, caused simultaneously reduced levels of LHCI 382
and LHCII (Fig. 1). In consequence, the comparatively strong decrease in LHCI and LHCII 383
content in the chaos mutant led to accumulation of free PSI and PSII core complexes in place 384
of the multiple PS-LHC complexes observed in wild-type chloroplasts (Figs 1 and 2). These 385
observations further support the idea that the cpSRP machinery acts non-selectively on the 386
post-translational targeting of LHCI and LHCII apo-proteins to thylakoid membranes (Richter 387
et al., 2010). 388
In contrast, the ch1-2 mutant, which is defective in Chl b synthesis, exhibited rather stable 389
LHCI complexes and wild-type-like PSI-LHCI supercomplexes, while LHCII content in ch1-2 390
was drastically reduced to a level comparable to that in chaos (Figs 1 and 2). These 391
observations are supported by the finding that the PSI antenna is larger than that of PSII in 392
ch1-2 (Fig. 3). 393
Two possible explanations for the preferential stability of LHCI rather than of LHCII are 394
proposed when availability of Chl b are limiting. Firstly, considering the varying specificity of 395
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23
LHCI and LHCII for Chl a and Chl b (Schmid, 2008), due to the enhanced promiscuity of LHCI, 396
the Chl b-binding sites of LHCI proteins could be filled by Chl a when Chl b is in short supply. 397
Indeed, in-vitro reconstitution analyses have shown that Chl a can in fact be integrated into 398
LHCI apo-proteins, such as LHCA2 and LHCA4, to form the stable LHCI (Schmid et al., 2002). 399
Furthermore, the Chl a-containing LHCI has been characterized in the ch1-1 mutant, which 400
lacks Chl b altogether (Havaux et al., 2007; Takabayashi et al., 2011). However, the Chl 401
a-containing LHCI was less tightly associated with PSI core complexes, indicating Chl b is 402
essential for the efficient energy transfer and stable assembly of PSI-LHCI supercomplexes 403
(Takabayashi et al., 2011). Thus, the residual amount of Chl b in ch1-2 (20% of the wild-type 404
level) might well be sufficient for the organization of functional PSI-LHCI supercomplexes (Figs 405
1, 2 and 3). 406
Secondly, we suggest that newly synthesized Chl b might be preferentially integrated into 407
LHCI rather than LHCII, particularly when only limited amounts of Chl b are available. This 408
hypothesis can be supported by our finding of the preferential accumulation of LHCI in ch1-2 409
(Figs. 1 and 2). So far, very little attention has been paid to the mechanisms that determine the 410
distribution of newly synthesized Chl to the various LHC and PS-LHC complexes. What 411
knowledge we do have is based on radioactive labeling with 14C. The radiolabelling 412
experiments with chlorophyll precursor molecules carried out on organisms exposed to high 413
light levels confirmed ongoing Chl synthesis in both higher plants (Beisel et al., 2010) and 414
cyanobacteria (Kopecna et al., 2012). Interestingly, in the cyanobacteria, the freshly 415
synthesized Chl was localized predominantly in PSI and to lesser extent in PSII (Kopecna et 416
al., 2012). In contrast, most of the fresh Chls produced in the chloroplasts of higher plants 417
were suggested to support the PSII repair cycle, since PSI is very stable under high-light 418
stress (Feierabend and Dehne, 1996). In this context, ch1-2 could be a useful tool for 419
assessing the relative affinities of LHCI and LHCII for Chl b during their biogenesis. 420
421
Modified State Transitions Observed in ch1-2 and chaos Imply the Flexible Association 422
of LHCs with two Photosystems 423
To balance the excitation status of PSI and PSII, state transitions enable the rapid and 424
efficient modification of the relative antenna size of the two photosystems in response to 425
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24
fluctuating light conditions (Allen and Forsberg, 2001; Rochaix, 2011; Goldschmidt-Clermont 426
and Bassi, 2015; Gollan et al., 2015). In the state I-to-state II transition, phosphorylated LHCII 427
proteins associate with PSI-LHCI to favor the absorption cross-section of PSI. However, 428
increased absorption cross-section of PSI and formation of LHCII-PSI complexes were not 429
detected in ch1-2 and chaos upon exposure to PSII-favoring light (Figs. 3 and 4), suggesting a 430
block of the state I-to-state II transition in both ch1-2 and chaos mutants. 431
According to the canonical model of state transitions, phosphorylation of LHCII is an 432
essential prerequisite for state I-to-state II transition and triggers the dissociation of LHCII from 433
PSII and promotes its lateral migration to PSI-LHCI-enriched regions of thylakoid membranes 434
(Allen, 1992; Rochaix, 2011). In this way, the missing formation of LHCII-PSI-LHCI complexes 435
in ch1-2 is associated with the lack of P-LHCII under state II light conditions (Figs. 5A and 6C), 436
which is explained by repression of STN7 activity. It is striking that in comparison with 437
wild-type and cpsrp mutant plants ch1-2 exhibited 2-fold larger antenna size of PSI than that of 438
PSII (Fig. 3). In contrast, chaos exhibited the similar LHCII antenna as that in ch1-2, but less 439
LHCI antenna (Figs. 1 and 2), that leading to the comparably balanced excitation state of PSI 440
and PSII. The phosphorylation of LHCII was observed in chaos in the state II conditions (Figs. 441
5A and 6E), suggesting the more activated STN7 in chaos mutant than that in ch1-2 mutant. 442
We assume that the electron transfer chain and the PQ pool were more oxidized in ch1-2 than 443
in wild-type and chaos mutants. In turn, oxidation of PQ pool in ch1-2 will lead to inactivation of 444
STN7. 445
As phosphorylation of LHCII is observed in chaos upon exposure to PSII light, balanced 446
distribution of excitation energy between PSI and PSII is likely to be required under state II 447
conditions. However, P-LHCII of chaos was associated with PSII complexes rather than with 448
PSI core complexes or a residual amount of intact PSI-LHCI supercomplexes (Fig. 6E). The 449
localization of P-LHCII could result from a failure to dissociate from PSII or an inability to dock 450
at PSI. The latter prospect is challenged by the elevated accumulation of LHCII-PSI core 451
complexes in the lhca4 mutant (Benson et al., 2015) and in the ΔLhca mutant (Bressan et al., 452
2016), which were adapted to state II conditions. Structural studies of LHCII-PSI-LHCI 453
complexes showed an opposite localization of P-LHCII and LHCI within the state-transition 454
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25
supercomplexes (Kouril et al., 2005; Drop et al., 2014). It is very unlikely that LHCI contributes 455
to stable docking of LHCII at PSI complexes. 456
We propose that an impaired dissociation of P-LHCII from PSII results from a strongly 457
reduced content of LHCII in chaos (Figs. 1 and 2). It is expected that a mobile P-LHCII pool is 458
limited to migrate towards PSI complexes and form LHCII-PSI complexes. Although free 459
phosphorylated LHCII trimers could be detected in chaos (Fig. 6E), the majority of LHCII 460
trimers were associated with dimeric PSII core complexes (Figs. 2 and 4). According to the 461
binding affinity of LHCII trimer to the PSII homodimer, S (strong), M (medium), and L (loose) 462
variants of LHCII trimers were found in the thylakoid membranes of higher plants (Dekker and 463
Boekema, 2005). It was indicated that the L-LHCII trimers could be associated with PSI, while 464
the S-LHCII and M-LHCII are unlikely to be involved in state transitions (Pietrzykowska et al., 465
2014). Thus, the failure to form P-LHCII-PSI complexes in chaos is proposed to be due to the 466
lack of a mobile LHCII pool. 467
In summary, the distinct accumulation of LHCI and LHCII complexes in ch1-2 and cpsrp 468
mutants not only underlines the requirement for coordination of Chl biosynthesis and the 469
post-translational integration of LHC apo-proteins into thylakoid membranes (Dall'Osto et al., 470
2015; Wang and Grimm, 2015), but also indicates the variable accumulation of LHCI and 471
LHCII complexes, when Chl b synthesis is compromised in ch1-2 mutant. Furthermore, the 472
detailed comparative analysis of state transitions in ch1-2 and chaos mutants provides 473
evidence for the flexible association of LHCs with the two photosystems. 474
475
MATERIALS AND METHODS 476
477
Plant Materials, Growth Conditions, and Light Treatment 478
The following Arabidopsis mutants were used in this study: chlorina1-2, which contains a 479
V274E mutation in the Rieske binding site (ch1-2; CS3120) (Espineda et al., 1999), the maize 480
Ds transposon-containing cpsrp43 mutant chaos (Klimyuk et al., 1999), the T-DNA insertion 481
lines ffc (cpsrp54, CS850421) (Pilgrim et al., 1998), cpftsy (SALK_049077) 482
(Tzvetkova-Chevolleau et al., 2007) and stn7/8 (Bonardi et al., 2005), and the chaos/ffc double 483
mutant (Hutin et al., 2002), together with the wild-type Arabidopsis thaliana ecotypes Columbia 484
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26
(Col-0) and Landsberg-0 (Ler-0). Wild-type and mutant Arabidopsis plants were routinely 485
grown at 22 °C and 70% humidity with 100 μM photons m-2 s-1 on a 16-h light/8-h dark 486
photoperiod. 487
488
Pigment Analysis 489
Chlorophylls were extracted from leaves with alkaline acetone (100% acetone:0.2 M NH4OH, 490
9:1) and analyzed using reversed-phase chromatography on an Agilent HPLC system as 491
described (Schlicke et al., 2014). 492
493
Isolation of Thylakoid Membranes 494
Thylakoid membranes were isolated from Arabidopsis plants grown in well-controlled 495
phytochambers or adapted to state I or state II conditions in the presence of 10 mM sodium 496
fluoride NaF as described (Jarvi et al., 2011). Chl concentration was determined as described 497
(Wellburn, 1994). 498
499
77K Fluorescence Emission Spectroscopy 500
Freshly isolated thylakoids equivalent to 10 μg chlorophyll ml-1 were resuspended in Chl 501
fluorescence buffer (20 mM HEPES pH 7.8, 60% glycerol, 300 mM sucrose, 5 mM MgCl2) and 502
frozen in liquid nitrogen. Chl a fluorescence emission was detected using a F-6500 fluorometer 503
(Jasco). The sample was excited at 475 nm wavelength. The emission spectra between 655 504
nm and 800 nm were recorded with a bandwidth of 10 nm. 505
506
2D LpBN-SDS-PAGE 507
LpBN-PAGE was performed essentially according to Jarvi et al. (2011). To comprehensively 508
analyze the PSI-LHC supercomplexes present in grana and unstacked thylakoids, freshly 509
isolated thylakoids equivalent to 0.5 μg chlorophyll μl-1 were solubilized with 1% β-DM at 4 °C 510
for 5 min. To detect the LHCII-PSI-LHCI supercomplexes formed during state transitions, 511
freshly isolated thylakoids were partially solubilized with 1% (w/v) digitonin at room 512
temperature for 15 min. For the second dimensional SDS-PAGE analysis, the excised 513
lpBN-PAGE lanes were soaked in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% 514
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27
glycerol, 0.002% bromophenol blue and 50 mM DTT) for 0.5 h at room temperature, and then 515
layered onto 12% SDS-PAGE gels containing 6 M urea. The gels were stained with 516
Coomassie Brilliant Blue G250 or used for immunoblot analyses. 517
518
Immunoblot Analyses 519
For immunoblot analysis, total thylakoid proteins normalized to equal Chl contents were 520
separated on 12% SDS-PAGE gels containing 6 M urea. After electrophoresis, proteins were 521
transferred to nitrocellulose membranes (GE Healthcare) and probed with specific antibodies 522
directed against the light-harvesting antenna proteins LHCA1 and LHCB1 (Agrisera), the PSI 523
core subunits D1 and CP43 (Agrisera), the Cyt b6f subunit Cyt f (Agrisera), the PSI core 524
subunits PsaA, PsaH and PsaL (Agrisera), the ATP synthase β subunit (ATPase β, Agrisera), 525
and phosphorylated thylakoid proteins (anti-P-Thr, New England Biolabs). Signals were 526
detected with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). 527
528
ACKNOWLEDGEMENT 529
We thank Dr. Danja Schünemann for all cpsrp mutants and for discussion of impaired 530
PSI-LHCI complexes in chaos mutant, and Dr. Dario Leister for the stn7/8 mutant. 531
532
FIGURE LEGENDS 533
Fig. 1. Characterization of Arabidopsis mutants with defects in chlorophyll b 534
biosynthesis and chloroplast SRP machinery. 535
A. Representative photograph of an 18-day-old chlorina1-2 (ch1-2) mutant and cpsrp mutants 536
including chaos (cpsrp43), ffc (cpsrp54), the chaos/ffc (cpsrp43/cpsrp54) double mutant and 537
the cpftsy mutant, and their corresponding wild-type progenitor plants (Ler-0 for chaos, Col-0 538
for all the others). Bar = 5 mm. B. Relative chlorophyll (Chl) contents and Chl a/b ratios in the 539
above plants. The total Chl a + b levels in the wild-type plants were set to 100%. The data 540
represent means ± SD of three biological replicates; C. Steady-state levels of LHC subunits 541
(LCHA1 for LHCI and LHCB1 for LHCII) and the ATPase β subunit in the thylakoid membranes 542
from the above plants were analyzed by immunoblotting. An equivalent of 1.5 μg of Chl was 543
loaded on the 12% SDS-urea-PA gel. Equality of loading was monitored by the level of the 544
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28
ATPase β subunit and by Ponceau red staining (Ponceau S). Three biological replicates were 545
performed, and similar results were obtained. 546
547
Fig. 2. Analyses of thylakoid membrane pigment-protein complexes. 548
A. Equal amounts of thylakoid membranes (8 μg of chlorophyll) from wild-type plants (Col-0 549
and Ler-0), ch1-2 and cpsrp mutant plants were solubilized with 1% (w/v) DM and first 550
separated by large-pore Blue Native electrophoresis (lpBN-PAGE). B. Individual lanes from 551
the lpBN-PAGE gel in A were then subjected to SDS-urea-PAGE in the second dimension. 552
Total proteins were visualized by staining with Coomassie Brilliant Blue. Identities of the 553
relevant proteins are indicated by arrows. The major PSI proteins, PsaA/B, as well as minor 554
proteins are circled. Two biological replicates were performed, and similar results were 555
obtained. 556
557
Fig. 3. Analysis of state transitions by low-temperature (77K) fluorescence emission 558
spectroscopy. Fluorescence emission spectra of thylakoid membranes were recorded at 559
77°K after exposure of wild-type plants (Col-0 and Ler-0) and the stn7/8, ch1-2, ffc, chaos 560
mutants to lighting conditions that favor either state I (black lines, far-red light of 730 nm) or 561
state II (gray lines, red light of 660 nm). The excitation wavelength was 475 nm, and spectra 562
were normalized with reference to peak height at 685 nm. Three biological replicates were 563
performed, and similar results were obtained. 564
565
Fig. 4. Analysis of state transitions by lpBN-PA gel electrophoresis. 566
A. Equal amounts of thylakoid membranes (9 μg of Chl) from wild-type plants (Col-0 and Ler-0) 567
and the stn7/8, ch1-2, ffc, chaos mutant plants, which had been adapted to state I light (far-red 568
light of 730 nm) or state II light (the red light of 660 nm), were solubilized with 1% (w/v) 569
digitonin and fractionated by lpBN-PAGE. B. Individual lanes from the lpBN-PA gel in A were 570
then subjected to SDS-urea-PAGE in the second dimension. Total proteins were visualized by 571
Coomassie Brilliant Blue staining. Identities of the relevant proteins are indicated by arrows. 572
The major PSI proteins, PsaA/B, as well as LHCII proteins in the LHCII-PSI-LHCII complexes 573
are circled. Two biological replicates were performed, and similar results were obtained. 574
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29
575
Fig. 5. Phosphorylation and steady-state levels of thylakoid proteins. 576
A. Representative anti-phosphothreonine (Anti-P-Thr) immunoblot showing the 577
phosphorylation of the PSII core proteins (P-D1, P-D2, and P-CP43) and the LHCII (P-LHCII) 578
proteins, and anti-LHCB1, anti-LHCA1, and anti-ATPase β immunoblots showing the 579
steady-state protein levels in the thylakoids of wild-type (Col-0 and Ler-0) and stn7/8, ch1-2, ffc, 580
and chaos mutant plants, which were adapted to state I light (far-red light, 730 nm) or state II 581
light (red light, 660 nm). Each sample contained 1 μg of Chl. To control for loading, the 582
thylakoid proteins were stained with Ponceau red (Ponceau S). Three biological replicates 583
were performed, and similar results were obtained. B. Immunoblots in A were analyzed with 584
Phoretix 1D software (Phoretix International). The relative amounts of LHCB1 and LHCA1 585
were normalized to the level of the β subunit of the ATP synthase (ATPase β). The relative 586
phosphorylation level of the LHCII proteins were further normalized to the protein levels of 587
LHCB1. Phosphorylation and protein levels in the mutant plants are shown relative to the 588
levels in the wild-type plants (100%). C. Steady-state protein levels in the thylakoids of wild 589
type (Col-0 and Ler-0) as well as stn7/8, ch1-2, ffc, and chaos mutant plants, which were 590
adapted to state II light (red light, 660 nm). Aliquots of 15 μg of total thylakoid proteins were 591
loaded on the gels. Description of thylakoid membrane protein complexes and their diagnostic 592
components are labeled on the left. Two biological replicates were performed, and similar 593
results were obtained. 594
595
Fig. 6. Distribution of phosphorylated LHCII proteins and PSII core subunits in the 596
thylakoid complexes. 597
Equal amounts of thylakoid membranes (9 μg of Chl) from wild-type plants (Col-0, A and Ler-0, 598
F) and stn7/8 (B), ch1-2 (C), ffc (D), chaos (E) mutants, which had been adapted to state II 599
light (red light, 660 nm), were solubilized with 1% (w/v) digitonin and separated by lpBN-PAGE. 600
Individual lanes from the lpBN-PA gel were subjected to SDS-urea-PAGE in the second 601
dimension, immunoblotted and probed with an anti-phosphothreonine antibody (Anti-P-Thr). 602
The phosphorylated LHCII (P-LHCII) and PSII proteins (P-D1, P-D2, and P-CP43) were 603
indicated by arrows. The proposed P-LHCII proteins associated with PSI-LHCI complexes are 604
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30
circled. 605
606
607
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