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The SEC6 protein is required for function of the contractile vacuole in Chlamydomonas 1
reinhardtii 2
Karin Komsic-Buchmann, Lisa Marie Stephan and Burkhard Becker 3
Botany, Cologne Biocenter, University of Cologne, Cologne, Germany 4
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Corresponding author: Burkhard Becker, Botany, Cologne Biocenter, University of Cologne, 6
Zülpicher Str. 47b, 50674 Cologne, Germany, Tel.: -49 221 470 7022; Fax: +49 221 470 7
5181; E-mail: [email protected] 8
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Running title: Contractile vacuole of Chlamydomonas 10
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© 2012. Published by The Company of Biologists Ltd.Jo
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JCS online publication date 16 March 2012
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Summery 12
Contractile vacuoles (CVs) are key players of osmoregulation in many protists. To investigate 13
the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory 14
mutants. 4 isolated mutant cell lines carried the same 33,641 b deletion rendering the cell lines 15
unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was 16
analyzed in detail. Mutant cells contained a variable CV morphology with most cells 17
displaying multiple small CVs. In addition enlarged 1 or 2 CVs or no light microscopically 18
visible CVs at all were observed. These findings suggest that the mutant is impaired in 19
homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants displayed a 20
long flagella phenotype. One of the affected genes is the only SEC6 homologue in 21
Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex 22
required for efficient exocytosis. Transformation of the Osmo75 mutant with CreSEC6GFP 23
construct rescued the mutant completely (osmoregulation and flagellar length). Rescued 24
strains overexpressed CreSEC6 (as GFP-tagged protein) and displayed a modified CV 25
activity. CVs were significantly larger, whereas the CV contraction interval remained 26
unchanged leading to increased water efflux rates. Electron microspical analysis of Osmo75 27
showed that the mutant is able to form the close contact zones between the PM (plasma 28
membrane) and the CV membrane observed during late diastole and systole. These results 29
indicate that the CreSEC6 is essential for CV function and required for homotypic vesicle 30
fusion during diastole and water expulsion during systole. In addition CreSEC6 is not only 31
necessary for CV function, but possibly influencing the CV cycle in an indirect way and 32
flagellar length control in Chlamydomonas. 33
Key-Words: Contractile vacuole; flagella; osmoregulation; SEC6; exocyst; Chlamydomonas 34
Introduction 35
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Contractile vacuoles (CVs) are osmoregulatory organelles found in many unicellular cell wall 36
less freshwater protists and some sponges (Allen and Naitoh, 2002). CVs are membrane 37
bound compartments that periodically accumulate (diastole) and expel water out of the cell 38
(systole), allowing cells to survive under hypotonic conditions. Based on structure and 39
behavior about 6 basic types of CVs have been described (Patterson, 1980). Despite this 40
structural diversity the basic mechanism seems to be conserved between different eukaryotes 41
as the same proteins/cellular processes have been implicated in CV function in Amoeba, 42
Dictyostelium, Paramecium, Trypanosoma and green algae [e.g. V-ATPase (Becker and 43
Hickisch, 2005; Fok et al., 2002; Heuser et al., 1993; Montalvetti et al., 2004; Nishihara et al., 44
2008; Robinson et al., 1998; Wassmer et al., 2005), aquaporin (Montalvetti et al., 2004; 45
Nishihara et al., 2008), vesicular transport (Becker and Hickisch, 2005; Buchmann and 46
Becker, 2009; Bush et al., 1994; Harris et al., 2001; Kissmehl et al., 2007; Schilde et al., 47
2006; Stavrou and O'Halloran, 2006), see Komsic-Buchmann & Becker (2012) for a summary 48
of identified proteins and cellular processes]. 49
The osmoregulatory role of CVs is supported in many experiments (Allen, 2000; Allen and 50
Naitoh, 2002) and it has been proposed that water flow into the CV is by osmosis. V-ATPase 51
and/or V-PPase drive secondary active transport systems allowing water to follow passively 52
through aquaporins. However, no significant acidification of the CV (as expected by a proton 53
pump mediated uptake system) has ever been observed. Therefore, HCO3- has been postulated 54
to be the most likely anion species to be continuously eliminated from the cell via the CV 55
(Robinson et al., 1998; Tominaga et al., 1998). This would be similar to the situation for water 56
transport in animal epithelia (Hoffmann, 1986; Zeuthen, 1992), but experimental evidence for 57
a role of bicarbonate in CV has never been presented. In contrast, experimental evidence 58
points to a role of phosphate in CV function in Trypanosoma and Chlamydomonas (Rohloff et 59
al., 2004; Ruiz et al., 2001) and K+ and Cl- have been identified as the major osmolytes in the 60
cytosol and CV in Paramecium (Stock et al., 2002). 61
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The structure and function of the CV from Chlamydomonas has been investigated in 62
some detail (Luykx et al., 1997a; Luykx et al., 1997b; Robinson et al., 1998). At the end of 63
diastole the contractile vacuole of Chlamydomonas has a spherical shape, expels the liquid 64
into the medium and the CV fragments into smaller vacuoles (systolic phase, see also Fig. 1 65
C). During diastole these smaller vacuoles swell and fuse with each other to form again the 66
spherical shaped vacuole at the end of a cycle (Luykx et al., 1997b, Fig. 1 A). Several 67
questions remain in Chlamydomonas and/or in general: Exocytotic pore-like structures were 68
identified in ciliates (McKanna, 1973) but have been very difficult to demonstrate in many 69
green algae (e.g. Buchmann and Becker, 2009; Luykx et al., 1997b). How the liquid leaves 70
the cell in these systems is not clear, but conspicuous intra-membrane particle arrays (up to 71
180 nm in diameter) have been observed in the plasma membrane overlying the CV region 72
(Weiss et al., 1977). These arrays apparently form only during systole and are often matched 73
by a similar array in the CV membrane opposing the PM array (Weiss et al., 1977). Both 74
array are connected by cytosolic electron dense material (Weiss et al., 1977; Fig. 1 A) and 75
similar cytosolic electron dense material has also been detected in another green alga 76
Mesostigma viride (Buchmann and Becker, 2009). A role for cytoskeletal elements during the 77
CV cycle could only be demonstrated in Dictyostelium (Tsiavaliaris et al., 2008), indicating 78
that force-generation during systole by cytoskeletal elements does not play any role in most 79
systems. Changes in membrane structure have been implicated in water expulsion during 80
systole in Paramecium (Allen and Naitoh, 2002), whether this is a general mechanism 81
remains to be seen. In addition, our knowledge how the CV cycle is controlled and adapted to 82
the need of the cell is at best fragmentary. Calcium, protein kinases and cAMP have been 83
implicated (Rohloff and Docampo, 2008), but in no system the CV is really understood. 84
Chlamydomonas is a well established protist model system (Grossman et al., 2003). 85
The genome of Chlamydomonas has recently been sequenced (Merchant et al., 2007). 86
Chlamydomonas can be transformed using several methods (Coll, 2006; Grossman et al., 87
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2003). Silencing of genes using RNAi has been successfully introduced in Chlamydomonas 88
and is continuously improved (Schroda, 2006) and several proteins have been expressed as 89
GFP-tagged constructs (e.g. Fuhrmann et al., 1999; Huang et al., 2007; Ruiz-Binder et al., 90
2002; Schoppmeier et al., 2005), allowing to observe the in-vivo dynamics of subcellular 91
structures and/or proteins. For this reason we have started a forward genetic approach to 92
analyze CV function in Chlamydomonas. Osmoregulatory mutants were isolated after 93
insertional mutagenesis, which showed defects in CV structure and function. Here, we 94
analyze a mutant, where membrane fusion events related to CV function are apparently 95
impaired. We show that the deletion of the single Chlamydomonas SEC6 protein accounts for 96
the observed phenotype, indicating a role for the SEC6 and most likely the exocyst complex 97
in CV function in Chlamydomonas. 98
Results 99
Characterization of the contractile vacuole of Chlamydomonas reinhardtii CC3395 100
We planned using Chlamydomonas reinhardtii strain CC3395 for the mutant screen, which 101
lacks a cell wall completely and is easily transformed. Therefore, we first characterized the 102
CV of this strain using light and electron microscopy (Fig. 1 A - D). As in other 103
Chlamydomonas strains the large round CV visible at late diastole develops from small 104
vacuoles (Fig. 1 A,), forms close contact zones with the PM at the end of diastole (Fig. 1 B), 105
which apparently persist during systole (Fig. 1 C). 106
CC3395 grows in media of different osmotic strength (Fig. 1 E, note that TAP/2 contains 107
only half of the mineral nutrients as the other media, see also Fig. 8 A). Cells displayed two 108
CVs at the anterior end in all media tested except TAP-SS (containing 120 mM sucrose 109
increasing the total osmotic strength of the medium to 204 mosM). In this medium less than 5 110
% of the cells displayed light microscopically visible CVs . Chlamydomonas displays light 111
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microscopical visible CVs only in hypotonic media, therefore, this result indicates that the 112
cytosolic osmolarity of Chlamydomonas CC3395 is approximately 200 mosM. Preliminary 113
data indicate that the cytosolic osmolarity varies with growth conditions and status of the cells 114
(unpublished own observations), therefore only cells 4-6 days after subculturing (end of log 115
phase, Fig. 1 E) were used in our analysis (see Material and Methods for details on cell 116
culturing). In TAP medium the average maximum diameter of the large round vacuole at the 117
end of the diastole was 1.78 ± 0.43 µm and the contraction interval 20.6 ± 5.3 s (n = 45). The 118
diastole lasts 19.4 ± 5.0 s and the systole 1.3 ± 0.5 s (see also Movie 1). From these results it 119
can be calculated that in TAP medium (64 mosM) a Chlamydomonas CC3395 cell expels on 120
average approximately 11.9 ± 8.75 µm³/min ● cell (approximately 2 % of the total cell 121
volume per minute). Water uptake in a cell is directly proportional to the cell surface area; we 122
therefore performed a linear correlation analysis between CV volume, CV period and water 123
efflux and the cell surface area of a cell. As is evident from Fig. 1 F all three factors show a 124
good correlation to the cell surface area (r² = 0.6568 (CV period – cell surface area), r² = 125
0.7907 (CV volume – cell surface area); r² = 0.7872 (efflux – cell surface area)). 126
Chlamydomonas cells increase their size during the cell cycle considerably. As the cells are 127
not synchronized in our cultures, we tried to normalize the CV data using the cell surface 128
areas determined for each cell from the videos used to characterize the CVs. We then 129
calculated the mean values and standard deviations for the normalized and non-normalized 130
data set (Fig. 1 G). As expected the normalized data seta, showed less variation compared to 131
the non-normalized data set for the CV volume and the water efflux from a cell (compare the 132
coefficients of variation indicated above the bars in Figure 1 G). In contrast, the standard 133
deviation obtained for the normalized data set was bigger for the contraction interval of the 134
CV, when compared to the non-normalized data set (Fig. 1 G). These results indicate that 135
cells use mainly variation of the size of the CV to adapt to the increasing water influx during 136
the cell growth, while the contraction period is apparently regulated by a different factor. 137
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Mutant screen 138
We used the mutant screen designed by Luykx et al. (1997a) in combination with insertional 139
mutagenesis using the hygromycin B resistance marker developed by Berthold et al. (2002) to 140
isolate osmoregulatory insertional mutants (see Material and Methods for details). 2858 141
hygromycin B-resistant clones were obtained on TAP plates containing 0.06 M sucrose (TAP-142
S, 144 mosM), and screened for failure to grow in TAP medium. 7 mutant cell lines failed to 143
grow at all in TAP medium. In addition 68 cell lines showed a different growth phenotype 144
(different growth rate, different color etc.) compared to cells grown in TAP medium 145
containing 0.06 M sucrose, but were still able to grow in TAP medium. Altogether we 146
obtained a total of 75 potentially osmoregulatory mutants (named Osmo1 to Osmo75 further 147
on). We concentrated our work on the 7 cell lines (Osmo12, 28, 32, 64, 66, 67, 75) showing a 148
strong phenotype (no growth in TAP medium, growth in TAP-S medium). 149
Osmo64, 66, 67, 75 carry the same insertion of the hygromycin B marker 150
RESDA-PCR (Gonzalez-Ballester et al., 2005) was used to determine the locus of insertion of 151
the hygromycin B resistance marker for Osmo12, 28, 32, 64, 66, 67 and 75. We obtained the 152
5’ and 3’ flanking sequences for Osmo64, 66, 67 and 75. All 4 isolated strains contain exactly 153
the same 33,641 b deletion (Fig. 2 A), indicating that the clones might have originated from 154
the same insertion event (possibly by cell division after the insertion of the marker gene, 155
during the recovery time after transformation). In contrast, we were only able to determine the 156
3’ insert flanking sequences for the other three mutants (Osmo28 Fig. 2 B; Osmo12 and 32 157
Fig. 2 C) showing a strong phenotype. Primer walking indicated that also in these strains large 158
deletions (> 9 kb) had occurred (Fig. 2 B and C). As the 5’ insert flanking sequences are 159
identical for Osmo12 and Osmo32, most likely, these two clones also originated from the 160
same insertion event. As currently the deletion size in Osmo12, 28 and 32 is not known; all 161
further work was carried out with Osmo75 as a representative of Osmo64, 65, 67 and 75. 162
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Characterization of Osmo75 163
We determined growth curves for Osmo75 in the same media used for characterization of the 164
parental strain and characterized the mutant cell lines by video and electron microscopy (Fig. 165
3). Fig.3 A shows the growth curves for Osmo75 in the different media (see also Fig. 8 A). As 166
is evident, the mutant is not able to grow in media of low osmolarity. Assuming that the 167
mutant cell lines possess a similar cytosolic osmolarity than the parental strain, Osmo75 cells 168
are able to grow under mild hypotonic conditions (144 mosM) but fail to grow or even die 169
under strong hypotonic conditions (≤ 64 mosM). 170
Video and electron microscopy was used to investigate whether the observed growth 171
defect is related to CV miss function. Video microscopy confirmed that indeed Osmo75 172
displayed an aberrant CV cycle (Movies 2 – 4). In all hypotonic media tested, all cells of the 173
parental strain have 2 CVs following a typical alternating CV cycle with a large round 174
vacuole at the end of the diastole (Fig. 1 D, Fig. 3 B). In contrast, all Osmo75 cells showed 175
CV dysfunctions (changes in the number of CVs, the size and the contraction interval of a 176
CV) in TAP-S medium (Fig. 3 B – L). However, the CV phenotype was quite variable in a 177
given Osmo75 cell population. Most of the cells showed multiple smaller CVs in the region 178
close to the basal bodies (61.3 %, Fig. 3 D and E, Movie 3). Surprisingly 23.7 % of the cells 179
do not display any light microscopically visible CVs (Fig. 3 F, Movie 4) at all. In 8.0 % of the 180
investigated cells one enlarged CV (Fig. 3 G) was visible and in 3.7 % two enlarged CVs 181
(Fig. 3 C, Movie 2) were visible. Finally, in 3.3 % of the examined cells a mixed morphotype 182
was detected; one enlarged CV and multiple smaller CVs (Fig. 3 H). 183
Electron microscopy confirmed the light microscopical observations (Fig. 3 I and J). 184
We could also detect the typical contact zones formed by the CV membrane with the PM 185
during systole, although water expulsion is clearly impaired in Osmo75 (see below, Fig. 4). 186
Often contact zones in Osmo75 appear to contain less electron dense cystosolic material 187
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between the PM and CV membrane than in CC3395 cells (compare Fig. 1 B with Fig. 3 K and 188
L, see also Fig. 8 H). 189
To investigate the behavior of individual CVs in the Osmo75 strain in more detail we 190
selected videos obtained from cells showing the phenotype one or two enlarged CVs (Fig. ) 191
and recorded the size of the CVs every 5 s in TAP-S medium. For comparison the size of 192
individual CVs in the parental strain were recorded too. CVs in CC3395 showed an oscillating 193
pattern. The diameter of a CV increases during diastole and rapidly decreases during systole; 194
at the end of systole generally no CV is visible in the light microscope (Fig. 4). However, it is 195
noteworthy that in TAP-S CVs of CC3395 cells do not always completely empty (Fig. 4), 196
whereas in TAP medium the CV of CC3395 always completely disappears (not shown). In 197
contrast, CVs of Osmo75 cells show irregular increases and decreases of the CV diameter or 198
appear for some time constant (Fig. 4), however total discharges rarely occurred. 199
200
Taken together the observed osmoregulatory phenotype indicates that in Osmo75 201
membrane fusion events during the CV cycle are impaired: 1. Multiple small vacuoles might 202
be caused by inefficient homotypic vacuolar fusion during diastole. 2. Enlarged CVs are 203
possibly caused by failure to terminate systole and achieve water expulsion. 204
Finally we noted that Osmo75 displayed an additional flagellar length phenotyope. 205
Flagella from the parental CC3395 strain had a length of 6.97 ± 1.05 µm (Fig. 5). In contrast 206
flagella of Osmo75 cells were much longer (9.63 ± 1.55 µm, Fig. 5). 207
Protein targeting to the CV is not impaired in Osmo75 208
To test whether protein targeting to the CV is impaired in Osmo75 we tried to develop 209
an GFP marker system for CV in Chlamydomonas. Aquaporins have been implicated in CV 210
function in several other organisms (Montalvetti et al., 2004; Nishihara et al., 2008). The 211
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genome of Chlamydomonas reinhardtii encodes only 2 putative aquaporins (Anderberg et al., 212
2011) CreMIP1 (Cre12.g549300, www.phytozome.net) and CreMIP2 (Cre17.g711250). RT-213
PCR showed that CreMIP1 but not CreMIP2 is expressed in vegetative cells (data not shown). 214
We reasoned that CreMIP1-GFP might be a useful marker to investigate whether protein 215
targeting to the CV is impaired in the Osmo75 cell line. In addition expression of 216
CreMIP1GFP might confirm that as in other systems aquaporins are localized to the CV. The 217
full length cDNA of CreMIP1 was cloned into the GFP expression vector pJR38 (Neupert et 218
al., 2009). Osmo75 and the UVM4 strain (which was specifically developed for GFP 219
expression in Chlamydomonas (Neupert et al., 2009)) were transformed with a linearized 220
ScaI/XbaI fragment of pJR38-MIP1GFP, coding for CreMIP1GFP and the APHVIII protein 221
(paromomycin resistance). Paromomycin resistant clones were selected and screened for GFP 222
expression. Fig. 6 shows the results of this experiment. Non-transformed cells showed some 223
background fluorescence in the GFP channel (Fig. 6 A). However, as is evident from Figs 6 B 224
– E CreMIP1GFP clearly localized to the CV in the UVM4 (Figs 6 B and C) and Osmo75 225
(Figs 6 D and E) strains, indicating that protein targeting of the CreMIP1GFP construct to the 226
CV is not impaired in Osmo75. In addition, using CreMIP1GFP in the UVM4 genetic 227
background we always observed that the membranes of the CV and PM apparently do not mix 228
with each other during the CV cycle (Fig. 6 C). 229
Rescue of Osmo75 230
Based on the available genome sequence (www.phytozome.net), the 33,641 b deletion in 231
Osmo75 affects 6 gene models. 4 putative proteins are deleted and two additional putative 232
proteins are truncated. Tab. 1 summarizes the available information on the 6 gene models. 233
Gene model Au9.Cre20.g759900.t1 encodes the only putative SEC6 protein in 234
Chlamydomonas. SEC6 proteins have been shown to be part of the exocyst complex (Bröcker 235
et al., 2010). The exocyst complex belongs to the group of multi-subunit tethering factors, 236
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required for efficient membrane fusion events and is involved in polarized secretion in many 237
eukaryotic systems. Membrane fusion events occur during the diastole and at the beginning of 238
systole during a CV cycle. The deletion of a protein similar to SEC6, possibly affecting 239
exocyst function, seems therefore a likely molecular cause for the observed phenotype of 240
Osmo75. For this reason we concentrated our work on this protein, named CreSEC6 further 241
on. 242
RT-PCR revealed that CreSEC6 is expressed in the parental strain (Fig. 7, lanes 1 - 4) under 243
all tested osmotic conditions, whereas we failed to amplify the same PCR-fragment from the 244
Osmo75 strain (Fig. 7, lane 5). Based on the augustus gene model published at 245
www.phytozome.net, we expected the full-length cDNA to be 2019 b (672 aa) long excluding 246
both UTRs. However, the isolated full-length cDNA was 2439 b (812 aa) long. Comparison 247
of the full-length cDNA with the transcript and protein sequence at www.phytozome.net 248
indicates that the predicted protein sequence at www.phytozome.net misses one exon. This 249
exon is also present in the published Arabidopsis sequence. Blast analysis of the full-length 250
cDNA sequence showed that CreSEC6 is 31 % identical (47 % similar) to the SEC6 from 251
Arabidopsis thaliana. 252
To confirm that CreSEC6 is indeed responsible for the observed phenotype of Osmo75 253
we tried to rescue the mutant with a CreSEC6GFP fusion protein using again pJR38 as 254
expression vector. 312 clones resistant to hygromycin B and paromomycin were obtained on 255
TAP plates. As Osmo75 cells do not grow in TAP medium, this already indicated a successful 256
rescue. For all clones investigated no defects in CV function could be observed by live cell 257
observations light microscopy and the long flagellar phenotope was nearly completely rescued 258
when analyzed in three randomly selected rescue cell lines (Fig. 5) 259
To characterize the rescued strains in more detail, 10 strains were randomly selected 260
(Osmo75-A5, A9, C11, D10, E3, F6, F9, G6, H5 and H7). RT-PCR analysis of all 10 selected 261
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cell lines showed that the CreSEC6GFP construct is expressed in all 10 strains (Fig. 7, lanes 6 262
- 15) at a higher level than the endogenous SEC6 in CC3395. Thus all rescue cell lines 263
represent SEC6-overexpressors. Although all 10 rescue strains grew at all osmotic conditions 264
tested, some strains did not grow as well on strong hypotonic media (e.g. A5 and D10) and 265
isotonic media (A5 and D10), possibly due to different insertion sites of the CreSEC6GFP 266
construct in the various strains. Detailed light microscopical analyses of the CV cycle 267
revealed interesting differences between the ten rescued Osmo75 strains investigated and the 268
parental strain. Wheras the contraction interval was not significantly altered in all 10 rescue 269
strains; the ratio of CV/cell surface area and water efflux/cell surface area increased 270
significantly (CV volume/cell surface area p ≤ 0.001 for 7 of 10 rescue strains; water efflux 271
/cell surface area p ≤ 0.001 for all ten rescue strains). Electron microscopy confirmed that the 272
CV structure was completely restored (Figure 8 E – I). The ultrastructure of the CV in the 273
rescued strains examined (G6: 8 E – I, C11 not shown) are indistinguishable from the parental 274
strain. 275
Discussion 276
The molecular mechanisms of CV function are still poorly understood. Over the last year 277
several proteins have been implicated in CV function in several systems (see Komsic-278
Buchmann and Becker, 2012 for a recent summary). Generally, proton pumps, SNAREs, Rab 279
proteins, and Calcium signaling have been shown to be important for CV function. The 280
current models suggest that water uptake into the CV is by osmosis, energized by proton 281
pumps and that aquaporins facilitate this process. While our knowledge about water uptake 282
into the CV has significantly increased over the last years, the mechanism of water expulsion 283
is not studied well in most systems. To increase our knowledge on CV function in green algae 284
and in general we choose a forward genetic approach using Chlamydomonas as a model 285
system and investigated the cellular localization of a Chlamydomonas aquaporin. 286
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Early genomic analyses indicated only the presence of a single clear aquaporin in the genome 287
of Chlamydomonas. A recent detailed analysis of algal MIPs indicated the presence of at least 288
a second isoform (Anderberg et al., 2011). However, RT-PCR indicates that CreMIP2 is not 289
expressed in C.reinhardtii, whereas CreMIP1 could be easily detected by RT-PCR. Using a 290
CreMIP-GFP construct we could clearly show that MIP1 is localized to the CV. In-vivo 291
observations of the CV indicated the CV membrane to be a stable compartment with no 292
intermixing of CV and plasma membrane. These results suggest that similar to the CV in 293
other systems, the membrane of the CV contains an aquaporin (Montalvetti et al., 2004; 294
Nishihara et al., 2008). In addition, as in many other systems the CV membrane and the 295
plasma membrane do not intermingle during the CV cycle (Patterson, 1981; Zanchi et al., 296
2010), suggesting that potential membrane fusion events follow the kiss-and-run mechanism. 297
For the mutant screen we selected Chlamydomonas CC3395, which misses a cell wall 298
completely. Analyses of the CV cycle in CC3395 showed that overall, the situation is very 299
similar to strain 137c (average diameter at end of systole, contraction interval), indicating that 300
the cell wall has only a minor effect on water uptake in Chlamydomonas. However, our 301
results indicate that the cytosolic osmolarity of Chlamydomonas cells is slightly higher in 302
CC3395 than in 137c. 303
Insertional mutants were generated and screened for CV dysfunction. Four of the obtained 304
mutants (Osmo64, 65, 67 and 75) show the same 33,641 b deletion indicating that the clones 305
might have originated from the same insertion event (possibly by cell division after the 306
insertion of the marker gene during the recovery time). Such large deletions are not 307
uncommon in Chlamydomonas after transformation (Gonzalez-Ballester et al., 2011) and 308
have been proposed do depend on the type of marker (large size, full plasmid) and the 309
transformation method used (Gonzalez-Ballester et al., 2011). However, as we obtained large 310
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deletions using a small linear DNA fragment, most likely the transformation method is more 311
important in this respect. 312
The 33,641 b deletion in Osmo75 includes the only SEC6 protein, encoded in the 313
Chlamydomonas genome. Characterization of the phenotype indicates that most likely 314
membrane fusion events during diastole (homotypic vacuolar fusion) and systole (exocytosis) 315
are not operating efficiently in the CV in Osmo75 leading to hypotonic sensitivity of the cells. 316
In addition cells displayed a long flagella phenotype. Rescue of the Osmo75 phenotype with a 317
CreSEC6GFP construct confirmed that indeed the deletion of CreSEC6 is responsible for the 318
observed defect in CV function and flagellar length in Osmo75. The SEC6 protein is part of 319
the exocyst complex, which belongs to the multi subunit tethering factors (MTCs, Bröcker et 320
al., 2010). MTCs are ancient facilitators of membrane fusion events and current knowledge 321
indicates that every membrane fusion event requires its own tethering factor (Koumandou et 322
al., 2007). The exocyst complex has been shown to be required for efficient exocytosis in 323
various systems (Bröcker et al., 2010; Zhang et al., 2010). In this respect the observed 324
phenotype in Osmo75 is surprising in two aspects: 1. we observed inefficient exocytosis 325
(leading to enlarged CVs) and that homotypic vacuolar fusion (leading to multiple smaller 326
CVs) are not taken place efficiently. Work in the yeast system indicates that homotypic 327
vacuolar fusion is mediated by the HOPS complex (Bröcker et al., 2010). The HOPS complex 328
in yeast consists of 6 different subunits (Bröcker et al., 2010) and 2 subunits have so far not 329
been found in the Chlamydomonas genome (Koumandou et al., 2007). Given the observed 330
phenotype in Osmo75 it is tempting to speculate that in Chlamydomonas SEC6 is also 331
involved in HOPS complex mediated homotypic vacuolar fusion. 2. the SEC6 deletion mutant 332
in Chlamydomonas is viable, whereas SEC6 is essential for growth in yeast (Potenza et al., 333
1992) and Arabidopsis T-DNA insertion lines that disrupt SEC6 expression fail to produce 334
homozygous progenys (Hala et al., 2008). The latter is caused by defects in pollen 335
germination and growth, indicating a major role in polar secretion in plants. However, in 336
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Chlamydomonas polar secretion seems not completely impaired, as the cells are able to form 337
longer flagella. This is in striking contrast to the requirement of the exocyst in ciliogenesis in 338
animals (Das and Guo 2011; Zuo et al., 2009). The exocyst localizes to the base of primary 339
cilia in MDCK epithelial cells (Rogers et al., 2004) and deletion of SEC10 abolishes 340
ciliogenesis (Zuo et al., 2009), while overexpression of SEC10 led to elongated primary cilia. 341
In contrast, deletion of SEC6 in Chlamydomonas caused elongated flagella, while 342
overexpression of a SEC6GFP did not change the flagellar length, The reason for the 343
difference in behavior between these two systems is currently not clear. 344
To our knowledge this is the first report of an involvement of SEC6 (and most likely the 345
exocyst complex) in CV function. Recently, Zanchi et al. (2010) reported that a secA mutant 346
in Dictyostelium discoideum develop a large vacuole, which was shown to be derived from 347
the CV. SecA is the Dictyostelium homologue of the yeast SEC1 and the mammalian Munc18 348
proteins (SM proteins), which are involved in vesicle docking during exocytosis and have 349
been shown to interact with the exocyst complex, pointing also to a role of the exocyst 350
complex in CV function in Dictyostelium. However, contrary to the SEC6 deletion in 351
Osmo75 the SecA mutation in Dictyostelium leads only to an enlarged CV and not to a 352
multiple CV phenotype, supporting the idea that the phenotype of Osmo75 indicates a 353
dysfunction of two different cellular processes (homotypic vacuolar fusion and exocytosis). 354
Interestingly, in a recent study Morgera et al. (2012) showed, that SEC6 regulates exocytosis 355
by interaction with SEC1. SEC1 (the yeast PM SM-protein (plasma membrane sec1/Munc18-356
like proteins) binds to the t-SNARE SEC9, inhibiting the formation of the SNARE-complex 357
required for exocystosis. SEC6 releases SEC1 from SEC9, thus allowing exocytosis to 358
proceed (Morgera et al., 2012). Given the function of the exocyst in other systems and these 359
new findings, it seems plausible, that the exocyst in Chlamydomonas is required for the 360
formation of the close contact zones between PM and CV membrane and polar secretion in 361
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flagellar biogenesis. SEC6 might be required to efficiently proceed further with water 362
expulsion in the CV cycle by releasing a similar block as the SEC1 block observed in yeast. 363
TMaterials and methods 364
Cell Cultures 365
The following strains were used in this study: Chlamydomonas CC 3395 (arg7-8 cwd mt1, 366
Shimogawara et al., 1998), UVM4 (cwd mt+ arg7, Neupert et al., 2009). Cells were cultured 367
in TAP medium (Gorman and Levine, 1965; the media for CC3395 and all derived mutant 368
cell lines were supplemented with additional arginine). To achieve different osmolarities the 369
medium was either diluted with aqua dest. (TAP/2), 60 mM or 120 mM sucrose was added for 370
TAP-S or TAP-SS, respectiveley. The osmolarity of all media was determined using a 371
freezing point depression osmometer (Osmomat 010, Gonotec, Berlin, Germany). All 372
transformants were always kept under selection pressure by addition of antibiotics to the 373
medium and transferred into new media latest every 6 weeks. Cells were cultured at 21°C 374
with a photon flux of 70 µMol/m² s and a 14/10 light/dark cycle. In all experiments five days 375
old cultures (+/- one day) were used with a cell density of 106 - 107cells ml-1. 376
Transformation of Chlamydomonas cells 377
All transformations were performed using the glass bead method by Kindle et al. (1990). For 378
the insertional mutagenesis Chlamydomonas CC3395 cells were transformed with the 379
HindIII-cassette of the pHyg3 (Berthold et al., 2002). Cells were allowed to recover first for 2 380
h in TAP followed by 16 h in TAP-S in the dark and plated on TAP-S plates containing 10 381
µg/ml hygromycin B (Roth, Karlsruhe, Germany). After transformation of UVM4 and 382
Osmo75 with the CreMIP1GFP fusion construct cells were recovered in the appropriate 383
media and plated onto plates containing paromomycin (Sigma, St. Louis, MO, USA, 10 g/ml). 384
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Transformed cells were screened for fluorescence of the GFP with the fluorescence 385
microscope (see below). 386
Screening for osmoregulatory mutants 387
Individual clones were picked and transferred into 96 well plates containing in each well 200 388
µl of TAP-S . After two to three weeks an aliquot was transferred into new microtiter plates 389
containing either TAP-S or TAP medium. Cell lines which showed a different growth in TAP 390
compared to TAP-S were selected and the screening process was performed in triplicates. 391
Determination of the insertion site 392
The insertion flanking regions were determined using the RESDA-PCR protocol of Gonzales-393
Ballester et al. (2005) and specific primers for the HindIII fragment of pHyg3 developed by 394
(Matsuo et al., 2008). 395
GFP-fusion constructs 396
Total RNA was isolated using TRI REAGENT (MRC, Cincinnati, OH, USA) following the 397
manufacture’s instruction. cDNA-synthesis was performed with the Revert Aid First Strand 398
cDNA Synthesis Kit (Fermentas, Burlington, Canada). In all PCR reactions dream taq 399
(Fermentas, Burlington, Canada) were used in combination with an enhancer for GC-rich 400
templates (Ralser et al., 2006). The complete cDNA of CreSEC6 (Cre20.g759900) and 401
CreMIP1 (Cre12.g549300) was amplified and cloned into pGEM-T-easy (Promega, Madison, 402
WI, USA). Subsequently, NdeI restriction sites were added to the coding sequences by PCR 403
and ligated into the NdeI-restriction site of pJR38 (Neupert et al., 2009) resulting in 404
CreSEC6GFP and CreMIP1GFP fusion constructs (primer, sequences and vector maps are in 405
the supplemental Table S1, supplemental Data S1 and supplemental Figs. S1 and S2). 406
Mutant rescue 407
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For rescue of the mutant phenotype Osmo75 cells were transformed with CreSEC6GFP. After 408
transformation cells were allowed to recover in TAP medium before plating the cells on solid 409
TAP medium without additional sucrose. 410
RT-PCR 411
Total RNA was isolated using the peqGOLD Plant RNA kit (peqlab, Erlangen, Germany). 412
cDNA-synthesis was performed with the Revert Aid First Strand cDNA Synthesis Kit 413
(Fermentas,Burlington, Canada), 1 µg total RNA was used for the synthesis. In the PCR 414
reactions dream taq (Fermentas, Burlington, Canada) were used in combination with an 415
enhancer for GC-rich templates (Ralser et al., 2006). The primer were designed using 416
quantprime (http://www.biomedcentral.com/1471-2105/9/465 ) and were specific for cDNA 417
(primer are listed in the supplemental Table S1). 418
Growth in different media 419
Cells for the determination of growth curves where cultured under reduced light conditions 420
(20 – 30 µMol/m² s). Two biological replicates were counted four times each using a 421
Neubauer hematocytometer. 422
Growth on media of different osmotic strength was also analyzed using a plate assay. The 423
number of cells of a culture were counted using a Neubauer hematocytometer. Cells were then 424
diluted with TAP medium to a concentration of 0.37 x 106 cells ml-1. 3 µl of the diluted cell 425
suspension were dropped in triplicates onto agar plates with different osmotic strength 426
(without antibiotics). Cells were grown for 21 days. 427
428
Light microscopy 429
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Light microscopy was performed as described in Buchmann & Becker (2009), except that we 430
used 7 µl cell suspension on each slide. For video microscopy pictures were taken every half 431
second. At least three cycles were analyzed per CV. The surfaces and volumes of the CVs 432
and cells were calculated as a prolate spheroid. The efflux rate per cell was calculated using 433
the size and period of the cell investigated. For measuring the flagella length cells were fixed 434
with 5 % of Lugol’s iodine. Linear regression analysis and significance tests (student t-test 435
with Welch correction) were don using the GraphPad-Prism 5 software (GraphPad Software 436
Inc., La Jolla, CA USA. Fluorescence microscopy was performed using a Nikon Eclipse 800 437
(Nikon GmbH, Düsseldorf, Germany) microscope equipped with a mercury short lamp 438
(Osram, Düsseldorf, Germany), Uniblitz shutter control (Vincent Associates, Rochester, NY), 439
a GFP filter set (480/40; 505; 535/50) and a Spot RT CCD digital camera (Diagnostic 440
Instruments). The images and videos were analyzed with the Metamorph imaging software, 441
version 6.3r4 (Universal Imaging, Corp.). 442
Electron microscopy 443
Cells were concentrated (500 g, 20°C, 15 min) and resuspended in HSM medium with a 444
appropriate amount of sucrose added to reach the respective osmotic strength and additional 445
HEPES (3 mM final concentration) before fixation simultaneously with glutaraldehyde and 446
aqueous osmium tetroxide (final concentration 1.25 % and 1 %). The first minute of fixation 447
was at room temperature and additional 30 minutes on ice. After fixation the cells were 448
washed once with fresh medium. To allow easier handling during the dehydration procedure, 449
the cell pellet obtained by centrifugation were cross linked with BSA as follows: Cells were 450
resuspended in BSA solution (30 % in medium) and transferred into BEEM capsules (Plano, 451
Marburg, Germany), pelleted (500 g, RT, 15 min) and overlaid with glutaraldehyde solution 452
(2.5 % in medium). Samples were incubated for 30 minutes on ice prior to removal of the 453
pellets. The cell pellets were incubated over night in a 1 % aqueous uranyl acetate solution at 454
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4°C. Samples were washed and dehydrated by an Ethanol series and embedded in Epon 812. 455
60 nm thin ultrathin sections were cut with a Leika-microtome EM UC7 and a diamante knife 456
(Diatome, 45° angle). Sections were stained with 2 % aqueous uranyl acetate and lead citrate 457
(Reynolds, 1963). Micrographs were taken with a transmission electron microscope (CM 10, 458
Phillips, Eindhoven, The Netherlands) and an digital camera (Orius SC200W 1, gatan, 459
Pleasanton, CA). Images were analyzed with Digital Micrograph and adobe photoshop CS4. 460
461
462
Acknowledgements 463
This work was supported by a DFG grant (Be1779/12-1) to BB. The authors thank R. Bock 464
(Golm, Germany) for providing plasmid pJR38 and the UVM4 strain and W. Mages 465
(Regensburg, Germany) for the plasmid pHyg3. The authors thank M. Schroda (Golm, 466
Germany), K.-F. Lechtreck (Athens, GA, USA) and J. Brown (Worcester, MA, USA) for 467
helpful discussions. In addition, we thank the following students: D. Langenbach, R. M. 468
Benstein, A.-K. Alteköster and K. Kehl, who helped to characterize the Osmo75 mutant. 469
Author Contributions 470
KK-B carried out all experiments except the rescue of Osmo75 and analyzed the data. LS 471
performed the rescue of Osmo75 using CreSEC6-GFP. BB conceived and designed the study, 472
analyzed the data and wrote the draft of the manuscript. All authors read and approved the 473
final manuscript. 474
475
Conflict of Interest 476
None 477
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478
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634
Figure Legends 635
Figure 1: The contractile vacuole of Chlamydomonas reinhardtii CC3395. A-C shows the 636
ultra structure of the CVs in CC3395. The two CVs are located close to the basal body (A). At 637
the end of diastole (B) the CV membrane form a contact zone with the plasma membrane 638
marked by cytosolic electron dense material between the membranes (arrows). In the systolic 639
phase the CV fragments into smaller vesicles (C). The contact zone persists apparently till the 640
end of systole (arrow). The bracelet a specialized PM region at the basis of the flagella (F) is 641
marked by an ellipse in A. M = mitochondrion, N = nucleus. D shows light microscopically 642
snap shots from a time-lapse. Numbers indicate the time passed since the end of last diastole. 643
The white arrow marks the CV, scale bar = 5 µm. E depicts the growth of CC3395 in four 644
different media (TAP/2, TAP, TAP-S and TAP-SS). The strain can grow in every medium 645
tested. The graph in F demonstrates the relation of the CV period, the CV volume and the 646
efflux of each CV to the cell surface (n = 45). The bigger the cell surface is, the longer the CV 647
period, the higher the CV efflux and the larger the CV volume. G shows the mean values and 648
the standard deviation of the data set of F as non-normalized and normalized to the cell 649
surface. The CV period shows higher variation in the normalized data set, whereas the 650
normalized data set for the CV volume and the water efflux from a cell shows less variation 651
compared to the non normalized data set. Numbers above the bars indicate the coefficient of 652
variation for the different data sets. 653
Figure 2: Insertion of the hygromycin B marker cassette caused deletions in the Osmo75 (A), 654
Osmo28 (B) and Osmo12 and 32 (C) mutants. The corresponding areas of the genome of 655
Chlamydomonas reinhardtii (www.phytozome.net) are shown. The deletion determined by 656
RESDA-PCR (see material and methods) for these mutants is indicated as grey bar. In 657
Osmo75 (A) four genes are completely deleted (Cre20.g759800.t1.2 – Cre20.g759950.t1.2) 658
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and two genes are truncated (Cre20.g759750.t1.2 and Cre20.g760000.t1.2) due to the 659
insertion of the marker cassette in the genome. In Osmo28 (B) and Osmo12 and 32 (C) only 660
one flanking sequence of the marker cassette could be determined. By primer walking a 661
minimal deletion of 9 kb could be detected. Vertical arrows indicate the identified insertion 662
site at the 3’ end of the marker cassette. Horizontal arrows indicate the positions of primers 663
which failed to amplify the 5’flanking region of the marker cassette. 664
Figure 3: The contractile vacuoles of the osmoregulatory mutant Osmo75. A depict the 665
growth of Osmo75 in four different media (TAP/2, TAP, TAP-S and TAP-SS). The strain can 666
grow in TAP-S and TAP-SS, but failed to grow or even died in TAP and TAP/2. (B - H) Cells 667
of Osmo75 show variable CV morphologies. The graph in B shows the proportion of the 668
different CV morphologies in the mutant and the parental strain (3 times 100 cells were 669
analyzed). (C-H) Examples for the different CV phenotypes. CVs are indicated by white 670
asteriks. The arrowhead in F marks the cytoplasmic region normally displaying a CV. Bar = 5 671
µm. I – L show electron micrographs of the CVs of Osmo75. In I 4 CVs are visible in one cell 672
(multiple CVs per cell). Two of them show contact zones with the plasma membrane (marked 673
by arrows and are shown enlarged in the inserts K and L). The mixed phenotype of Osmo75 is 674
depicted in J, one enlarged CV and multiple smaller CVs. 675
Figure 4: Osmo75 fails to expel liquid from the cells efficiently. The diameter of individual 676
CVs was determined every 5 seconds and used to calculate the surface area of individual CVs 677
in the parental strain CC3395 and Osmo75. Each line displays a different CV. Whereas CVs 678
in the parental strain show a reiterate pattern; CVs of Osmo75 cells show a complete irregular 679
behavior. 680
Figure 5: Flagellar length of Chlamydomonas reinhardtii CC3395, the mutant Osmo75 and 681
three rescued strains (Osmo75-SEC6GFP). Significant differences to CC3395 are indicated 682
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by asterisks (* p ≤ 0.05, *** p ≤ 0.001). Significant difference of the rescued strains to 683
Osmo75 are indicated by # (### p ≤ 0.001). (n = 37, 37, 29, 33 and 32) 684
Figure 6: Expression of CreMIP1GFP in the UVM4 and Osmo75 background. A 685
untransformed UVM4 cell. B and C UVM4 cell transformed with CreMIP1GFP. D and E 686
Osmo75 cell transformed with CreMIP1GFP. B and D overview of the whole cell. C and E 687
time lapse of the CV region of the cells shown in B and D respectively. Numbers indicate the 688
time (in seconds). Exposure time for individual frames was 2.2 s. PH = phase contrast. Bar = 689
5 µm. 690
Figure 7: Expression of SEC6 in various Chlamydomonas strains. RT-PCR was performed 691
for the parental strain in 4 different media of different osmotic strength and Osmo75 (upper 692
row) and 10 rescue strains of Osmo75 (two lower rows). The gene targeted by the primers 693
used are indicated on the top. Lane numbers refer to the different templates used and are 694
explained on the right. Centrin was used as loading control. Expected length: SEC6 – 126 bp, 695
SEC6GFP – 222 bp and centrin – 94 bp. 696
Figure 8: Characterization of the rescued strains Osmo75-SEC6GFP. A depicts the growth of 697
CC3395, Osmo75 and 10 randomly selected rescued strains Osmo75-SEC6GFP (A5 to H7) 698
on agar plates with different osmotic strength ranging from strong hypotonic (32 mosM, 699
TAP/2) to isotonic (204 mosM, TAP-SS). The graph in B compares the CV period of CC3395 700
(n = 45) with the CV periods of the 10 rescued strains Osmo75-SEC6GFP A5 to H7 (n = 20). 701
Only E3 and F6 show a significant different period as CC3395 (* p ≤ 0.05). The other mutants 702
show a similar period as CC3395. C compares the CV volume relative to the cell surface area 703
of CC3395 (n = 45) in the rescued strains Osmo75-SEC6GFP A5 to H7 (n = 20). Only two 704
rescued strains C11 and D10 show a similar CV volume as CC3395, the others all differ 705
significantly (* p ≤ 0.05, *** p ≤ 0.001). D depicts the CV efflux relative to the CV surface 706
area of CC3395 (n = 45) and the rescued strains Osmo75-SEC6GFP A5 to H7 (n = 20). The 707
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efflux of all rescued strain is significant higher than the efflux of CC3395 (*** p ≤ 0.001). E – 708
I show electron micrographs of one rescued strain, Osmo75-SEC6GFP-G6. At the end of 709
diastole the CV forms contact zones with the plasma membrane (E – arrows, and enlarged in 710
H). Two CVs are visible in the cell shown in F, the left CV at mid diastole and the right CV in 711
early diastole. The contact zones seem to persist till the end of the systolic phase (G – arrows, 712
and enlarged in I). 713
714
Supplementary Material 715
716
Supplemental Figure S1 717
Vector map of pJR38-CreMIP1-GFP 718
Supplemental Figure S2 719
Vector map of pJR38-CreSEC6-GFP 720
Supplemental Table S1 721
Primer used in this work 722
Supplemental Data S1 723
Full length of the coding sequence of CreSEC6. 724
Movie 1 725
Chlamydomonas reinhardtii CC3395. The two CVs are located close to the flagella. Scale bar 726 5 µm. 727
Movie 2 728
One cell of the mutant strain Osmo75 showing two enlarged CVs. Scale bar 5 µm. 729
Movie 3 730
One cell of the mutant strain Osmo75 showing multiple CVs. Scale bar 5 µm. 731
Movie 4 732
One cell of the mutant strain Osmo75 showing no light microscopically visible CVs. Scale bar 733 5 µm. 734
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735
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Tables
Table 1: (Putative) proteins of Chlamydomonas affected in the Osmo75 strain.
Name of model Annotation Deletion/Truncation Expression
Cre20.g759750.t1 Phytoene
dehydrogenase
139 b truncation of the 3’
end of the 3’UTR
+
Cre20.g759800.t1 SpoU rRNA
Methylase family
Deletion
Cre20.g759850.t1 CGI-12 PROTEIN-
RELATED
Deletion
Cre20.g759900.t1 Exocyst complex
component Sec6
Deletion +
Cre20.g759950.t1 Protein of unknown
function (DUF789)
Deletion +
Cre20.g760000.t1 None 1089 b truncation of the
5’end
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