A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii

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A cytochrome b5-containing plastid-located fatty acid desaturase from 1

Chlamydomonas reinhardtii 2

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Running Title: Cytb5 containing plastid fatty acid desaturase 4

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Simone Zäuner1, Wibke Jochum2, Tara Bigorowski3 and Christoph Benning# 6

Department of Biochemistry and Molecular Biology, Michigan State University, East 7

Lansing, MI 48824, USA 8

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1Present address: Institute of Molecular Physiology and Biotechnology of Plants 10

(IMBIO), University of Bonn, 53115 Bonn, Germany 11

2Participant of the 2010 DAAD Rise in North America Undergraduate Program 12

(http://www.DAAD.de) 13

3Participant in the 2010 Michigan State University Plant Genomics Summer 14

Undergraduate Program (http://www.plantgenomics.msu.edu). 15

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#Corresponding author Note: Christoph Benning 17

Email: [email protected] 18

Phone: 517-355-1609 19

Fax: 517-353-9334 20

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00079-12 EC Accepts, published online ahead of print on 4 May 2012

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SUMMARY 24

Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green 25

algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn-2 position. 26

While many genes necessary for the introduction of acyl chain double bonds have been 27

functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic 28

comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from 29

Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features 30

of a membrane-bound desaturase including three histidine boxes and a transit peptide for 31

chloroplast targeting. But distinguishing it from other known plastid desaturases, it has a 32

N-terminal cytochrome b5 domain. Cytochrome b5 is the primary electron donor for ER 33

desaturases and is often fused to the desaturase domain in desaturases modifying the 34

carboxyl end of the acyl group. Difference absorbance spectra of the recombinant 35

cytochrome b5-domain of CrΔ4FAD showed that it is functional in vitro. Green 36

fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in 37

Chlamydomonas. Interestingly, overproduction of CrΔ4FAD in Chlamydomonas did not 38

only increase 16:4 acyl groups in cell extracts, but specifically increased the total amount 39

of MGDG. Vice versa, the MGDG amount was lowered in lines with reduced CrΔ4FAD. 40

These data suggest a link between MGDG molecular species composition and 41

galactolipid abundance in the alga and a specific function for this fatty acid in MGDG. 42

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INTRODUCTION 45

Membranes in biological systems play a major role as barriers enclosing cells and their 46

organelles within. The lipid composition of a specific membrane is characteristic and 47

reflects its function. Membrane lipid fatty acid composition of plants and algae is not 48

static but can adapt to changes in the environment. In particular (poly)unsaturated fatty 49

acids are important in maintaining membrane fluidity and thus enhance temperature and 50

stress tolerance, e.g. (47). Unsaturated fatty acids are also precursors of signaling 51

molecules such as jasmonate and, thus, are important for plant defense (15). 52

Polyunsaturated fatty acids in algae are also of applied interest since algae are the 53

primary producers of ω3-long-chain polyunsaturated fatty acids (long chain PUFAs) such 54

as eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) or docosahexaenoic acid (DHA, 55

22:6Δ4,7,10,13,16,19), which are important for human nutrition (33). Efforts are ongoing to 56

produce these fatty acids in crop plants to increase their nutritional value (45). 57

Fatty acid desaturases can be grouped into two enzyme classes: Soluble Acyl-ACP-58

desaturases catalyze the first step in PUFA biosynthesis (introduction of a cis-double 59

bond between C9 and C10) and are exclusively found in photosynthetically active plastids. 60

The crystal structure of these non-heme diiron enzymes and their active site is known 61

(11,19). 62

The second and larger group of desaturases is comprised of membrane-bound 63

enzymes: They can be found in all eukaryotes and a few prokaryotes. No crystal structure 64

is currently available, but it is proposed that the reaction mechanism is similar to that 65

reported for the acyl-ACP-desaturases (41). Membrane-bound desaturases most likely 66

contain four membrane-spanning domains and three histidine-box-motifs (HX3-4H, HX2-67


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3HH and (H/Q)X2-3HH) which are crucial for activity (38). In plants, membrane-bound 68

fatty acid desaturases are present in the plastid and the endoplasmic reticulum (ER). The 69

electron donors for plastid desaturases are typically ferredoxins, while ER enzymes use a 70

cytochrome b5. Many ER-associated desaturases are directly fused to a cytochrome b5 71

domain. Typical “front end” desaturases, which introduce a double bond three carbons 72

closer to the carboxy-terminus of the acyl chain relative to a preexisting double bond, 73

predominantly contain an N-terminal cytochrome b5 (39,40). No plastid desaturase 74

carrying a cytochrome b5-fusion has been previously described. However, free forms of 75

cytochrome b5 have been reported in the (outer) chloroplast envelope (17,22), but no 76

cytochrome b5 was detected in a recent chloroplast proteome study of Arabidopsis (14). 77

The plastid lipid monogalactosyldiacylglycerol (MGDG) in the green alga 78

Chlamydomonas reinhardtii occurs preferentially as a molecular species containing α-79

linolenic acid 18:3Δ9,12,15 in the sn-1 position and (4Z,7Z,10Z,13Z)-hexadeca-4,7,10,13-80

tetraenoic acid (16:4) in sn-2 position of the glycerol backbone (10,34). This 16:4 acyl 81

group````` is found only in trace amounts in other membrane lipids. Genes necessary for 82

the introduction of the double bonds in lipid acyl groups in Chlamydomonas have been 83

identified and characterized (6,16,36) except for this MGDG-specific Δ4-desaturase. 84

Desaturation in the Δ4-position of acyl groups in complex lipids also occurs during 85

the synthesis of DHA in the ER, and the respective desaturase has been characterized 86

(Supplemental Fig. 1). Genes encoding this activity have been cloned and characterized 87

from various algae such as Euglena gracilis (Meyer et al. 2003), Thalassiosira 88

pseudonana, (44) or species of the genus Pavlova (Rebecca), (43,50) or 89

Thraustochytrium (31). All enzymes mentioned above are ER-located and contain an N-90


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terminal cytochrome b5 domain. Alternatively, Schizochytrium, a close relative of 91

Thraustochytrium is able to synthesize DHA by a desaturase-independent polyketide 92

synthase (PKS) pathway (24). However, taking into account that no PKS gene cluster has 93

been detected in the Chlamydomonas genome (23), it is likely that the 16:4 acyl group in 94

MGDG is synthesized exclusively by stepwise desaturation of the 16:0 acyl group. 95

Therefore, we reasoned at the onset of this study that the candidate enzyme had to be 96

similar to a membrane-bound desaturase and not PKS. 97

A possibly related desaturase was assumed to be the ER-located Δ4-desaturase from 98

Euglena gracilis, because it was shown to be active in the synthesis of very long chain 99

fatty acids but could also act on shorter C16-acyl groups (25). Its activity is strongly 100

dependent on a preexisting Δ7-double bond as would be expected for the 101

Chlamydomonas MGDG-specific Δ4-desaturase. Here we report the identification and 102

characterization of the Δ4-desaturase from Chlamydomonas, which is an unusual plastid 103

located cytochrome b5 fusion protein. 104

105

MATERIALS AND METHODS 106

Bioinformatics 107

BLASTP searches (3) were conducted in the Chlamydomonas genome V4 (23). The gene 108

model was manually corrected using RNAseq-data from our lab (26) and GreenGenie2 109

(18). The model was confirmed by RT-PCR and followed by TA cloning into pGEM-T™ 110

Easy (Promega, Madison, WI) and sequencing. The sequence is deposited under 111

GenBank™ Accession Number: JN089704. 112


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The hydropathy plots were generated with an algorithm according to (46) using the 113

TOPPRED program (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred). Protein 114

alignments were generated with the Clustal algorithm (42). Phylogenetic analysis was 115

computed by the PHYLIP package (version 3.69) using the neighbour-joining method 116

and comparison of 1000 bootstrap replicates. TreeView was employed for visualization. 117

118

RNA isolation and cDNA preparation from Chlamydomonas 119

RNA was isolated from 5 mL mid log phase cultures using the RNeasy Plant Mini Kit 120

(Qiagen, Valencia, CA) including the optional on-column DNase digest. 0.5 µg total 121

RNA was reverse-transcribed with Superscript III (Invitrogen, Carlsbad, CA) and Oligo-122

dT primers at 50°C in a 20 µL reaction volume throughout all experiments. 0.5 µL cDNA 123

was used for estimation of mRNA abundance by PCR and amplification of the full-length 124

coding sequence. 125

126

PCR conditions 127

Primers used to amplify the full length coding sequences and the cytochrome b5 domains 128

can be found in Supplemental Table 1. All constructs mentioned before were amplified 129

with Phusion polymerase (Finnzymes, Woburn, MA) using the supplied GC buffer (HF 130

buffer for the cytochrome b5 gene from Arabidopsis), 2 % DMSO (Chlamydomonas 131

genes only) and the following conditions: Initial denaturation was carried out at 98°C for 132

2 minutes, followed by 30-35 cycles with 10 seconds at 98°C, 10 seconds at 65°C (52°C 133

for Arabidopsis cytochrome b5) and 20 seconds per kb at 72°C. The last cycle was 134

followed by 1 minute of final elongation at 72°C. For the estimation of RNA abundance 135


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RT-PCR internal fragments of CrΔ4FAD, MGD1, DGD1 and RACK1 were amplified 136

using GoTaq-Flexi DNA polymerase (Promega, Madison, WI) according to the 137

manufacturer’s instructions. RACK1 is a control gene with stable high expression under 138

normal growth conditions, commonly used for normalization of Chlamydomonas mRNA 139

(2). The required number of cycles was determined individually for each gene. The band 140

intensity in gel images was estimated with Image J (1). Detailed information on primer 141

sequences, cycle numbers and fragment sizes can be found in Supplemental Table 2. 142

143

Cultivation and transformation of Chlamydomonas 144

Chlamydomonas cells were grown in Tris-Acetate-Phosphate (TAP) medium (34) at 145

22°C and continuous light until mid-log phase (OD560 ≈ 1-1.5). Two different cell wall-146

less strains were used in this study; dw15 (courtesy of Arthur Grossman) and UVM4 147

which is based on strain cw15. Strain UVM4 was especially designed for efficient 148

expression of nuclear transgenes (29). When growing dw15 as a control, the medium had 149

to be supplemented with 100 µM arginine. Agar-plates additionally contained 0.8 % 150

Phytoblend (Caisson Laboratories Inc, North Logan, UT). For selection of transgenic 151

lines plates also contained 10 µg/mL paromomycin (Sigma-Aldrich, St. Louis, MO). 152

Transformation of Chlamydomonas was carried out as described in (21) with the 153

following modifications: 30 mL of mid-log phase culture grown in TAP, were harvested 154

(1500 x g, 3 min, RT, swinging bucket rotor), and the cells resuspended in 500 µL fresh 155

TAP-medium. The cells were then transferred to a glass vial containing ≈ 300 mg acid-156

washed glass beads (Ø 0.4-0.5 mm). After addition of 1 µg linearized desalted plasmid 157

DNA the cells were agitated using a standard vortexer for 15 seconds. 5 mL TAP 158


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medium was added and the cells were regenerated over night in the dark under shaking 159

(22°C). The following day, cells were harvested as above and resuspended in 3 mL fresh 160

TAP medium. After careful mixing with 3 mL Top-Agar (0.5 % Phytoblend in TAP-161

medium, cooled to ≤ 45°C) cells were spread on TAP-Agar containing 10 µg/mL 162

paromomycin. Plates were kept at room temperature (≈ 24°C under continuous light) 163

until colonies appeared (≈ 6-7 days). Single colonies were then transferred to a fresh 164

TAP-plate containing 10 µg/mL paromomycin and continued to grow as described. 165

Single colonies were used to inoculate 10 mL of TAP-medium (in a 25 mL flask) and 166

grown as above for biochemical and microscopic analyses. 167

168

Construction of the artificial microRNA vector 169

Artificial microRNA constructs were designed as described (28) using the vector 170

pChlamiRNA3int. 1 µg plasmid DNA was linearized with KpnI-HF (New England 171

Biolabs, Ipswich, MA) and purified (PCR-purification Kit; Qiagen) prior to 172

transformation of Chlamydomonas strain dw15 – three constructs were tested for 173

functionality by RT-PCR analysis of transgenic Chlamydomonas lines. Only data of the 174

most effective oligomer (5’-TCACTAAAGGATGACAACCTA-3’) is shown. 175

176

Construction of the Chlamydomonas CrΔ4FAD-overexpression/localization vectors 177

The cDNA-sequence encoding CrΔ4FAD was cloned into the NdeI site of pJR38 (29) in 178

frame with the codon optimized EGFP-gene. Prior to transformation of strain UVM4, 1 179

µg plasmid DNA was linearized with XbaI (NEB) and desalted (PCR Purification Kit, 180

Qiagen). 181


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Analysis of lipid composition and chlorophyll content 182

5 mL of mid-log phase cell cultures were harvested (1500 x g, 3 min, RT, swinging 183

bucket rotor) and lipids extracted as described by Bligh and Dyer (4). The organic phase 184

was transferred to a fresh glass tube and dried under N2 stream. The lipids were dissolved 185

in 100 µL Chloroform. Usually 10 µL were converted to their fatty acyl methyl esters 186

(FAMEs) as described (35). Lipids contained in 30 µL were separated by TLC (48), 187

MGDG and DGDG scraped and also converted to FAMEs. FAMEs in hexane were 188

analyzed by gas liquid chromatography (GC) on a HP6890-machine with a DB-23 189

column (both Agilent Technologies, Santa Clara, CA) and the following temperature 190

settings: 140°C for 2 min, increase to 160°C at 25°C /min, further increase to 250°C at 191

8°C/min. 250°C was held for 4 min followed by a temperature decrease back to 140°C at 192

38°C/min. All samples were run at constant Helium flow of 1.5 mL/min and a split ratio 193

1:30 (for total fatty acids) or 1:10 (for TLC-purified lipids). For identification of FAMEs 194

by gas chromatography mass spectrometry the same model instrument was coupled to a 195

model 5975B MS detector (all Agilent Technologies) using similar running conditions. 196

The content of chlorophyll a and b from Chlamydomonas cultures was determined for 197

acetone extracts as described before (30). 198

199

Expression of the cytochrome b5 domain in E. coli 200

The soluble cytochrome b5-portion (corresponding to amino acids 48-147) of CrΔ4FAD 201

was cloned between the EcoRI and XhoI site of pDsRed-plw01-His (20) and expressed in 202

E. coli BL21 (DE3)-cells (Novagen, Rockland, MA). A free form of cytochrome b5 from 203

Arabidopsis (At1g26340) and the cytochrome b5 domain of the ER-located fatty acid Δ5-204


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desaturase from Chlamydomonas (amino acids 1-99 of CrFAD13, (16)) were expressed. 205

30 min prior to induction with 0.4 mM IPTG the heme-precursor γ-aminolevulinic acid 206

was added (final concentration 0.5 µM) as described previously (40). After induction cell 207

growth was continued at 28°C for 4 h. Cells were cooled on ice, resuspended in Lysis 208

buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.5) and lyzed by 209

sonication (10 cycles à 10 seconds, interrupted by 10 seconds on ice). After 210

centrifugation at 10,000 x g the supernatant was mixed with Ni2+-nitrilotriacetic acid 211

agarose (Qiagen) and incubated with shaking at 4°C for 60 minutes. The resin was 212

washed twice with washing buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 213

pH 7.5) before the DsRED-fusion proteins were eluted (50 mM Tris-HCl, 300 mM NaCl, 214

250 mM imidazole, pH 8.0). Protein purity was confirmed by acrylamide gel 215

electrophoresis and Coomassie staining. 216

217

Cytochrome b5-Assay 218

An absorbance spectrum between 400 and 600 nm was recorded for 250 µg purified 219

protein (air oxidized) in assay buffer (100 mM Tris-HCl, 0.5 mM EDTA, pH 8.0) at room 220

temperature as described (40). The samples were then reduced with sodium dithionite or 221

dithiothreitol (DTT) and immediately measured again as above. Redox-difference spectra 222

were calculated for all treatments. 223

224

Confocal microscopy 225

Confocal laser scanning microscopy images were taken at the Center of Advanced 226

Microscopy at Michigan State University on an Olympus FluoView FV1000 Confocal 227


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Laser Scanning Microscope (Olympus, Center Valley, PA) configured on the Olympus 228

IX81 fully automated inverted microscope. The GFP was excited using the 488nm Argon 229

laser line, while the green fluorescence was collected using a 505-525nm band pass filter. 230

Simultaneously, red chlorophyll autofluorescence was also collected using the 488nm 231

laser line for excitation and a 650nm long pass filter, while transmitted laser light was 232

used to record the bright field image. Images were recorded using the 60x UPLAPO oil 233

objective (NA 1.42). 234

235

RESULTS 236

Identification of a Δ4-desaturase candidate gene 237

Chlamydomonas reinhardtii strains used in this study contain 16:4 acyl groups in MGDG 238

(Figure 1A) as previously reported for other strains (10). High amounts of 16:4 acyl 239

groups were detected in MGDG, while other lipids such as DGDG contained only trace 240

amounts. In addition, 16:4 acyl groups were present in triacylglycerol (TAG) of cells 241

cultured under nitrogen-deprivation (Figure 1B), conditions that induce TAG 242

biosynthesis in Chlamydomonas (7,27,49). 243

A phylogenetic comparison was used to identify the desaturase required for 16:4 acyl 244

groups in MGDG and TAGs. Searches in the Chlamydomonas genome V4 using protein 245

sequences for the Δ4-desaturases from Euglena gracilis (AAQ19605) and Thalassiosira 246

pseudonana (AAX14506) as queries identified a candidate sequence (protein ID 35253 in 247

the Chlamydomonas genome V4). The predicted protein consisted of 1675 amino acids 248

(coding sequence 5025 bp) and contained desaturase and cytochrome b5-domains. Since 249

the size of the coding sequence of cytochrome b5 fusion desaturases usually varies 250


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between 1,400 and 1,600 bp, and all commonly relevant domains were encoded in the 251

first 1,500 bp of the candidate sequence, the size of the gene model was questioned. 252

GreenGenie2 (18) was used to correct the gene model and the coding sequence was 253

verified by mapping transcriptomics data (26) to the genome. The updated coding 254

sequence was 1551 bp (516 amino acids), the gene 4812 bp and consisted of 10 exons 255

(Figure 2). Primers were designed based on this new model, and the PCR amplified 256

genomic sequence was subjected to sequencing for confirmation. The corrected coding 257

sequence is deposited under GenBank™ Accession Number: JN089704. No information 258

about 5’- and 3’-untranslated regions was obtained. Figure 2 shows the hydrophobicity 259

plot and a schematic structural overview of the likely desaturase, tentatively designated 260

CrΔ4FAD. A complete protein alignment of CrΔ4FAD with known Δ4-fatty acid 261

desaturases can be found in Supplemental Figure 2. Supplemental Figure 3 shows a 262

regioselectivity dendrogram of desaturase-like sequences from organisms of different 263

classes including CrΔ4FAD (A) and a schematic structural comparison of the major 264

desaturase classes (B). 265

Distinguishing this enzyme from other described desaturases, the CrΔ4FAD sequence 266

contained both a putative chloroplast transit peptide and an N-terminal cytochrome b5 267

domain suggesting a chloroplast location. 268

269

CrΔ4FAD-GFP is targeted to the chloroplast in Chlamydomonas 270

The presence of a likely chloroplast transit peptide suggested that this protein is located 271

in the plastid. To corroborate whether CrΔ4FAD is indeed imported into the plastid, we 272

expressed a Green Fluorescence Protein (GFP)-tagged version in Chlamydomonas and 273


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analyzed the transgenic lines by confocal laser scanning microscopy. Chlamydomonas 274

cells producing GFP alone showed a strong fluorescence signal in the cytosol, whereas 275

the GFP-signal overlapped with the chlorophyll autofluorescence signal in cells 276

expressing CrΔ4FAD-GFP (Figure 3A and B). Due to the high fluorescence background 277

in Chlamydomonas we were not able to determine whether the protein is located in the 278

envelope or thylakoid membranes. 279

280

Modulation of CrΔ4FAD expression in Chlamydomonas alters MGDG content 281

A CrΔ4FAD-GFP fusion construct was introduced into Chlamydomonas strain UVM4 282

which is optimized for the expression of nuclear transgenes (29). Transgenic lines were 283

screened for enhanced CrΔ4FAD-transcript levels by PCR as well as increased protein 284

abundance by immunoblotting using a GFP antibody (Figure 4B). 285

To repress CrΔ4FAD-activity in Chlamydomonas, an artificial microRNA construct 286

was generated targeting specifically the 5’ part of the identified coding sequence and 287

introduced into the cell wall-less strain dw15. Transformants had reduced CrΔ4FAD-288

transcript levels (Figure 4A). 289

Because in the polar lipid fraction the 16:4 acyl group is almost exclusively found in 290

MGDG, we initially analyzed only the fatty acid composition of this lipid for decreased 291

or increased 16:4-levels in CrΔ4FAD-knockdown and overexpression lines. No obvious 292

alterations in the fatty acid composition of MGDG were observed. Interestingly, the total 293

fatty acid profile of the overexpression lines showed a statistically significant increase in 294

16:4 and 18:3Δ9,12,15 acyl groups (with a concomitant decrease in the corresponding 295

saturated acyl groups, e.g. 16:0, Figure 4C). Vice versa, the knock down lines had a 296


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decrease in 16:4 and 18:3Δ9,12,15 acyl groups (Figure 4D). The decrease in the 18:3Δ9,12,15 297

acyl groups was initially surprising. However, this is explained by the decreased amount 298

of MGDG present in knockdown lines (Figure 5A). Vice versa, overexpression of 299

CrΔ4FAD led to an increase in total MGDG and a corresponding increase in the 300

predominant acyl groups present in this lipid. 301

Digalactosyldiacylglycerol (DGDG), the second prominent galactolipid is derived 302

from MGDG but contains only trace amounts of 16:4 (10). The fatty acid composition of 303

DGDG was not affected by changes in CrΔ4FAD expression, but we observed a slight 304

increase in the total DGDG amount in the knockdown lines (Figure 5B). Transcript levels 305

of the galactolipid synthesis genes MGD1 and DGD1 were not altered (Figure 4A). 306

The increase in total galactolipid amount in the overexpression lines was 307

accompanied by an increase in chlorophyll content per cell (Figure 5D), but did not affect 308

the chlorophyll a/b ratio (Supplemental Figure 4). 309

310

The Cytochrome b5 domain of CrΔ4FAD is active in vitro 311

To test functionality of the cytochrome b5 domain, DsRED-fusion constructs were 312

expressed in E. coli and the proteins purified by their C-terminal His-Tag. Redox 313

difference spectra were calculated from the absorbance of the air-oxidized and dithionite-314

reduced samples (redox potential ΔE = -660 mV). The observed absorbance difference 315

maxima at 425, 527 and 557 nm (Figure 6A) were similar to those of other fatty acid 316

desaturase cytochrome b5 domains (12,40). They were also detected in protein extracts 317

from cells producing DsRED fused to a free cytochrome b5 from Arabidopsis and the 318

cytochrome b5 domain of the ER-localized desaturase FAD13 from Chlamydomonas, but 319


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not in the DsRED control (Figure 6 B,C). The weaker reducing agent dithiothreitol (DTT, 320

∆E = -330 mV) was still able to reduce DsRED-AtCytb5 and DsRED-CrFAD13Cytb5, 321

although at lower levels, but not DsRED-Cr∆4Cytb5 (Figure 6 D-F). 322

323

DISCUSSION 324

Although a large number of different fatty acid desaturases have been characterized from 325

a variety of organisms including algae, some of the more specialized enzymes are still 326

being discovered. One example is the recent identification of the plastid 327

phosphatidylglycerol specific desaturase FAD4 from Arabidopsis, which was shown to 328

fall into a distinct class of fatty acid desaturases (9; Supplemental Figure 3). The MGDG-329

specific Δ4 desaturase from Chlamydomonas, CrΔ4FAD, described here is another 330

example. While we were able to predict it by phylogenetic analysis in the genome of 331

Chlamydomonas, it contained surprising features setting it apart from other known 332

desaturases. Notably an N-terminal cytochrome b5 domain was present in what we 333

determined, is a plastid localized fatty acid desaturase. Plastid localization of the enzyme 334

was predicted based on the presence of a transit peptide and due to the fact that the 335

enzyme appeared to be highly specific for the exclusively plastid localized galactolipid 336

MGDG. 337

Different approaches to confirm the activity by expression of the cDNA in yeast or 338

Arabidopsis were not productive at this time and modification of CrΔ4FAD abundance in 339

Chlamydomonas did not alter the fatty acid composition of MGDG per se, but affected 340

the amount of MGDG in the cells. Apparently, in Chlamydomonas the availability of the 341

16:4 acyl group in MGDG limits overall MGDG content in the thylakoid membrane. 342


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This result provides evidence for the presence of a regulatory or compensatory system in 343

Chlamydomonas that governs membrane lipid homeostasis. A different compensatory 344

effect was observed in previous attempts to suppress the activity of the betaine lipid 345

synthase Bta1 of Chlamydomonas using an RNAi approach (34). Rather than reducing 346

the relative amount of betaine lipid, the cells reduced growth to maintain a constant 347

overall lipid composition. Considering both observations, it appears that strong 348

compensatory mechanisms are in place in Chlamydomonas that maintain membrane lipid 349

homeostasis at the level of the lipid class, as observed for betaine lipid, and the molecular 350

species composition of individual lipids as is the case for MGDG shown here. 351

While plastid localization for CrΔ4FAD was expected, the presence of a cytochrome 352

b5 domain was not. In fact, the deduced protein sequence contained all structural features 353

of a membrane-bound “Front End” desaturase typically associated with the ER: besides 354

an N-terminal cytochrome b5-domain, three histidine-box motifs were identified, which 355

have been shown to be essential for activity in comparable enzymes (38). Like in all 356

“Front End” desaturases identified so far, one of the histidines in the third conserved box 357

is replaced by a glutamine (motif QXXHH). Replacement of the corresponding glutamine 358

residue with a histidine in the Δ6-desaturase from borage abolished activity (37). 359

Cytochrome b5-fusions typically occur at the N-terminus in “Front End” desaturases and 360

at the C-terminus of Δ9-desaturases in fungi and red algae. Only a few “Front End” 361

desaturases without a cytochrome b5 are known, e.g. the Δ6-desaturase from the 362

cyanobacterium Synechocystis sp. PCC 6802 (32). 363

The exclusive presence of 16:4 acyl groups in Chlamydomonas under nutrient replete 364

conditions in the sn-2 position of MGDG suggests strong substrate specificity for 365


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CrΔ4FAD in vivo or high specificity of substrate channeling towards the active site of the 366

enzyme. Another example of a highly specific plastid desaturase is FAD4, responsible for 367

the synthesis of 16:1Δ3(E) esterified to the sn-2 position of phosphatidylglycerol (9). In a 368

second study involving transgenic plants producing bacterial diacylglycerol kinase in the 369

plastid thereby causing channeling of different lipid molecular species into the FAD4 370

substrate phosphatidylglycerol, the Δ3(E)-double bond was also found in C18 fatty acids 371

(8). This demonstrates that FAD4-activity is lipid specific but independent of the length 372

of the acyl chain. On the other hand, Heilmann et al. (13) forced a new substrate on the 373

normally plastid localized desaturase FAD5 by altering subcellular targeting to the ER. 374

Instead of the native substrate MGDG, the double bonds were introduced into the acyl 375

chains of phosphatidylcholine albeit with altered regioselectivity. 376

We identified CrΔ4FAD by sequence comparison with a known Δ4-desaturase 377

involved in DHA biosynthesis. The activity of the Δ4-desaturase from Euglena was 378

shown to be dependent on a preexisting Δ7-double bond, but like FAD4 independent 379

from the acyl chain length (25). Given that the Δ4-double bond is found in small amounts 380

also in 16:2Δ4,7 and 16:3Δ4,7,10 (Figure 1) CrΔ4FAD may also be dependent on Δ7-381

unsaturated substrates. Elucidation of substrate preferences was not possible due to the 382

lack of an in vitro system. It seems possible that the major limitation may be the 383

availability of a suitable electron donor. In the ER, electrons are transferred to 384

cytochrome b5 by a specific oxidoreductase. This leads to the question for the nature of 385

the primary electron donor of CrΔ4FAD. We did not find evidence for a cytochrome b5 386

oxidoreductase, which could be imported into the plastid of Chlamydomonas. 387

Furthermore, our in vitro expression data of the cytochrome b5 domain suggest that 388


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CrΔ4FAD requires a stronger reducing agent than free cytochrome b5 in the ER. All 389

three, CrΔ4Cytb5, CrFAD13Cytb5 and AtCytb5 could be fully reduced by dithionite ∆E 390

= -660 mV), but only AtCytb5 and CrFAD13Cytb5 also by DTT (∆E = -330 mV). The 391

redox potential of DTT is within the same range as that of NADH (∆E = -315 mV). 392

Taken into account that the redox potentials of ferredoxins from Chlamydomonas vary 393

between -390 and -425 mV (5), it seems likely that CrΔ4Cytb5 receives electrons from a 394

ferredoxin rather than NAD(P)H. 395

The presence of this unusual desaturase in Chlamydomonas chloroplasts and the 396

apparent correlation of 16:4 acyl group content and MGDG amounts raise the question 397

for the function of this particular molecular species of MGDG. Absent of an efficient 398

system for the complete inactivation of genes in Chlamydomonas, it will be difficult to 399

answer this question by creating a null-mutant. But it seems clear based on the analysis of 400

the transgenic lines with altered CrΔ4FAD abundance that exquisite compensatory 401

mechanism are in place in Chlamydomonas to adjust the abundance of MGDG and its 402

molecular species composition. 403

404

ACKNOWLEDGEMENTS 405

The authors would like to thank Melinda Frame (Center for Advanced Microscopy at 406

MSU) for help with the confocal imaging and Beverley Chamberlin (Mass Spectrometry 407

Facility, MSU) for the GC-FID/MS analyses. This work was funded by a DAAD Postdoc 408

Research Fellowship (http://www.daad.de) to S.Z., the National Science Foundation 409

supporting T.B. during a summer internship program, and by a grant from the Air Force 410

Office of Scientific Research to C.B. 411


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594 595

596


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FIGURE LEGENDS 609

Figure 1. Fatty acyl profile of MGDG (A) and TAG (B) isolated from Chlamydomonas 610

strains dw15 (black) and UVM4 (grey). Hexadecatetraenoic acid (16:4) is the major fatty 611

acid in the sn-2 position of MGDG, but was also recovered in TAG (5 % in dw15, 8 % in 612

UVM4). Lipids were separated by thin layer chromatography, converted to their fatty 613

acid methyl esters and quantified by GC-FID. Fatty acids are displayed by number of 614

carbons:number of double bonds. The Δ-numbers indicate the position of the double 615

bonds in the acyl chain. Means and standard deviations of three biological replicates are 616

shown. 617

618

Figure 2. Protein topology prediction for CrΔ4FAD. The hydropathy plot (A) was 619

generated using the TOPPRED program (http://mobyle.pasteur.fr/cgi-620

bin/portal.py?#forms::toppred) and is based on the algorithm by von Heijne et al. (46). 621

(B) Schematic overview of CrΔ4FAD topology. cTP – chloroplast transit peptide; Cytb5 622

– cytochrome b5 domain; TMD – membrane spanning domains, each composed of two 623

transmembrane helices; the three histidine boxes and the heme binding motif are 624

indicated. 625

626

Figure 3. CrΔ4FAD-GFP localizes to the chloroplast in Chlamydomonas. Free GFP is 627

localized in the cytosol of Chlamydomonas strain UVM4 (A) in contrast to CrΔ4FAD-628

GFP which is found in the plastid (B). Scale bars are 5 µm. 629

630


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Figure 4. Knockdown and overexpression of CrΔ4FAD in Chlamydomonas. (A) 631

Knockdown lines (dw15-Δ4-KD) show reduced CrΔ4FAD expression compared to the 632

empty vector control (dw15). CrΔ4FAD-expression is elevated in strain UVM4-Δ4-GFP 633

expressing CrΔ4FAD-GFP. UVM4-GFP – control, UVM4 expressing GFP alone. The 634

expression of the control genes MGD1, DGD1 and RACK1 was not affected in any of the 635

lines. cDNA was prepared from equal amounts of total RNA was used as PCR template 636

with varying cycle numbers depending on each gene: 34 for CrΔ4FAD, 30 for MGD1 and 637

DGD1, 24 for the control gene RACK1. (B) Immunoblot showing the expression of the 638

CrΔ4FAD-GFP-fusion construct in three independent lines. Chlamydomonas cells were 639

harvested, mixed with SDS-sample buffer and directly loaded on a denaturing gel. 640

Proteins were then transferred to a PVDF membrane and subjected to an immunoblot 641

analysis using an anti-GFP antibody. (C) The fatty acid composition in Chlamydomonas 642

cells overexpressing CrΔ4FAD is changed: Compared to the empty vector control (black 643

bars), the levels of 16:4 and 18:3∆9,12,15 are higher in overexpression lines (grey bars). (D) 644

Total fatty acid composition in Cr∆4FAD knock down lines. Compared to the empty 645

vector control (black bars) knock down lines (light grey bars) show decreased levels of 646

16:4 and 18:3∆9,12,15 acyl groups. For C and D total fatty acids were converted to their 647

FAMEs and quantified by GC-FID. Significant changes in the fatty acid composition 648

between the two lines are marked with an asterisk (P < 0.05; non-paired two-sample t-649

test). 650

651

Figure 5. CrΔ4FAD expression specifically affects the MGDG-content in 652

Chlamydomonas (A) but not DGDG (B). The total fatty acid content remains unchanged 653


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(C), but the higher total galactolipid content in the overexpression lines leads to an 654

increased chlorophyll content (D). Lipids for (A-C) were isolated from a defined amount 655

of cells, separated by TLC and quantified as FAMEs. Pigments (D) were isolated, their 656

absorbance spectra detected in 80 % acetone. Asterisks indicate significant changes 657

between knockdown and overexpression lines and their corresponding controls (P < 0.05, 658

non-paired two-sample t-test). Data were obtained from at least six independent 659

experiments. Abbreviations are as defined for Figure 4. 660

661

Figure 6. Redox difference spectra of dithionite (A-C) and dithiothreitol (DTT; D-F) 662

reduced recombinant cytochrome b5. For (A-C) spectra were calculated from the 663

absorbance of air oxidized and dithionite reduced DsRED fusion proteins. DsRED-664

CrΔ4Cytb5 (A), DsRED-CrFAD13Cytb5 (B) and DsRED-AtCytb5 (C) show difference 665

maxima at 425 (424), 528 (527) and 558 (559) nm respectively, which is in line with 666

previous observations ((40), (12)). For (D-F) spectra were calculated from the absorbance 667

of air oxidized and DTT- reduced DsRED fusion proteins. The characteristic shape of the 668

cytochrome b5 difference spectrum is still maintained in DsRED-CrFAD13Cytb5 (E) and 669

DsRED-AtCytb5 (F), although the difference between oxidized and DTT-reduced forms 670

is much less pronounced compared to dithionite. In contrast, DsRED-CrΔ4Cytb5 cannot 671

be reduced with DTT (D), indicating that CrΔ4Cytb5 requires a stronger reductant than 672

DTT (ΔE= -330 mV, comparable with NADH; ΔE(dithionite)= -660 mV). 673


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Documents

A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii