MARGEN-00190; No of Pages 8 Marine Genomics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Marine Genomics journal homepage: www.elsevier.com/locate/margen

Ryo Yoshinaga, Megumi Niwa-Kubota 1, Hiroaki Matsui, Yusuke Matsuda ⁎

i n f o

3 15 4 16 5 17 6 7

Article history: Received 7 October 2013 Received in revised form 24 December 2013 Accepted 24 January 2014 Available online xxxx

8 9 10 11 12 13 14

Keywords: Iron response Marine diatom Promoter Cis-element Transcriptional regulation CO2 response

a b s t r a c t

It is well established that iron is one of the major constraints of primary productivity of marine diatoms in world oceans. In the present study, changes in the transcript levels of the 20 iron related genes were profiled in the marine diatom Phaeodactylum tricornutum during an early stage of acclimation from iron replete to ironlimited conditions. The results clearly showed that the profiles differ depending on genes, suggesting the occurrence of several modes of iron-responsive regulation at the transcriptional level. Upstream DNA sequences of iron starvation induced protein1 (Isi1), ferrichrome binding protein1 (FBP1), and flavodoxin (Fld) genes were isolated, fused with the GUS reporter gene, uidA, and transformed into P. tricornutum. Obtained transformants were subjected to the GUS reporter assay and the result clearly revealed that the GUS activity of all transformants was significantly increased upon iron limitation. Iron responsive Cis-elements in each promoter region were determined by the promoter truncation technique, demonstrating the occurrence of the critical iron-responsive regulatory regions of about 30 bp in the promoter regions of three genes, Isi1, FBP1, and Fld. Interestingly, these sequences were similar with each other revealing two conserved motifs, A; A(A/C)G(G/C)C(G/-)C(A/G)TG; and B; CACG TG(T/C)C, which are homologous to the iron responsive Cis-element in the green alga, Chlamydomonas reinhardtii. The impairment of the motif B in the Isi1 promoter resulted in the loss of iron response and the core regulatory region of the FBP1 promoter conferred an iron response on the constitutive cytomegalovirus promoter, PCMV, indicating that these conserved promoter sequences are iron-responsive elements. Finally, the inductive regulation of these promoters under iron-limited conditions was dissipated specifically by 5% CO2, strongly suggesting the participation of CO2 in the transcriptional regulation of the iron-related gene promoters. © 2014 Published by Elsevier B.V.

P

a r t i c l e

R

E

C

T

E

D

2

R O

Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Hyogo 669-1337, Japan

F

Q1

Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum

O

1

37

R

36

1. Introduction

39

Iron is the fourth most abundant element on the earth. However, the concentration of irons is extremely low at the surface area of oceans since the iron supplied from rivers is mostly scavenged by clay and organic particles within the coastal area, which hinders irons reaching to the outer ocean. The iron supply to the open ocean from the land then relies exclusively on silt particles that are carried by the wind. As a result, oceans far from the continents lose the iron supply from the land, which causes a severe iron limitation, forming the High-Nutrient LowChlorophyll (HNLC) area of world oceans (Martin et al., 1989; Coale et al., 1996; Boyd et al., 2000; Tsuda et al., 2003). Iron is involved in numerous housekeeping metabolisms in a form as iron–sulfur clusters and hemes, which are involved in redox chains of respiration and photosystem, nitrate/sulfate reduction pathways, nitrogen fixation, and radical

42 43 44 45 46 47 48 49 50 51

U

40 41

N C O

38

⁎ Corresponding author. Tel.: +81 79 565 8559; fax: +81 79 565 8542. E-mail address: [email protected] (Y. Matsuda). 1 Present address: KOSÉ Corporation, 3-6-2 Nihonbashi, Chuo-ku, Tokyo 103-8251, Japan.

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 35 34

scavenging systems. Iron is thus the transition metal of the highest biological demand (Raven, 1988; Briat et al., 1995; Briat, 1999; Strzepek and Harrison, 2004). Marine diatom is one of the most successful marine phytoplanktons, and is responsible for up to 20% of the annual global primary production (Tréguer et al., 1995; Falkowski et al., 2000). The genome of the marine pennate diatom, Phaeodactylum tricornutum holds a homologue of the ferric reductase (FRE) gene (Bowler et al. 2008), which is known to be a part of the redox-type-iron transport complex in the budding yeast, Saccharomyces cerevisiae. But FRE in yeast functions forming a complex with ferroxidase (FET) and iron permease (FTR) (Eide, 1998; Kosman, 2003), both of which are absent in the P. tricornutum genome (Bowler et al. 2008). In sharp contrast, the genome of the marine centric diatom, Thalassiosira pseudonana holds these genes for the redox-type-iron transport complex, which are significantly homologous to those in yeast (Armbrust et al., 2004). Moreover, the iron storage protein, ferritin occurs in the marine pennate species, P. tricornutum, Pseudo-nitzschia granii, and Pseudonizchia multiseries (Marchetti et al., 2008), while it is absent in the centric species, T. pseudonana, which instead possesses enterobactin, an iron chelating siderophore (Armbrust et al., 2004). It is postulated that ferritin confers on these pennate species a significant

1874-7787/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.margen.2014.01.005

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

52 53 54 55 56 57 58 59 60 61 62 Q2 Q3 63 64 65 66 67 68 69 70 71 72

96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138

The marine diatom P. tricornutum Bolin (UTEX642) was obtained from the University of Texas Culture Collection of algae (Austin, MN) and cultured in artificial seawater, which was supplemented with the half-strength of the Guillard's “f” solution (F/2ASW) under continuous illumination (50–75 μmol m−2 s−1) and constant aeration with ambient air (0.039% CO2) at 20 °C. For iron limitation experiments, cells were washed 3 times with the iron deprived medium and inoculated to F/2ASW without or with 12 μM of supplemental iron in the presence of 12 μM 2,2′,2″,2″-(Ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA). To make iron limitation severer, cell culture at the mid-logarithmic phase was diluted by filtering out the cells without any replenishment of new medium, which was done every 3 days for up to 10 days. In some experiments, diatom cells were cultured under the iron limited condition with 5% CO2, or atmospheric air (0.039% CO2) at pH 9.0 or at pH 6.5.

141 142

O

F

140

R O

94 95

2.1. Culture conditions of marine diatom

143 144 145 146 147 148 149 150 151 152 153 154

2.2. Quantification of transcript levels of iron-responsive genes

155

Total RNAs were extracted from the P. tricornutum (UTEX642) cells grown under iron-replete and iron-limited conditions as described above using a RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's protocol. cDNAs were synthesized from RNAs using the reverse transcriptase, ReverTra Ace® (TOYOBO). To standardize the transcript level, the endogenous glyceraldehyde-3-phosphate dehydrogenase C2 (gapC2) gene was used as the constitutive maker gene. Semiquantitative RT-PCR and quantitative RT-PCR (qRT-PCR) were carried out by a set of primers shown in Table S1. The semi-quantitative RT-PCR was carried out with a GoTaq Green Master Mix (Promega). cDNAs of the iron responsive genes were amplified using the primer pair (Table S1) under PCR conditions as follows: heating at 94 °C for 2 min followed by 27–31 cycles of denaturing at 94 °C for 30 s, annealing at 67 °C for 30 s, and elongation at 72 °C for 30 s. 5 μL PCR mixture were applied to 2% (wt/vol) Tris-borate-EDTA (TBE) agarose gel electrophoresis, and product was visualized by 0.5 μg mL−1 ethidiumbromide on a transilluminator. The standard curve of Isi1, FBP1, Fld, gapC2, and actin for qRT-PCR was drawn with the known amount of template using plasmids containing the Isi1, FBP1, Fld, gapC2, and actin cDNAs. Each transcript level was calculated using each standard curve from the value of Threshold Cycle (Ct) that is the intersection point of threshold and amplification curve. The level of the Isi1, FBP1, and Fld transcripts was normalized by the level of the gapC2 transcript. The level of the gapC2 transcript was further compared with that of the actin gene in order to double check the stability of the quantitative marker gene. Quantitative RT-PCR was carried out with a Thermal Cycler Dice® Real Time System II (TaKaRa) and GeneAce SYBR® qPCR Mixα No ROX (Nippon Gene) under PCR conditions as follows; heating at 95 °C for 10 min followed by 45 cycles of denaturing at 95 °C for 30 s, annealing at 60 °C for 1 min, and elongation at 72 °C for 30 s.

156

2.3. Preparation of transformation constructs

186

A series of upstream truncation was carried out at the position −1021, −781, − 495, −231, −87, −53, −21, +5, + 20, +54, +83, and + 121 relative to the transcription-start site of the Isi1 cDNA and these truncated constructs were ligated to the GUS reporter gene, uidA at the position + 214 relative to the transcription-start site of the Isi1 cDNA. Similarly, the upstream sequence of the FBP1 gene was truncated at the position −1097, −877, −627, −576, −431, −354, −286, and −257, and each construct was ligated with uidA at +9 relative to the transcription-start site of the FBP1 gene; the upstream sequence of the Fld gene was truncated at − 688, − 378, − 232, + 41, + 185, + 205, + 234, and + 268 and was ligated to uidA at + 407 relative to the transcription-start site of the Fld gene. All these truncations were done

187

P

92 93

139

D

90 91

2. Materials and methods

T

88 89

C

86 87

E

84 85

R

82 83

R

80 81

O

79

C

77 78

N

75 76

competence to survive in the severer iron limitation (Marchetti et al., 2008). These data strongly suggest that strategies to tolerate to the severe iron starvations are different among suborders of diatoms. Proteins participating to the uptake and the accumulation of irons are, in general, responsive to the environmental iron concentrations in many living organisms. The ferritin gene in Arabidopsis thaliana, AtFer1 is induced by iron limitations via the function of its iron-dependentregulatory sequence (IDRS) (CACGAGGCCGCCAC) in its promoter region (Petit et al., 2001). Similarly, in the promoter region of the siderophore synthesizing enzyme gene, the iron deficiency specific gene 2 (IDS2) in the barley, Hordeum vulgare, there are two iron-deficiency-inducible elements, IDE1 and IDE2 (CATGC), which are respectively targeted by the IDE-binding factors 1 and 2 (Kobayashi et al., 2003; 2007; Ogo et al., 2008). The transcription of the iron deficiency-induced ferroxidase gene, FOX1 in the green alga, Chlamydomonas reinhardtii is regulated by the two iron responsive elements, FeRE1 (CACACG at − 87 to − 82) and FeRE2 (CACGCG at −65 to −60) (Deng and Eriksson, 2007). Furthermore, it was reported that the other elements are also required for the upregulation of the copper chaperon gene, ATX1 (elements denoted AtxFeRE1 and 2: GNNGCNNTGGCATNT) and the ferric transporter gene, FEA1 (elements denoted FeaFeRE1 and 2: TGGCA) in response to iron limitation in C. reinhardtii (Deng and Eriksson, 2007; Fei and Deng, 2007). The promoter of the iron permease gene in C. reinhardtii, FTR1 also have the similar elements to FeaFeRE, FtrFERE1, and FtrFERE2 {TG(G/-)CA} which work for the upregulation of FTR1 in response to iron limitation (Fei et al., 2010). Interestingly, the FTR1 promoter also possesses an element FtrFeRE3 (AGTAACTGTTAAGCC), which works for down-regulation in response to iron limitation (Fei et al., 2010). Iron response and photosynthesis probably have a tight relationship. The expression of the ferritin gene in C. reinhardtii increased concomitantly with the degradation of the photosystem I (PSI), but the knock down of the ferritin gene resulted in the suppression of the PSI degradation, indicating that the iron accumulation in part governs the turnover of the PSI (Busch et al., 2008). In the marine diatom, Pseudo-nitzschia sp., the maximum quantum yield (Fv/Fm) in the PSII increased synchronously with the increase in the ferritin mRNA level (Marchetti et al., 2008), strongly suggesting the occurrence of the direct support mechanism of the iron-based electron-transport chain in the PSII by the iron accumulation capacity. In the marine diatom, P. tricornutum, iron limitation suppressed the expressions of phosphoribulokinase (PRK) and pyrenoidal β-carbonic anhydrases, PtCA1 and PtCA2 (Allen et al., 2008). This transcriptomic analysis by Allen et al. (2008) suggests that the decay of the Calvin cycle closes the photosystem and the mitochondrial alternative oxidase (AOX) may play a critical role to scavenge the photochemical energy in the photosystems. Detailed mechanisms of the iron signal perception and the iron responsive transcriptional regulation in diatoms are not well known yet. Recently, Lommer et al. (2012) reported that there was a conserved palindromic motif, ACACGTGC, in the promoter region of low-iron regulated genes, iron-starvation-induced protein1 (ISIP1), flavodoxin A (FLDA), and fructose-bisphosphate-aldolase3 (FBA3) in the genomes of marine diatoms, Thalassiosira oceanica, P. tricornutum, and Fragilariopsis cylindrus, suggesting a critical role of this motif in the iron-dependent gene regulation. As another intriguing aspect, the relationship between the iron limitation responses and the transcription of photosynthesis apparatus (Allen et al. 2008) strongly suggests the occurrence of a crosstalk between iron and CO2 signals. In the present study, based upon the expression-sequence tag (EST) database of P. tricorntum, we have selected 20 iron-responsive gene candidates, whose function can be grouped into metal uptakes, signal transduction, amino acid metabolism, heme biosynthesis, stress response, and functionally unsolved iron-starvation-induced proteins. Detailed profiles of transcript accumulation of these genes were determined during the early acclimation stage to the iron-starvation. Three evidently iron-responsive promoters were then isolated and their functions were characterized.

U

73 74

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

E

2

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185

188 189 190 191 192 193 194 195 196 197 198

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

210 211

2.4. Transformation of P. tricornutum

213 214

226 227

The air-grown cells of P. tricornutum were harvested at midlogarithmic phase (optical density at 730 nm of 0.3 to 0.4). Approximately 5 × 107 cells were spotted as a plaque of 2.5 cm diameter on the surface of the F/2ASW agar Plate. 500 μg tungsten microcarrier (JAPAN NEW METALS; particle size: 0.79 μm) was coated with 1.0 mg of plasmid DNA containing 1.0 M CaCl2 and 16 mM spermidine. The PDS-1000/He Biolistic Particle Delivery System (Bio-Rad) was used for the microprojectile bombardment of the microcarrier. The bombardment was done at 10.7 MPa to the cells in the chamber under the negative pressure of 91.4 kPa with a target distance of 6 cm. Bombarded cells were maintained on the plate for 1 day in the dark and were then suspended in 2 mL of F/2ASW. This cell suspension was plated on agar plates containing 100 mg mL− 1 Zeocin (Takara Bio) in F/2ASW and allowed to form colonies for 3 to 4 weeks under continuous illumination.

228

2.5. Screening of the GUS-expressing transformants

229 230

Zeocin-resistant clones were suspended in 60 mL of a GUS extraction buffer 1 (50 mM sodium phosphate [pH 7.0], 10 mM β-mercaptoethanol, 0.1% [wt/vol] sodium lauryl sarcosine, and 0.1% [vol/vol] Triton X-100) containing 1 mM 5-bromo-4-chloro-3-indolyl-β-D-GlcA cyclohexyl ammonium salt (X-Gluc) (Sigma-Aldrich) on 96-well plates and were incubated overnight at 37 °C. Transformants expressing GUS, which displayed the dark-blue-colored chromogenic reaction product were inoculated and used for further experiments.

231 232 233 234 235 236

C

224 225

E

223

R

221 222

R

219 220

N C O

217 218

T

212

215 216

2.6. Quantification of the GUS activity

238

Cells under changing iron conditions were harvested by centrifugation at 3000 xg at 25 °C, resuspended in 0.3 to 1.0 mL of a GUS extraction buffer 2 [50 mM sodium phosphate (pH 7.0), 10 mM β-mercaptoethanol, and 0.1% (vol/vol) Triton X-100], and disrupted by a sonicator (Ultrasonic disruptor model UD-201; TOMY Seiko Co. Ltd.); 5 cycles of disruption for 10 s, pause for 30 s in ice bath. The crude lysate was then centrifuged at 13,000 rpm for 5 min at 4 °C. Supernatant (lysate) (18 μL) was added to 882 μL of a GUS reaction buffer [10 mM p-nitrophenyl β-D-glucuronide (PNPG) in 50 mM phosphate buffer] and incubated at 37 °C. The reaction was terminated by the addition of 80 μL 0.5 M Na2CO3 to the 200 μL aliquot of the reaction solutions every 1 or 5 min after starting the reaction, and the absorbance of p-nitrophenol (PNP) released from the substrate was measured at 405 nm. Protein concentrations in each lysate were measured by the Bradford method (Bio-Rad) according to the manufacturer's protocol. The standard curve of PNP was drawn with 0.2– 8.0 nmol PNP (Wako), which gave a range of absorbance at 405 nm from 0.011 to 0.454. Concentrations of total lysate protein in the reaction mixture were adjusted to produce PNP within the range of the above standard curve in the reaction for 4 min. The GUS activity was calculated as the rate of PNP production per min per mg total lysate protein.

241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257

U

237

239 240

According to the array assay by Allen et al. (2008), 20 iron responsive genes were selected (Table 1). That is, genes related to the uptakes and homeostasis of iron and metals, ferric reductase1 (FRE1), FRE2, ferrichrome binding protein (FBP1), flavodoxin (Fld), iron-sulfur cluster assembly protein (IscA1), and ferritin (FTN); genes related to signaling and transcription, Ca2+-dependent protein kinase (CDPK1), and cellular repressor of E1A-stimulated genes (CREG1); a gene related amino acid metabolism, copper tyrosinase (TYR1); genes related to chlorophyll and heme biosynthesis, uroporphyrinogen decarboxylase1 (HemE1), HemE2, and coproporphyrinogen III oxidase (HemF1); genes related to stress response, heat shock protein20A (HSP20A), HSP20B, and HSP20C; and unknown function genes, iron starvation induced protein1 (Isi1), Isi2A, Isi2B, and Isi3. These genes and protein ID are listed in Table 1.

260 261

3.2. Quantification of transcript levels of iron limitation responsive genes

273

Changes in the transcript levels of the 20 genes (Table 1) in the P. tricornutum were profiled by the semi-quantitative RT-PCR using the 3 independent batches of cell culture during the initial stage (up to 10 days) of acclimation to iron-limited condition. The exposure of diatom cells to this short iron limited conditions for up to 10 days did not significantly slow down the growth rate nor reduced the photosynthetic capacity (Supplemental Fig. S1). Cells revealed these physiological damages after 10 days (Supplemental Fig. S1). Transcript levels of 12 genes, FRE1, FRE2, FRE3, FBP1, Fld, IscA1, CREG1, TYR1, Isi1, Isi2A, Isi2B, and Isi-3, were gradually increased with an increase in the duration of the irondeprived treatment (Fig. 1a). Among these 12 genes, 7 genes, FRE1, FRE3, Fld, CREG1, Isi2A, Isi2B, and Isi3, responded to iron limitation at the relatively early stage (from 3 days of iron deprivation), while the induction of other 5 genes, FRE2, FBP1, IscA1, TYR1, and Isi1, started from 5 to 7 days treatment (Fig. 1a). On the contrary, the transcript levels of FTN were decreased under iron limited condition (Fig. 1a). CPDK, all Hem genes, and all HSP genes revealed an initial increase in the

274 275

F

208 209

259

O

206 207

3.1. Screening of iron responsive genes

R O

205

258

P

203 204

3. Results

D

201 202

by PCR using the longest promoter constructs, PIsi1 (−1021 to +214), PFBP1 (− 1097 to +9), or PFld (−688 to + 407) as a template. These PCR products were phosphorylated and inserted into the blunt-ended site of the transformation vector pFcpApGUS (Harada et al., 2005), a derivative of the general P. tricornutum transformation vector pPha-T1, which is equipped with the bleomycin-resistant gene cassette, bler (Zaslavskaia et al., 2000). The replacement of the specific motif sequence with the NotI restriction site was carried out by the modified inverse PCR method using a vector plasmid pFcpApGUS containing PIsi1 (−1021 to +214), PFBP1 (−1097 to +9), or PFld (−688 to +407) as a template, and primers attached with NotI restriction site. Primers used for the isolation of promoter sequences and promoter truncations/ replacements are listed in Tables S2 and S3.

E

199 200

3

263 264 265 266 267 268 269 270 271 272

276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 t1:1 t1:1

Table 1 List of iron response genes tested in this study. Gene

Protein ID

Fe and metal uptakes and homeostasis FRE1 (ferric reductase 1) FRE2 (ferric reductase 2) FRE3 (ferric reductase 3) FBP1 (ferrichrome binding protein 1) Fld (flavodoxin) IscA1 (iron-sulfur cluster assembly protein 1) FTN (ferritin)

54486 46928 54940 46929 23658 14867 16343

Signaling and transcription CDPK1(Ca2+-dependent protein kinase 1) CREG1 (cellular repressor of E1A-stimulated gene 1)

54954 51183

Amino acid metabolism TYR1 (copper tyrosinase) Chlorophyll and heme biosynthesis HemE1 (uroporphyrinogen decarboxylase E1) HemE2 (uroporphyrinogen decarboxylase E2) HemF1 (coproporphyrinogen III oxidase)

262

40017 20757 19188 15068

Stress response HSP20A (heat shock protein 20A) HSP20B (heat shock protein 20B) HSP20C (heat shock protein 20C)

35138 54150 36981

Unknown Isi1 (iron starvation induced protein 1) Isi2A (iron starvation induced protein 2A) Isi2B (iron starvation induced protein 2B) Isi3 (iron starvation induced protein 3)

55031 54465 54987 47674

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

+Fe (day) 0 7 10

-Fe 0 1 2 3 4

+Fe

5 6

7 8 9 10

(day) 0 7 10

FRE1

HemE1

FRE2

HemE2

FRE3

HemF1

FBP1

HSP20A

Fld

HSP20B

IscA1

HSP20C

FTN

Isi1

CDPK1

Isi2A

CREG1

Isi2B

TYR1

Isi3

-Fe 0 1 2 3 4

5 6

R O

gapC2

b

0.7

0.05

1.5 1

0.04 0.03 0.02

+Fe

0

-Fe

+Fe

T

0

E

0.01

0.5

0.5

P

2

86.7

0.6

(Fld / gapC2)

(FBP1 / gapC2)

2.5

26.9

D

176.6

3

(Isi1 / gapC2)

Relative transcript level

7 8 9 10

F

a

O

4

0.4 0.3 0.2 0.1 0

-Fe

+Fe

-Fe

298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315

R

construct and is barely quantitative. However, the GUS activity ratios (−Fe/+Fe) clearly shows whether or not the introduced promoters respond to the external iron, which enabled us to assess the iron response with some accuracy (Fig. 2). The GUS activity ratios were thus presented

O

296 297

C

294 295

transcript levels followed by a steep decrease and oscillation during the later stage of the iron-limitation treatment (Fig. 1a). Three genes, Isi1, FBP1, and Fld which showed a clear and simple induction profile by the iron-limitation treatment were selected for further experiment. These three genes also revealed high sequence identity compared to their counterparts in other algae and bacteria. Quantitative RT-PCR showed that the levels of Isi1, FBP1, and Fld transcripts were, respectively, 176.6, 26.9, and 86.7 times in 7 days of iron limitation compared to those in the iron-replete cells (Fig. 1b). The level of the transcript of the quantitative maker gene, gapC2 did not show a significant change between iron-replenished and iron-limited conditions as referenced by the level of the actin gene (Supplemental Fig. S2).

N

292 293

U

291

R

E

C

Fig. 1. Changes in the levels of transcript of iron-related genes. a, Time course of transcript levels of 20 genes of the P. tricornutum under the iron-replete (+Fe) (for 0, 7, 10 days) or ironlimited (−Fe) (for 0–10 days) condition. b, qRT-PCR of Isi1, FBP1, and Fld under the iron-replete condition (+Fe) for 7 days (white column) or iron-limited (−Fe) condition for 7 days (black column). Transcript levels are expressed as a relative value to the gapC2 transcript. The semiquantitative RT-PCR and qPCR were duplicated with the 3 independent batches of culture and the typical results are shown for the semiquantitative RT-PCR. The relative transcript levels in qRT-PCR are the mean value with SD. Induction ratios (the transcript level in −Fe cells over + Fe cells) are indicated at the top of the diagram.

GUS activity (mmol g-1 protein min-1) 0 PIsi1

57.6

uidA

3.6 9.8

PFBP1

+Fe - Fe 212.6

+9

-1097

3.3. GUS reporter assay of iron responsive promoters Three reporter constructs, Pisi1 (− 1021–+ 214), PFBP1 (− 1097–+ 9), and PFld (− 688–+ 407) were transformed into the P. tricornutum cells and the GUS expressing transformants were obtained from all transformations. Our previous study showed that the heterologous expression of the GUS in the P. tricornutum cells was primarily regulated at the transcriptional levels and the level of the uidA mRNA and the GUS activity agreed well (Sakaue et al., 2008). The levels of the GUS reporter activity expressed in transformants varied significantly depending upon clones (data not shown). This is presumably due to position effects and copy number of the promoter::uidA constructs integrating into different parts of the genome. The absolute activity of the GUS thus does not necessarily reflect the activity of a single promoter

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

+214

-1021

29.5

uidA 102.4 37.2

-688

+407 PFld

PCMV

uidA

uidA

148.8 17.9 0.8

n=3

Fig. 2. Activities of isolated iron response promoters. The GUS activity was measured for lysates of transformants carrying uidA fusions downstream of PIsi1 (− 1021–+ 214), PFBP1 (−1097–+9), PFld (−688–+407), or PCMV. White and black bars indicate the iron replete (+Fe) and iron limited (−Fe) cells, respectively. Values are mean with SD of three replicates. The ratio of the GUS activity in iron limited cells over the iron replete cells is indicated at the right side of the bar. The measurement was carried out with 2–4 clones for each transformation line. Each vertical bar aside the Y axis represents a set of measurement for one clone.

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

316 317 318 319

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

320 321 322 323 324 325 326 327 328 329 330

5

a

for the GUS promoter assays hereafter. The result of the GUS reporter assay in this study clearly showed that all these three promoters are iron responsive (Fig. 2). Three reporter constructs revealed the significant levels of the GUS expression when the cells grown in the iron-limited environment, while promoter activities was efficiently suppressed under the iron-replete conditions (Fig. 2). Repressions by the iron-replete condition were stronger in PFBP1 (− 1097–+ 9), and PFld (− 688–+ 407) compared to that of Pisi1 (− 1021–+ 214) (Fig. 2). In sharp contrast, the GUS activity in cell lysate extracted from transformants carrying the cytomegalovirus promoter (PCMV) did not show difference under changing iron conditions (Fig. 2).

GUS activity (mmol g-1 protein min-1) 0 +214 uidA

-1021 PIsi1

0.1 3.8

-495

+Fe - Fe

1.9

-231

2.4

-87

2.2

-53

10.8

-21

4.6

+5

10.2

+20

2.0

+54

3.4. Upstream truncation assay of 3 iron responsive promoter

332

The function of three promoters, PIsi1 (−1021 to +214 relative to the transcription-start site), PFBP1 (−1097 to +9) and PFld (−688 to + 407) was investigated by a series of upstream truncations of these promoters fused with the GUS reporter gene, uidA. Of the eleven truncation constructs of Pisi1, nine constructs (− 781 to + 214, throughout + 54 to + 214) revealed a clear iron response (Fig. 3a). Likewise, among the seven upstream truncation constructs of PFBP1, six upstream constructs (−877 to +9 throughout −286 to +9) showed a clear iron response (Fig. 3b). The upstream truncations of PFld also showed a clear iron response until promoter region was cut to + 205 (Fig. 3c). These data strongly suggest that relatively short sequences about 30 bps (between + 54 and + 83 in Pisi1; − 286 and − 257 in PFBP1; + 205 and + 234 in PFld) are the critical regulatory region to respond to the environmental iron concentrations. It should also be noted that the truncation of the region −354 to −286 of PFBP1 caused a decrease in the induction value from 63.9 to 8.1 (Fig. 3b). The same was true for the truncation of the region from −232 to +41 of PFld by which induction ratio was decreased about 10 times (Fig. 3c), strongly suggesting the occurrence in these regions of critical elements required to obtain an enough dynamic range of iron response of these promoters.

343 344 345 346 347 348 349 350 351

3.5. Determination of critical iron responsive Cis-elements

353

373

The loss-of-function assays by promoter truncation technique have screened critical regions of about 30 bp as a candidate of the iron-responsive core regulatory region. Interestingly, the sequence alignment of these regions among three promoters showed a striking similarity (Fig. 4a); that is, there are two well conserved motifs in these regions, motif A, A(A/C)G(G/C)C(G/-)C(A/G)TG, and motif B, CACGTG(T/C)C, and the distance between motifs A and B varied depending upon promoters (Fig. 4a). Motifs A and B were then individually or concurrently replaced by the NotI restriction site and the promoter activity was reported by the GUS. In all promoters, the GUS expression levels under iron-limited condition tended to be reduced compared to the case of the intact promoter (Figs. 2 and 4). However, iron response of these partly impaired promoters was still evident except for Pisi1 with the impaired motif B (Fig. 4b–d). Pisi1 with the NotI-replaced motif B showed a constitutive activity independent of the iron condition (Fig. 4b). The dynamic range of the iron response in other manipulated constructs was also reduced but the iron response was still evident when motif A or B was individually replaced by the NotI sequence (Fig. 4b–d). On the other hand, the concurrent replacement of both motifs A and B in PFBP1 and PFld revealed a significant reduction of iron response of these promoters (Fig. 4c).

374 375

3.6. Gain-of-function of PCMV by the conserved iron responsive region of PFBP1

376

The sequence of PFBP1 (−286 to −252) (Fig. 4a) was fused with the PCMV::uidA construct (Fig. 5a) and transformed into the P. tricornutum cells. PCMV::uidA construct was not iron responsive but the addition of upstream PFBP1 (− 286 to − 252) conferred on this constitutive

360 361 362 363 364 365 366 367 368 369 370 371 372

377 378 379

R

R

N C O

358 359

U

356 357

E

352

354 355

O

0

+9 PFBP1

-877 -627 -576

0.1

R O

-1097

0.8

0.2

0.3

0.4

0.7

uidA

29.5 50.5 63.0 36.2

P

341 342

b

-431

30.1

-354

63.9

D

339 340

1.1

PCMV

-286

8.1 -257

E

337 338

0.9

+121

T

335 336

+83

C

333 334

7.0

F

331

0.4 9.8

-781

0.8 PCMV

0.8

c -688

+407 PFld

0

0.05

0.1

0.25

0.3

uidA

190.2

-378

n.d.

-232

93.8 +41

9.7 +185

3.1 +205

4.0 +234

0.5 +268

1.0 PCMV

0.8

Fig. 3. Loss-of-function assay of iron-responsive promoters by upstream truncations. A series of upstream truncation constructs of PIsi1 (a), PFBP1 (b), and PFld (c) were fused with the uidA gene (left half). White and black bars indicate the iron-replete (+Fe) and iron limited (−Fe) cells, respectively. Values are mean with SD of three replicates. The ratio of the GUS activity in iron limited cells over the iron replete cells is indicated at the right side of the bar. n.d. indicates not detected.

promoter sequence a clear iron response (Fig. 5a), indicating that 380 the sequence of PFBP1 (− 286 to − 252) includes iron responsive 381 Cis-elements. 382 3.7. CO2 and pH responses of the iron responsive promoters

383

Physiological and transcriptomic evidences have shown the close relations between iron limitation and photosynthesis (Busch et al., 2008; Marchetti et al., 2008; Allen et al., 2008) which prompted us to investigate the functional crosstalk between iron and CO2 signals. Interestingly, the induction of all three iron-responsive promoters upon iron

384 385

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

386 387 388

6

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

a

A PIsi PFBP PFld

a

B

0

+54 TTTGAAGCCGCATG---------TCA--GCACGTGTCCACGTCCTG +88 -286 ----ACGGCGCGTGGTAGCCACGTTACGGCACGTGCCCT------- -252 +205 TGCCACGCC-CATGA--GTTTCCTTG--ACACGTGCCAAC------ +239

b

GUS activity (mmol g-1 protein min-1)

-286 -252 PFBP1 IR PCMV

uidA

PCMV

uidA

0.02

0.04

N B

0.06

0.08

0.10

uidA

b

15.4

GUS activity (mmol g-1 protein min-1) 0

NotI

+214 A N

0 -1097

0.01

-1097 PFBP1 uidA

0.03

0.04

-688 PFld

PCMV uidA

+9 A N

4.4 uidA

+9 N N

2.8

0

0.02

0.04

0.06

4.3

E

uidA

+407 PFld

uidA

49.4

+407 N N

uidA

4.8

O

PFld

R

-688

A N

R

-688

N B

C

+407 PFld

U

N

C

Fig. 4. Determination of iron-responsive Cis-elements of three promoters. a, Sequence alignment of the critical regulatory regions of three iron-responsive promoters. Conserved base sequences are highlighted in white character in black background. Two conserved motifs are boxed (A and B). b, Motifs A and/or B were substituted by the NotI restriction site (left) and the GUS reporter assay was carried out with transformants carrying these substituted constructs. White and black bars indicate the iron replete (+Fe) and iron limited (−Fe) cells, respectively. The measurement was carried out with 1 – 2 clones for each transformation line. Each vertical bar aside the Y axis represents a set of measurement for one clone. Values are mean with SD of three replicates. The ratio of the GUS activity in iron limited cells over iron replete cells is indicated at the right side of the bar.

396 397 398

0.8

Fig. 5. Gain-of-function assay of PCMV by the core PFBP1 sequence (−286 to −252) and the response of iron-responsive promoters to CO2 and pH. a, The constitutive PCMV was fused with the core PFBP1 sequence (−286 to −252) at its upstream and the GUS reporter assay was carried out. White and black bars indicate iron replete (+Fe) and iron limited (−Fe) cells, respectively. b, The iron-responsive promoter activity was characterized in high CO2 grown cells and air-grown cells in acidified medium. White and black bars respectively indicate iron-replete (+Fe) and iron limited (−Fe) cells grown under the pH condition at 9.0 in the atmospheric air. Pink and red bars respectively indicate + Fe and −Fe cells grown under the pH condition at 6.5 in 5% CO2. Pale green and green bars respectively indicate + Fe and −Fe cells grown under the pH condition at 6.5 in the atmospheric air. Acidification treatment was carried out only to the PIsi1 assay. Values are mean with SD of three replicates. The ratio of the GUS activity in iron-limited cells over the ironreplete cells is indicated at the right side of the bar.

T

1.5

-688

164.2

1.3

E

uidA

d

32.7

D

17.4 -1097

Air/pH9.0

0.3 +Fe / Air / pH9.0 - Fe / Air / pH9.0 +Fe / 5%CO2 / pH6.5 - Fe / 5%CO2 / pH6.5 +Fe / Air / pH6.5 - Fe / Air / pH6.5

P

-1097

0.8

Air/pH9.0

uidA

2.1 uidA

6.4

PFBP1

7.8

Air/pH9.0 CO2/pH6.5

CO2/pH6.5

+9 N B

0.02

0.2 14.5

0.6

Air/pH6.5

c

PFBP1

CO2/pH6.5

uidA

1.0

PFBP1

uidA

R O

PIsi1

0.1

Air/pH9.0

F

-2021 PIsi1

1.6 -1021

+Fe

0.8

+Fe - Fe

GCGGCCGC CGCCGGCG

394 395

0.20

- Fe

4.9

+214

PIsi1

392 393

0.15 5.7

O

-1021

390 391

0.10

GUS activity (mmol g-1 protein min-1) 0

389

0.05

limitation was dissipated when cells were grown in 5% CO2 (Fig. 5b). In weakly buffered conditions, the ample inflow of 5% CO2 into the medium can acidify the culture medium. In order to confirm whether or not CO2 directly represses the iron-responsive genes or the acidification of the medium mediates it, transformants carrying PIsi1::uidA were cultured in atmospheric air with adjusted pH at 6.5 under iron deprived or replete condition. This simple acidification of the medium without the enrichment of CO2 did not affect the iron response of PIsi1 (Fig. 5b), strongly suggesting the direct participation of CO2 to the ironresponsive transcriptional control.

4. Discussion

399

In the present study, the changes in transcript levels of 20 genes (Table 1) during an early acclimation stage to iron limitation were profiled. These genes were previously reported to be upregulated upon iron limitation by a gene allay analysis in the P. tricornutum (Allen et al., 2008). Our results clearly showed that there are multiple types of the iron-responsive transcriptional regulation (Fig. 1), strongly suggesting the existence of multiple mechanisms of the iron-signal transduction under the iron limited environment. Most of iron/metal uptake/homeostasis genes and Isi genes, together with CREG1 and TYR1 revealed a gradual increase in transcript levels toward the prolonged iron limitation stage, allowing an assumption that the iron limitation signals are transmitted to the promoter via changes in some physiological states which are caused by the iron starvation. It is also interesting that the induction timing varies depending on genes in this group (Fig. 1). This supports the above assumption and perhaps diatom cells prepare for iron starvations in response to different physiological states under the iron-limited environment. Among these relatively slow induction genes, three pairs, FRE2 and FBP1, CREG1 and Isi2B, and TYR1 and Isi1 were clustered on the P. tricornutum genome (Allen et al., 2008) and the induction timing of each gene pairs appeared to be similar (Fig. 1), suggesting that each gene cluster is functionally related and is under the same regulatory mechanism of transcription. For example, FBP1 has a significant sequence similarity to the iron-siderophore uptake ABC system-binding component in the thermophilic bacteria, Symbiobacterium thermophilum IAM14863 (Braun and Hantke, 1991). The clustering of the FRE2 and FBP1 genes suggests the occurrence of an iron acquisition system

400

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492

514

O

F

Acknowledgments

R O

446 447

P

444 445

493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513

We thank Ms. Nobuko Higashiuchi for her technical assistance and Ms. Miyabi Inoue for her skilful secretarial assistance. This work was supported by Grant-in-Aid for Scientific Research B (grant no. 24310015 to Y. M.), by Grant-in-Aid for Challenging Exploratory Research (grant no. 24651119 to Y. M.) from Japan Society for the Promotion of Science about my English (JSPS), and by MEXT-Supported Program for the Strategic Research Foundation for the Advancement of Environmental Protection Technology and for Development of Intelligent Self-Organized Biomaterials.

515 516

References

524

Allen, A.E., LaRoche, J., Maheswari, U., Lommer, M., Schauer, N., Lopez, P.J., Finazzi, G., Fernie, A.R., Bowler, C., 2008. Whole-cell response of the pinnate diatom Phaeodactylum tricornutum to iron starvation. PNAS 105, 10438–10443. Armbrust, E.V., Berges, J.A., Bowler, C., Green, B.R., Martinez, D., Putnam, N.H., Zhou, S., Allen, A.E., Apt, K.E., Bechner, M., Brzezinski, M.A., Chaal, B.K., Chiovitti, A., Davis, A.K., Demarest, M.S., Detter, J.C., Glavina, T., Goodstein, D., Hadi, M.Z., Hellsten, U., Hildebrand, M., Jenkins, B.D., Jurka, J., Kapitonov, V.V., Kroeger, N., Lau, W.W., Lane, T.W., Larimer, F.W., Lippmeier, J.C., Lucas, S., Medina, M., Montsant, A., Obornik, M., Parker, M.S., Palenik, B., Pazour, G.J., Richardson, P.M., Rynearson, T.A., Saito, M.A., Schwartz, D.C., Thamatrakoln, K., Valentin, K., Vardi, A., Wilkerson, F.P., Rokhsar, D.S., 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306, 79–86. Bowler, C., Allen, A.E., Badger, J.H., Grimwood, J., Jabbari, K., Kuo, A., Maheswari, U., Martens, C., Maumus, F., Otillar, R.P., Rayko, E., Salamov, A., Vandepoele, K., Beszteri, B., Gruber, A., Heijde, M., Katinka, M., Mock, T., Valentin, K., Verret, F., Berges, J.A., Brownlee, C., Cadoret, J.P., Chiovitti, A., Choi, C.J., Coesel, S., Def Martino, A., Detter, J.C., Durkin, C., Falciatore, A., Fournet, J., Haruta, M., Huysman, M.J., Jenkins, B.D., Jiroutova, K., Jorgensen, R.E., Joubert, Y., Kaplan, A., Kröger, N., Kroth, P.G., La Roche, J., Lindquist, E., Lommer, M., Martin-Jézéquel, V., Lopez, P.J., Lucas, S., Mangogna, M., McGinnis, K., Medlin, L.K., Montsant, A., Oudot-Le Secq, M.P., Napoli, C., Obornik, M., Parker, M.S., Petit, J.L., Porcel, B.M., Poulsen, N., Robison, M., Rychlewski, L., Rynearson, T.A., Schmutz, J., Shapiro, H., Siaut, M., Stanley, M., Sussman, M.R., Taylor, A.R., Vardi, A., von Dassow, P., Vyverman, W., Willis, A., Wyrwicz, L.S., Rokhsar, D.S., Weissenbach, J., Armbrust, E.V., Green, B.R., Van de Peer, Y., Grigoriev, I.V., 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456, 239–244. Boyd, P.W., Watson, A.J., Law, C.S., Abraham, E.R., Trull, T., Murodoch, R., Bakker, D.C.E., Bowie, A.R., Buesseler, K.O., Chang, H., Charette, M., Croots, P., Dowining, K., Frew, R., Gall, M., Hadfield, M., Hall, J., Harvey, M., Jameson, G., Laroche, J., Liddicoat, M., Ling, R., Maldonald, M.T., Mckay, R.M.L., Nodder, S., Pickmere, S., Pridmore, R., Rintoul, S., Safi, K., Sutton, P., Strzepek, R., Tanneberger, K., Turner, S., Waite, A., Zeldis, J., 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization. Nature 407, 695–702. Braun, V., Hantke, K., 1991. Genetics of Bacterial Iron Transport. In: Winkelmann, G. (Ed.), Handbook of microbial iron chelates (Boca Raton). Briat, J.F., 1999. Plant ferritin and human iron deficiency. Nat. Biotechnol. 17, 621. Briat, J.F., Fobis-Loisy, I., Grignon, N., Lobréaux, S., Pascal, N., Savino, G., Thoiron, S., von Wirén, N., van Wuytswinkel, O., 1995. Cellular and molecular aspects of iron metabolism in plants. Biol. Cell. 84, 69–81.

525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 Q5 559 560 561 562 563

D

442 443

at its − 500 and − 400 bps relative to the initial codon (data not shown). Similarly, PFBP1 and PFld also possess multiple CCRE-like sequences in its promoter regions (data not shown). Interestingly, the low-CO2 inducible carbonic anhydrase gene promoters in P. tricornutum, Pptca1 and Pptca2 possess motifs A and B-like sequences (data not shown), strongly suggesting that iron and CO2 signals may differentially control the transcriptional activity of promoters of genes relating to the iron and CO2 acquisition. In this study, the early-stage transcriptional responses of the marine diatom P. tricornutum to iron limitation were shown to be divided into at least two types. The fact that the exposure of diatom cells to this short iron deprived conditions for up to 10 days caused little physiological damage (Supplemental Fig. S1) indicates that 10 days of iron limitation did not give a severe iron starvation on cells. However, drastic changes in the transcriptional activity of numerous genes were started already in the very early stage of iron limitation. Detailed mechanisms of the gene expression controls and physiological changes in the early stage iron limitation will provide critical information for our understanding on how marine diatom cells prepare for the iron starvation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.margen.2014.01.005.

E

440 441

T

438 439

C

436 437

E

434 435

R

433

R

431 432

N C O

429 430

combining the capture system of Fe3+ by siderophore with the transport system across the plasmalemma by reduction into Fe2+. In sharp contrast, the quick response of mRNA accumulations upon iron limitation was observed in mRNA accumulations of genes for chlorophyll/heme biosynthesis and stress responses, suggesting that these genes are more directly regulated by extracellular iron limitation signals. These gene expressions decayed after the initial stage of iron limitation and showed an oscillation at the later stage (Fig. 1). This profile of the chlorophyll biosynthesis genes and the stress response genes probably reflect the highly stressed physiology of the P. tricornutum cells at the initial stage of iron limitation which is followed by a moderately stressed stage after cells start strengthening the iron uptake and the accumulation by acclimating to iron limitation. The GUS reporter assay for the upstream-truncated promoters clearly displayed that the conserved short sequences about 30 bps close to the transcription start site of PIsi1, PFBP1, and PFld are the core-regulatory regions for the iron response (Figs. 3 and 4a). However, the specific replacement assays of the A and/or B motifs gave partly discrepant results (Fig. 4b–c), that is, PIsi1 completely lost iron response by impairing the motif B, while Pisi1 singly impaired in the motif A, and other promoters, PFBP1 and PFld with the impairment of either motif A or B still maintained a significant iron response, even though the dynamic range of iron response was reduced. On the other hand, a concurrent replacement of both motifs A and B in PFBP1 and PFld resulted in a significantly reduced iron responsibility and the putative core 35 bps of PFBP1 (−286 to −252) clearly conferred a capacity to respond to iron on PCMV (Fig. 5a), indicating that this conserved region functions as the iron-responsive element. The results of the concurrent replacement of motifs A and B in PFBP1 and PFld, and the gain-of-function assay of PCMV by the PFPB1 core region strongly suggest that motifs A and B may cooperate. The distance between motifs A and B differs among these three promoters (Fig. 4a) which may enable several modes of interaction between motifs A and B. Also the short promoter region including motifs A and B may work with the other upstream elements to fully activate these promoters upon iron limitation. Given this consideration, it is interesting to consider that there might be some critical elements in the regions −354 to −286 of PFBP1 and −232 to +41 of PFld to acquire an enough dynamic range of induction (Fig. 3b, c). The sequence of motif A is not previously reported thus it is likely a diatom specific sequence, while the motif B sequence was very similar to the sequence reported as the iron-responsive Cis-element of the FOX1 promoter, C(A/G)C(A/G)C(G/T), in the green alga, C. reinhadtii (Deng and Eriksson, 2007). Also in sharp agreement with our data, this sequence was previously predicted to be a candidate for the iron-responsive element at the bioinformatics bases in T. oceanica, P. tricornutum, and F. cylindrus (Lommer et al., 2012). The activation of three promoters, Pisi1, PFBP1, and PFld, by iron limitation only occurred under the atmospheric CO2 condition, where the CO2 availability for photosynthesis is limited. In sharp contrast, a simple acidification without the enrichment of CO2 did not result in the dissipation of iron response, indicating that the increase in inorganic carbon in the bulk medium could call off the induction of iron responsive genes at least at the early stages of acclimation to iron limitation. Iron limitation is supposed to be accompanied by oxidative stresses most probably due to the slowdown of the Calvin cycle. Ascorbic acid metabolism and biosynthesis pathways of α-tocopherol are also stimulated in iron limitations (Allen et al., 2008). This information agrees with the data in this study that the transcript accumulations of heme biosynthesis and general stress response genes occurred at the initial iron limitation stages (Fig. 1a). The enhanced spontaneous CO2 supply to the Calvin cycle under high CO2 conditions probably mitigates the oxidative effects of the iron starvation stresses on photosynthesis as far as the Calvin cycle is active. CO2 might be a direct regulatory signal on the iron-responsive promoters tested in this study. In fact, PIsi1 possesses the diatom CO2/ cAMP-responsive Cis-elements, CCRE1 and CCRE2 (Ohno et al., 2012),

U

427 428

7

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

517 518 519 520 521 522 523

P

R O

O

F

M., LaRoche, J., 2012. Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation. Genome Biol. 13, R66. Marchetti, A., Parker, M.S., Moccia, L.P., Lin, E.O., Arrieta, A.L., Ribalet, F., Murphy, M.E.P., Maldonado, M.T., Armbrust, E.V., 2008. Ferritin is used for iron storage in bloomformin marine pinnate diatoms. Nature 457, 467–470. Martin, J.H., Gordon, R.M., Fitzwater, S., Broenkow, W.W., 1989. Phytoplankton/iron studies in the Gulf of Alaska. Deep Sea Res. 36, 649–680. Ogo, Y., Kobayashi, T., Itai, N.R., Nakanishi, H., Kakei, Y., Takahashi, M., Toki, S., Mori, S., Nishizawa, N.K., 2008. A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants. J. Biol. Chem. 283, 13407–13417. Ohno, N., Inoue, T., Yamashiki, R., Nakajima, K., Kitahara, Y., Ishibashi, M., Matsuda, Y., 2012. CO2-cAMP-responsive cis-elements targeted by a transcription factor with CREB/ATF-like basic zipper domain in the marine diatom Phaeodactylum tricornutum. Plant Physiol. 158, 499–513. Petit, J.M., Wuytswinkel, O.V., Briat, J.F., Lobréaux, S., 2001. Characterization of an irondependent regulatory sequence involved in the transcriptional control of AtFer1 and ZmFer1 plant ferritin genes by iron. J. Biol. Chem. 276, 5584–5590. Raven, J.A., 1988. The iron and molybdenum use efficiencies of plant growth with different energy, carbon and nitrogen sources. New Phytol. 109, 279–287. Sakaue, K., Harada, H., Matsuda, Y., 2008. Development of gene expression system in a marine diatom using viral promoters of a wide variety of origin. Physiol. Plant. 133, 59–67. Strzepek, R.F., Harrison, P.J., 2004. Photosynthetic architecture differs in coastal and oceanic diatoms. Nature 431, 689–692. Tréguer, P., Nelson, D.M., Bennekom, A.J., DeMaster, D.J., Leynaert, A., Quéquiner, B., 1995. The silica balance in the world ocean: a reestimate. Science 268, 375–379. Tsuda, A., Takeda, S., Saito, H., Nishioka, J., Nojiri, Y., Kudo, I., Kiyosawa, H., Shiomoto, A., Imai, K., Ono, T., Shimamoto, A., Tsumune, D., Yoshimura, T., Aono, T., Hinuma, A., Kinugasa, M., Suzuki, K., Sohrin, Y., Noiri, Y., Tani, H., Deguchi, Y., Tsurushima, N., Ogawa, H., Fukami, K., Kuma, K., Saino, T., 2003. A mesoscale iron enrichment in the western subarctic Pacific induces a large centric diatom bloom. Science 300, 958–961. Zaslavskaia, L.A., Lippmeier, J.G., Kroth, P.G., Grossman, A.R., Apt, K.E., 2000. Transformation of the diatom Phaeodactylumtricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J. Phycol. 36, 379–386.

N

C

O

R

R

E

C

T

E

Busch, A., Rimbauld, B., Naumann, B., Rensch, S., Hippler, M., 2008. Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii. Plant J. 55, 201–211. Coale, K.H., Johnson, K.S., Fitzwater, S.E., Gordon, R.M., Tanner, S., Chavez, F.P., Ferioli, L., Sakamoto, C., Rogers, P., Millero, F., Steinberg, P., Nightingale, P., Cooper, D., Cochlan, W.P., Landry, M.R., Constantinou, J., Rollwagen, G., Trasvina, A., Kudela, R., 1996. A massive phytoplankton bloom induced by an ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean. Nature 383, 495–501. Deng, X., Eriksson, M., 2007. Two iron-responsive promoter elements control expression of FOX1 in Chlamydomonas reinhardtii. Eukaryot. Cell 6, 2163–2167. Eide, D.J., 1998. The molecular biology of metal ion transport in Saccharomyces cerevisiae. Annu. Rev. Nutr. 18, 441–469. Falkowski, P.G., Scholes, R.J., Boyle, E., Canadell, J., Canfield, D., Elser, J., Gruber, N., Hibbard, K., Hogbeg, P., Linder, S., MacKenzie, A.F., Moore, B.I.I.I., Pedersen, T.F., Rosenthal, Y., Seitzinger, S., Smetacek, V., Steffen, W., 2000. The global carbon cycle: a test of our knowledge of Earth as a system. Science 290, 291–296. Fei, X., Deng, X., 2007. A novel Fe deficiency responsive element (FeRE) regulates the expression of atx1 in Chlamydomonas reinharditii. Plant Cell Physiol. 48, 1496–1503. Fei, X., Eriksson, M., Li, Y., Deng, X., 2010. A Novel Negative Fe-Deficiency-Responsive Element and a TGGCA-Type-Like FeRE Control the Expression of FTR1 in Chlamydomonas reinhardtii. J. Biomed. Biotechnol. 2010, 1–9. Harada, H., Nakatsuma, D., Ishida, M., Matsuda, Y., 2005. Regulation of the expression of intracellular β-carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum. Plant Physiol. 139, 1041–1050. Kobayashi, T., Nakayama, Y., Itai, R.N., Nakanishi, H., Yoshihara, T., Mori, S., Nishizawa, N.K., 2003. Identification of novel cis-acting elements, IDE1 and IDE2, of the barley IDS2 gene promoter conferring iron-deficiencyinducible, root-specific expression in heterogeneous tobacco plants. Plant J. 36, 780–793. Kobayashi, T., Ogo, Y., Itai, R.N., Nakanishi, H., Takahashi, M., Mori, S., Nishizawa, N.K., 2007. The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants. PNAS 104, 19150–19155. Kosman, D.J., 2003. Molecular mechanisms of iron uptake in fungi. Mol. Microbiol. 47, 1185–1197. Lommer, M., Specht, M., Roy, A.S., Kraemer, L., Andreson, R., Gutowska, M.A., Wolf, J., Bergner, S.V., Schilhabel, M.B., Klostermeier, U.C., Beiko, R.G., Rosenstiel, P., Hippler,

U

564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599

R. Yoshinaga et al. / Marine Genomics xxx (2014) xxx–xxx

D

8

Please cite this article as: Yoshinaga, R., et al., Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum, Mar. Genomics (2014), http://dx.doi.org/10.1016/j.margen.2014.01.005

600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635