# The Myb-like transcription factor Phosphorus Starvation Response (PtPSR) controls conditional P acquisition and remodeling in marine microalgae

Amit Kumar Sharma, Alice Mühlroth, Juliette Jouhet, Eric Maréchal, Leila Alipanah, Ralph Kissen, Tore Brembu, Atle Bones, Per Winge

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Amit Kumar Sharma, Alice Mühlroth, Juliette Jouhet, Eric Maréchal, Leila Alipanah, et al.. The Myb-like transcription factor Phosphorus Starvation Response (PtPSR) controls conditional P acquisition and remodeling in marine microalgae. New Phytologist, 2020, 225, pp.2380-2395. 10.1111/nph.16248. hal-02333579

HAL Id: hal-02333579

https://hal.science/hal-02333579

Submitted on 9 Oct 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

# The Myb-like transcription factor phosphorus starvation response (PtPSR) controls conditional P acquisition and remodelling in marine microalgae

Amit Kumar Sharma¹, Alice Mühlroth¹, Juliette Jouhet², Eric Maréchal², Leila Alipanah¹, Ralph Kissen¹, Tore Brembu³ ID, Atle M. Bones¹ and Per Winge¹ ID

¹Cell, Molecular Biology and Genomics Group, Department of Biology, Norwegian University of Science and Technology, 7491, Trondheim, Norway; ²Laboratoire de Physiologie Cellulaire Végétale, Centre National de la Recherche Scientifique, Commissariat à l'Energie Atomique, Institut National de la Recherche Agronomique, Université Grenoble Alpes, 38000, Grenoble, France; ³Department of Biotechnology and Food Science, Norwegian University of Science and Technology, 7491, Trondheim, Norway

Authors for correspondence:
Per Winge

Tel: +47 73 59 62 29

Email: per.winge@ntnu.no

Atle M. Bones

Tel: +47 73 59 86 92

Email: atle.m.bones@ntnu.no

Received: 15 April 2019

Accepted: 29 September 2019

New Phytologist (2020) 225: 2380-2395

doi: 10.1111/nph.16248

**Key words:** alkaline phosphatase (AP), diatom, lipid remodelling, Myb transcription factor, phosphate stress response, phosphorus, reshuffling of phosphate containing compounds.

## Summary

* Phosphorus (P) is one of the limiting macronutrients for algal growth in marine environments. Microalgae have developed adaptation mechanisms to P limitation that involve remodelling of internal phosphate resources and accumulation of lipids.

* Here, we used *in silico* analyses to identify the P-stress regulator PtPSR (*Phaeodactylum tricornutum* phosphorus starvation response) in the diatom *P. tricornutum*. *ptpsr* mutant lines were generated using gene editing and characterised by various molecular, genetics and biochemical tools.

* PtPSR belongs to a clade of Myb transcription factors that are conserved in stramenopiles and distantly related to plant P-stress regulators. PtPSR bound specifically to a conserved cis-regulatory element found in the regulatory region of P-stress-induced genes. *ptpsr* knockout mutants showed reduction in cell growth under P limitation. P-stress responses were impaired in *ptpsr* mutants compared with wild-type, including reduced induction of P-stress response genes, near to complete loss of alkaline phosphatase activity and reduced phospholipid degradation.

* We conclude that PtPSR is a key transcription factor influencing P scavenging, phospholipid remodelling and cell growth in adaptation to P stress in diatoms.

## Introduction

Phosphorus (P) is one of the most important macronutrients for algae and is a limiting factor of marine primary production in some parts of the world's oceans (Sarthou et al., 2005; Elser et al., 2007). It can be found in its inorganic form ($P_i$, orthophosphate) or in dissolved organic phosphorus (DOP) (Van Mooy et al., 2009). Recent studies have revealed underlying common molecular mechanisms of stramenopiles to deal with P stress that can be divided into global and specific P-stress responses (Dyhrman et al., 2012; Cruz de Carvalho et al., 2016; Cañavate et al., 2017; Mühlroth et al., 2017; Alipanah et al., 2018). In microalgae, specific P-stress responses involve P transportation, P scavenging and degradation of alternative P sources such as DOP (Dyhrman, 2016). Global P-stress responses include cell division arrest, reduction of the RNA pool and redirection of main carbon fluxes towards carbon storage molecules such as chrysolaminarin or triacylglycerol (TAG, Mühlroth et al., 2013; Brembu et al., 2017). Even though a few molecular P-stress mechanisms are

known, key transcriptional regulators have not been identified in stramenopiles.

Transcriptome and lipidome studies in the diatoms *Phaeodactylum tricornutum* and *Thalassiosira pseudonana*, as well as the eustigmatophyte *Nannochloropsis oceanica*, have provided detailed insights of gene regulation patterns during P limitation (Dyhrman et al., 2012; Cruz de Carvalho et al., 2016; Mühlroth et al., 2017; Alipanah et al., 2018). P acquisition is activated by transcriptional induction of genes encoding high and low affinity $P_i$ transporters, phosphodiesterases and phosphatases (Donald et al., 1997; Shimogawara et al., 1999). Phosphodiesters such as phospholipids (PLs) and nucleic acids are degraded by extracellular and intracellular glycerophosphoryl diester phosphodiesterases (GDPDs) and nucleotidases, which are poorly studied in stramenopiles (Dyhrman et al., 2012; Baldwin, 2013). A recent study in *N. oceanica* showed that expression of cellular and extracellular GDPDs was induced as part of an early P-stress response, suggesting that degradation of membrane-bound PLs was likely part of the P-stress-inducible PL-recycling scheme (Mühlroth
et al., 2017). This PL-recycling scheme includes degradation of PLs, reduction of PL biosynthesis and synthesis of P-free lipid classes (Martin et al., 2011; Cañavate et al., 2017). Phosphatases involved in P acquisition include phosphomonoesterases (PMEs) and alkaline phosphatases (APs) degrading phosphomonoesters, enabling the cell to obtain orthophosphate by degrading intracellular and extracellular DOP sources (Flynn et al., 1986; Theodorou et al., 1991; Mühlroth et al., 2017; Alipanah et al., 2018). The P-stress-inducible APs are the most commonly encountered phosphatases and are found in a wide range of phytoplankton. AP activity measurements are therefore used as a biomarker for P limitation in the marine environment and single cultures (Dyhrman et al., 2012; Lin et al., 2012; Feng et al., 2015; Cañavate et al., 2017). Several putative APs identified in diatoms have either bacterial origin or are similar to the characterised low P-inducible APs of the green alga *Chlamydomonas reinhardtii* (Dyhrman et al., 2012; Alipanah et al., 2018). APs in *C. reinhardtii* are either membrane bound, which allows AP detection in intact concentrated cells or extracellular, allowing AP activity measurements in the filtrate (Quisel et al., 1996; Xu, 2006; Li et al., 2012; Dyhrman, 2016).

Long-term P-stress-induced processes classically involve tran-
scription factors (TFs) that are often lineage-specific and key
players in gene expression. However, apart from *in silico* studies,
little information is known regarding nutrient stress-induced TFs
in stramenopiles (Rayko *et al.*, 2010; Matthijs *et al.*, 2016, 2017;
Thiriet-Rupert *et al.*, 2016). The myeloblastosis (Myb) family of
TFs is widespread in eukaryotic organisms and is a large, func-
tionally diverse family (Dubos *et al.*, 2010; Rayko *et al.*, 2010).
The Myb-related TFs PHOSPHORUS STARVATION
RESPONSE 1 (PSR1) and PHOSPHATE STARVATION
RESPONSE1 (PHR1) were found to act as important regulators
of P-stress responses in *C. reinhardtii* and *Arabidopsis thaliana*,
respectively (Wykoff *et al.*, 1999; Rubio *et al.*, 2001). PSR1 and
PHR1 control the expression of P transporters, enzymes involved
in DOP degradation and P scavenging, and P starvation-specific
secreted polypeptides under low Pᵢ conditions (Dubos *et al.*,
2010). Myb TFs are divided into several classes depending on the
adjacent repeats of the Myb DNA-binding domain (Dubos *et al.*,
2010; Chiou & Lin, 2011). Phylogenetic analysis showed that
PHR1 and PSR1 share the same Myb DNA-binding domain
with a SHLQKYR motif located at its C-terminal part (Wykoff
*et al.*, 1999; Rubio *et al.*, 2001; Thiriet-Rupert *et al.*, 2016). In
addition to the Myb DNA-binding domain, PHR1 and PSR1
also share similarities in a C-terminal, predicted coiled-coil (CC)
domain, which is a glutamine-rich sequence potentially involved
in protein-protein interactions (Dubos *et al.*, 2010; Chiou &
Lin, 2011). Under low Pᵢ, PSR1 and PHR1 form homodimers,
probably through their CC domain, that bind an imperfect palin-
dromic DNA sequence termed the PHR1-binding sequence
(P1BS) present in the promoters of Pᵢ starvation-responsive genes
(Wykoff *et al.*, 1999; Rubio *et al.*, 2001).

The marine pennate diatom *P. tricornutum* has become a
model species for molecular studies in diatoms, due to its well
characterised genome and development of advanced molecular
engineering techniques (Falciatore *et al.*, 1999; Bowler *et al.*,

2008; Karas et al., 2015; Nymark et al., 2016). The recently
established CRISPR/Cas9 gene editing system in P. tricornutum
opens up possibilities for efficient reverse-genetic pipelines to
identify key players in metabolic pathways and transcriptional
regulation (Nymark et al., 2016; Sharma et al., 2018). In the pre-
sent study, we used available transcriptome data to identify the P-
stress-induced Myb-related TF PtPSR (P. tricornutum phospho-
rus starvation response). ptpsr mutants generated by gene editing
were subjected to chemical, biochemical, physiological and tran-
scriptional analyses to investigate its role as a regulator of P limi-
tation responses.

**Materials and Methods**

Growth conditions

Axenic *P. tricornutum* wild-type cells (PtWT) (Bohlin clone Pt1
8.6, CCMP632) were grown in liquid culture as described previ-
ously under constant light (100 µmol photons m⁻² s⁻¹), at 20°C
with shaking (150 rpm) (Nymark *et al.*, 2009). Before experi-
ments, the cells (PtWT and mutants) were acclimatised by culti-
vation in continuous light under shaking (150 rpm) for 2–3 wk;
cells were diluted 10-fold every second day to maintain exponen-
tial growth. The experimental cultures were inoculated to
c. 1 × 10⁵ cells ml⁻¹ in either f/2 medium (36.38 µM PO₄³⁻, +P
cells) or f/2 medium with reduced P concentration (0.6 µM
PO₄³⁻, –P cells). The mutant lines (*ptpsr 1_3.4*, *ptpsr 1_3.10*,
*ptpsr 2_2.2*, *ptpsr 2_2.3*) and PtWT were grown in four biologi-
cal replicates under both –P and +P conditions. For validation
of the AP activity experiment, RNA samples at 48 h were taken
to confirm reproducibility of the experimental setup under P lim-
itation (data not shown). For cell counts, a NovoCyte flow
cytometer (ACEA Biosciences, San Diego, CA, USA) was used
with light excitation at 488 nm, a forward angle light scatter
(FSC, 0 ± 13), and the fluorescence filter FL3 with absorption at
670 nm as a threshold. Cultures were regularly tested for axenity
by plating on agar plates with f/2 medium and 1 g l⁻¹ peptone
followed by incubation for 3–5 d at room temperature. Axenic
growth was confirmed throughout the experiments.

Vector construction and sgRNA design

The CRISPR/Cas9 vector pKsdiaCas9_sgRNA (Addgene ID: 74923, Watertown, MA, USA) and pPtPuc3_Cas9_sgRNA (Addgene ID: 109219) were used to produce InDels in *Phatr2_47256* encoding PtPSR in *P. tricornutum* (Nymark *et al.*, 2016; Sharma *et al.*, 2018). The CRISPR/Cas9 vector, pKsdiaCas9_sgRNA, is delivered via biolistic method and plasmid is integrated into the host genomic DNA, while pPtPuc3_Cas9_sgRNA is delivered via a bacterial conjugation method and remains as an episome inside the cells (Nymark *et al.*, 2016; Sharma *et al.*, 2018; Slattery *et al.*, 2018). A custom-made Perl script that search for unique PAM sites with low homology to other genomic loci that might produce off-target effect was used to design single guide RNA (sgRNA) adapters for targeting two regions in *PtPSR* gene. *PtPSR* PAM 1 (target site 1)
was located 5' of the Myb DNA-binding domain so that out-of-
frame InDels would produce a nonfunctional protein lacking the
Myb domain, and *PtPSR* PAM 2 (target site 2) targeted the con-
served domain 2. The adapter sequences (followed by PAM
sequence: 5'-NGG-3') were selected as described in Nymark
*et al.* (2016) by using an in-house program based on a Perl script.
The adapters targeting *PtPSR* were ligated into the
*pKSdiaCas9 sgRNA* and *pPtPuc3 Cas9 sgRNA* plasmids as
described previously by Nymark *et al.* (2016, 2017). *PtPSR*
PAM1 lines were generated using *pKS diaCas9 sgRNA* plas-
mids (Nymark *et al.*, 2016) and *PtPSR* PAM2 lines using
*pPtPuc3 diaCas9 sgRNA* with transformation via biolistic bom-
bardment and bacterial conjugation, respectively (Sharma *et al.*,
2018).

Transformation

*Phaeodactylum tricornutum* cells were transformed as described previously (Falciatore *et al.*, 1999; Nymark *et al.*, 2017). For delivery by conjugation to *P. tricornutum*, cells were transformed using *Escherichia coli* (DH10β) as previously described (Sharma *et al.*, 2018; Slattery *et al.*, 2018). *E. coli* cells with conjugative pT-MOB plasmid and *pPtPuc3_diaCas9 sgRNA* plasmid were grown at 37°C to OD₆₀₀ 0.8–1.0, harvested by centrifugation (10 min, 3000 g) and resuspended in 500 µl SOC media (Karas *et al.*, 2015). *Phaeodactylum tricornutum* cells were grown for 4 d on 50% f/2-Si agar plates and then scraped off, resuspended and adjusted to 5 × 10⁸ cells ml⁻¹. For the conjugation, 200 µl each of *P. tricornutum* and *E. coli* cells were mixed and plated on 50% seawater (SW) f/2 with 5% LB agar plates and incubated in darkness at 30°C for 90 min. Plates were transferred to constant light (100 µmol photons m⁻² s⁻¹) at 20°C. After 2 d, *P. tricornutum* cells were transferred to selection plates. Colonies were selected from selection plates after 2 wk and transferred to f/2 liquid medium containing 50 µg ml⁻¹ zeocin or on new selection plates.

Screening primary *ptpsr* mutant colonies and isolation of *ptpsr* mutant lines

*Phaeodactylum tricornutum* mutations at PAM1 and PAM2 sites were identified by high-resolution melting (HRM) analysis followed by Sanger sequencing of PCR amplicons using primers from Supporting Information Table S1, as described by Nymark *et al.* (2016, 2017). Pure mutant lines were obtained as previously described (Nymark *et al.*, 2016; Sharma *et al.*, 2018). Primary mutant colonies were diluted (c. 200 cells ml⁻¹), and 250 µl of cells were transferred to selection plates (50% SW f/2, 100 µg ml⁻¹ zeocin, 1% (w/v) agar). Mutants grown on selection plates were picked and checked for target mutations as described above. Clean biallelic clones were identified by HRM and the PCR product was cloned into the pCR4-TOPO TA vector (Invitrogen). At least six PCR clones from each colony were sequenced to get an overview of the different InDels present within a colony. Allelic InDels and mutations were verified using single nucleotide polymorphisms (SNPs) observed in the gene (Sharma *et al.*, 2018).

Alkaline phosphatase assay

*Phaeodactylum tricornutum* cells were harvested for cellular AP activity (cAP) measurements by centrifugation (15 min, 4000 *g* and 1 min, 16 000 *g*). The supernatant was removed and replaced by fresh seawater to obtain a cell concentration of 7 × 10⁶ cells ml⁻¹ 0.5 ml concentrated cell suspension was added to 0.5 ml Tris-HCl–pNPP solution (100 mM Tris-HCl and 10 mM *p*-nitrophenyl phosphate (pNPP) were suspended in sterile seawater and adjusted to pH 9.5). After 2 h incubation in dimmed light at room temperature, presence of pNP (*p*-nitrophenol) was measured by absorption at 405 nm. As a blank, 0.5 ml concentrated cell suspension was added to 0.5 ml Tris-HCl–pNPP solution immediately before measuring. A dilution series of pNP standard was measured at 405 nm and together with the cell count used to determine the AP activity (mM conversion of pNP per 10⁶ cells). External AP activity (eAP) was measured using the supernatant of centrifuged cell samples and measured as described above.

Phosphorus determination

Samples for dissolved Pᵢ were collected as supernatant after cen-
trifugation of algal cells and stored at −20°C until analysis. Pi
determination was done according to the standard colorimetric
method by the reaction of phosphomolybdic acid at 880 nm with
the Analytical Flow Solution IV analyser (Hansen & Koroleff,
1999). For cellular P, defined volumes of the cell cultures (10–
50 ml) were filtered on a *c*. 1-µm Whatman GF/C filter and
stored at −20°C until analysis following Norwegian standard
NS6878 (2004).

RNA isolation and quantitative real-time PCR

Depending on cell density, 100–250 ml of four biological repli-
cates of each mutant line were quickly harvested through vacuum
filtration on 0.65-µm Durapore membrane filters (DVPP; Merck
Millipore, Darmstadt, Germany) at time points 2, 24 or 48 h.
Cells were washed from filters using 1 ml of the same growth
medium and then centrifuged at 16 000 g for 1 min at 4°C. The
supernatant was discarded, and the pellet was flash frozen in liquid
nitrogen and stored at –80°C. Total RNA was isolated using the
Spectrum Plant Total RNA Extraction Kit (Sigma-Aldrich) and
quantified using a NanoDrop spectrophotometer (Thermo Fisher
Scientific, Wilmington, NC, USA). Reverse transcription of total
RNA was performed from 1000 ng RNA using the QuantiTect
Reverse Transcription Kit (Qiagen) and oligo(T) and random
primers. Four biological replicates from each line, time point and
treatment were used to perform quantitative real-time PCR (qRT-
PCR) on a LightCycler 480 using the LightCycler 480 SYBR
green I master kit (Roche Applied Science, Indianapolis, IN,
USA). The standard method included a preincubation for 5 min
at 95°C, followed by 50 cycles of amplification consisting of 10 s
at 95°C, 10 s at 58°C and 15 s at 72°C. Both genes of interests
(GOIs) and housekeeping genes used for qRT-PCR were chosen
based on the microarray data from previous P limitation experi-
ments in *P. tricornutum* and *T. pseudonana* (Table S2) (Dyhrman
et al., 2012; Alipanah et al., 2018). LINREG PCR 11.1 software was used to determine primer efficiency, and QBASEPLUS software for identifying the Ct value (Ramakers et al., 2003; Ruijter et al., 2009). Means of the individual biological samples (n = 4) were calculated and used to determine the ratio and the respective log2 value, using the QBASEPLUS software (Biogazelle, www.qbaseplus.com) and one-way ANOVA analysis. For ptpsr mutants, regulation of selected genes at each time point was calculated using PtWT as reference under the same conditions.

## Phylogenetic analysis of Myb DNA-binding domain

Protein alignments were generated using MEGA 7.0.26 (Kumar et al., 2016). A number of substitution matrices were evaluated (MEGA 7.0.26) and the best one selected. Maximum likelihood (ML) trees were created with MEGA 7.0.26 using the LG model as the amino acid substitution model, with gamma-distributed mutation rates among amino acid sites and complete deletion of gaps and missing data. MEGA 7.0.26 was also used to create bootstrapped neighbour-joining (N-J) (Saitou & Nei, 1987) trees using the Jones-Taylor-Thornton (JTT) model as the amino acid substitution model. In total, 1000 nonparametric bootstrap inferences were executed both for ML and N-J trees. The trees were refined using Affinity Designer (Serif (Europe) Ltd, Nottingham, UK).

## PtPSR recognition motif screen

The Multiple EM for Motif Elicitation (MEME) web server was used for identifying potential transcription factor binding sites in promoters from 84 selected genes, using the 0-order model to normalise for biased distribution of bases (Bailey et al., 2006). The promoters were selected from genes that showed a specific and robust induction under P-limited conditions, that is the GEO dataset GSE66063 (Alipanah et al., 2018).

## Electrophoresis mobility shift assay

Full-length PtPSR was cloned into pBADM-20(+) (Addgene) to generate a fusion protein with an N-terminal His-tag. The recombinant protein was expressed in E. coli BL21-CodonPlus (DE3)-RIPL (Stratagene, La Jolla, CA, USA), purified on HisTrap column (GE Healthcare, Pittsburgh, PA, USA) and treated with Tobacco Etch Virus (TEV) protease to cleave off the tag. For the Electrophoresis Mobility Shift Assay (EMSA), three 51 bp DNA oligos (Integrated DNA Technologies, Leuven, Belgium) were used: one containing three PtPSR binding motifs and labelled with IRDye700 ('probe'), a similar unlabelled one ('competitor') and one unlabelled where the PtPSR binding motifs were mutated ('mutated competitor'). Double-stranded probes, DNA-binding reactions and EMSA were performed as described by Nakata & Ohme-Takagi (2014) using the Odyssey Infrared EMSA Kit (Li-Cor Biosciences, Lincoln, NE, USA).

## SDS-PAGE

Tris-glycine SDS-PAGE mini gels were run using Precision Plus Protein Dual Color Standards (Bio-Rad) as the molecular weight

marker and stained with SimplyBlue SafeStain (Life Technologies).

## Lipid contents and composition

Lipid contents and class was determined as described in Mühlroth et al. (2017). Algae cultures were harvested by centrifugation (15 180 g, 10 min) and subsequent filtration. The filter was resuspended in 0.5 ml seawater, centrifuged (20 000 g, 1 min) and the pellets were stored at -80°C. Lipids were extracted and quantified by mass spectrometric analysis according to Jouhet et al. (2017).

## Results

### Identification and characterisation of PtPSR and phylogenetic analysis of the Myb DNA-binding domain

Myb TFs in plants and green algae are known components of a central regulatory system controlling transcriptional responses to P limitation (Jyoti et al., 2019). When we investigated the transcriptional response to P limitation in genes encoding TFs of the diatom *P. tricornutum*, using a publicly available dataset (Alipanah et al., 2018), the most responsive TF at 48 and 72 h after P deprivation was a transcript encoding a Myb-like TF annotated as PtMyb1R_SHAQKYF5 (*Phatr2_47256*), here re-named PtPSR. *PtPSR* transcript induction was more than four times stronger compared with other Myb-like genes (Fig. 1a). We also analysed a transcriptome dataset of P limitation in the eustigmatophyte *N. oceanica* (Mühlroth et al., 2017). Two *N. oceanica* transcripts encoding Myb-like TFs closely related to *PtPSR* (No_10770 and No_8993 in the genome assembly by Vieler et al. (2012)) displayed a similar early induction under P limitation, suggesting that the role of this TF is conserved among stramenopiles (Fig. S1). *Nannochloropsis* spp. harbour 35 Myb and Myb-related TF genes (15 R2R3 Myb, 8 R1R2R3 Myb and 12 Myb related); three of these are related to PtPSR (Hu et al., 2014). PtPSR belongs to a family of nine Myb-like TFs in *P. tricornutum* that is characterised by a single Myb DNA-binding domain-containing a SHAQKYF-like motif (Rayko et al., 2010). SHAQKYF-like Mybs in diatoms can be further divided into three classes. Class I SHAQKYF-like Mybs are related to *Arabidopsis* ARR and PHR1-like proteins, class II are weakly related to Arabidopsis CCA1 and LHY proteins while class III are diatom-specific (Fig. S2; Rayko et al., 2010). PtPSR (PtMyb1R_SHAQKYF5) belongs to class I together with PtMyb1R_SHAQKYF8 (*Phatr2_50328*), which showed lower induction than PtPSR at 72 h under P limitation (Fig. 1a). PtPSR encodes a 575 amino acids (AA) long protein with four conserved domains (CDs) of unknown function and the Myb DNA-binding domain at 219 to 275 AA (Fig. 1b). A putative nuclear localisation signal (NLS, GGGSSRSSKVRRRKGK, amino acid residues 198–212) predicted by LOCALIZER 1.0 (Sperschneider et al., 2017) is present upstream of the Myb DNA-binding domain. The CD2 domain is conserved in diatoms and contains a predicted helical structure. The CD3 domain showed similarity
<figure><img src="image_4.png" /></figure>

**Fig. 1** The Myb transcription factor *PtPSR* is strongly induced at the transcript level by P limitation. (a) Transcriptional responses of all *Phaeodactylum tricornutum* Myb TFs significantly regulated (*, P* < 0.01) by P depletion after 48 and 72 h. The ratios were log₂ transformed. Raw data from Alipanah *et al.* (2018). Myb nomenclature is taken from Rayko *et al.* (2010). (b) Domain organisation of *PtPSR*. CD, conserved domain; NLS, nuclear localisation signal. (c) Unrooted maximum likelihood (ML) tree based on a protein alignment of *PtPSR* and related class I *SHAQKYF* TFs. The overall topologies for ML and Neighbour-Joining (NJ) trees are the same. Black circles indicate nodes supported by ML and NJ bootstrap values (1000× sampling, > 60%). (d) 100% conserved *PtPSR* recognition motif (expect value (e) = 4.8 × 10⁻⁶¹) based on the promoter sequence of 84 P-stress-inducible genes (Supporting Information Fig. S1) analysed with MEME (Bailey *et al.*, 2006).

in several diatoms and was identified as a putative CC domain using both COILS and Parcoil2 CC prediction programs (Lupas *et al.*, 1991; McDonnell *et al.*, 2006). Similar CC domains are found in Myb-CC TFs in plants including PHR1 in Arabidopsis. However, no clear function could be identified for the CDs. A phylogenetic analysis of the Myb domain indicated that Subgroup I *SHAQKYF*-like Mybs are distributed throughout stramenopiles (Fig. 1c).

In order to identify genes exhibiting a P limitation-specific transcript response, we collected publicly available transcriptome datasets for response to silicon limitation (Sapriel *et al.*, 2009), cadmium exposure (Brembu *et al.*, 2011), N limitation (Alipanah *et al.*, 2015) and P limitation (Alipanah *et al.*, 2018). The promoter regions of the 84 genes strongest induced by 72 h of P stress in the dataset from Alipanah *et al.* (2018), but little affected or not in the other datasets (Fig. S3) were analysed for motifs using the MEME web server (Bailey *et al.*, 2006). Our analysis revealed a highly conserved motif containing 8-bp sequence 5'-

YGAATCTH-3' (Fig. 1d) within 81 of these genes, with up to six copies of the motif in the promoter region of a single gene, such as the alkaline phosphatase AP1 *Phatr2_49678* (Fig. 2). This enriched motif was similar to the recognition motifs of Arabidopsis P-stress-inducible Myb TFs, HYPERSENSITIVITY TO LOW PHOSPHATE-ELICITED PRIMARY ROOT SHORTENING1 (HRS1, At1g13300, *P*-value: 1.1 × e⁻⁰²) and its homologues HRS1 HOMOLOG2 (HHO2, At1g68670, *P*-value: 4.7 × e⁻⁰⁴) and HRS1 HOMOLOG6 (HHO6, At1g49560, *P*-value: 2.6 × e⁻⁰⁴) (Fig. S4), suggesting that this could be the recognition motif for PtPSR, which itself has four of these conserved motifs in its promoter. Transcription factor binding sites are known to be conserved through evolution in both plants and animals (Nitta *et al.*, 2015).

We performed an EMSA analysis to verify the binding of recombinant PtPSR to the predicted recognition motif using IRDye-labelled DNA probes containing three GAATCT motifs (Fig. S5a). PtPSR binds specifically to the GAATCT sequences,
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Strongly P stress-inducible genes contain *PtPSR* recognition motifs. Eighty-one of the 84 strongest upregulated genes under P stress contained putative *PtPSR* recognition motifs in promoter region in *Phaeodactylum tricornutum*. Transcriptome data were derived from microarray data (Sapriel *et al.*, 2009; Brembu *et al.*, 2011; Alipanah *et al.*, 2015; Alipanah *et al.*, 2018). (O), number of *PtPSR* recognition motif within the promoter region of the gene; (-), genes without *PtPSR* recognition motif; green asterisks, genes validated by qRT-PCR.</figcaption></figure>

as signal was lost from the PtPSR/DNA complex when the unla-
belled competitor DNA probe was added but not when an unla-
belled DNA probe with mutated motifs was added (Fig. S5b).
Also, the unrelated protein A. thaliana NSP1 (Kong *et al.*, 2012),
expressed and purified under similar conditions as PtPSR, did
not bind the probes. For some reason, PtPSR bound to the probe
did not migrate into the native gel, while migration of PtPSR was
not prevented on SDS-PAGE (Fig. S5c).

Generation of *ptpsr* mutants

*ptpsr* mutant cell lines were generated using CRISPR/Cas9-based
gene editing. Two sgRNAs were designed targeting the N-termi-
nus between the CD1 and Myb domains (PAM1) and the CD2
domain (PAM2), respectively (Fig. 3a). Among the transfor-
mants obtained, two mutants for each target site were further
analysed. Both of the PAM1 mutants, *ptpsr* 1_3.4 and *ptpsr*
1_3.10, carried large deletions in allele 1 and rearrangements in
allele 2, resulting in a complete loss of the Myb DNA-binding
domain (Figs 3a,b, S6). None of the PAM1 lines harboured
wild-type alleles. A 504 bp deletion and 2 bp insertion in *ptpsr*
1_3.4 introduced a premature stop codon due to out-of-frame
InDels. A 537 bp in-frame deletion of *ptpsr* 1_3.10 resulted in
deletion of the Myb domain while all other CDs remained intact
(Fig. 3c). To test for off-target effects, loci showing the highest
probability for off-target by CRISPR/Cas9 (4 bp mismatch
against sgRNA) were sequenced (Table S3). The sequencing data

showed no mutation at the predicted off-target regions (one for
each sgRNA) tested in *P. tricornutum* (data not shown). In the
PAM2 mutant *ptpsr 2_2.3*, editing of both alleles of *PtPSR*
resulted in the loss of CDs except CD1 and the Myb DNA-bind-
ing domain, due to a 14 bp deletion in allele 1 and a 1 bp inser-
tion in allele 2. *ptpsr 2_2.2* had a 14 bp deletion in allele 1
resulting in an out-of-frame mutation. However, allele 2 of *ptpsr
2_2.2* contained a point mutation in CD2, leading to an
exchange of a 100% conserved leucine to phenylalanine (L373F).
In conclusion, the *PtPSR* PAM2 mutants had an active and prob-
ably functional Myb DNA-binding domain, but lacked CD3
and CD4, whereas the *PtPSR* PAM1 mutants exhibited an inac-
tive Myb DNA-binding domain.

Effect of P limitation on *ptpsr* mutants

*ptpsr* mutants and PtWT were cultivated under P-deplete (−P)
and replete (+P) conditions. Cell growth and cellular P were
monitored to observe the effect of mutations in the *PtPSR* gene.
AP activity and the dynamic regulation of P-stress response genes
were analysed.

**P household and P transport in ptpsr mutants** Cell growth and specific growth rate (μ) presented in Fig. 4(a) and 4(b), respectively, showed no significant difference between *ptpsr* mutants and PtWT under +P conditions. After 48 h of −P cultivation, all mutant lines showed reduced μ compared with PtWT; however,
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

20 bp target site 2 PAM2

PtWT PtPSR: 5' AGAAAAAGAGAAAAAATTCTCCGTTGGGGACGTCCATGAGTTATC-TCATGGGCCTTTTTTTCTCGTTGCGGCAACAACTG 3'

PtPSR 2_2.3 A1: AGAAAAAGAGAAAAAATTCTCCGTTGGGGACGTCCATGC----------------CTTTTTTTCTCGTTGCGGCAACAACTG -14 bp
PtPSR 2_2.3 A2: AGAAAAAGAGAAAAAATTCTCCGTTGGGGACGTCCATGAGTTATCCTCATGGGCCTTTTTTTCTCGTTGCGGCAACAACTG +1 bp

PtPSR 2_2.2 A1: AGAAAAAGAGAAAAAATTCTCCGTTGGGGACGTCCATGC----------------CTTTTTTTCTCGTTGCGGCAACAACTG -14 bp
PtPSR 2_2.2 A2: AGAAAAAGAGAAAAAATTCTCCGTTGGGGACGTCCATGAGTTATT-TCATGGGCCTTTTTTTCTCGTTGCGGCAACAACTG C>T

(c)

<figure><img src="image_8.png" /></figure>

**Fig. 3** Alignment of *Phaeodactylum tricornutum* wild-type (PtWT) and *ptpsr* mutant lines. (a) Representation of putative conserved domains in the *PtPSR* protein in comparison to designed mutants. The block size of the conserved domain does not represent the actual size of conserved domains. PAM sites and results of mutants are indicated for the specific mutants. (b) Partial gene sequence alignment of PtWT *PtPSR* compared with the *PtPSR* mutants (*ptpsr* 1_3.4, *ptpsr* 1_3.10, *ptpsr* 2_2.2, *ptpsr* 2_2.3). The 20-bp target sites 1 and 2 are indicated in red and green, respectively, with their corresponding PAM sequence in blue. PAM site 1 (CCT) refer to the reverse strand. The size of each deletion/insertion and the name of the allele are indicated at the right and left, respectively. Rearrangements in allele 2 of the *PtPSR* *PAM1* mutant lines were identified by high throughput sequencing (*). For a more detailed presentation of the rearrangements see Supporting Information Fig. S6. (c) PCR-amplified DNA fragments of the target sites.
µ was significantly reduced only in *ptpsr 1_3.4* and *1_3.10* (Fig. 4b). Cellular P (cP) was reduced in all mutant lines except *ptpsr 2_2.2* after 48 h in +P conditions (Fig. 4c). This trend was strengthened by –P cultivation; all mutant lines (except *ptpsr 1_3.10*) showed significantly reduced cP after 24 and 48 h (Fig. 4c). The cP content of *ptpsr 1_3.4* under –P conditions was 1/7 of PtWT cells at 48 h. Dissolved P (dP) in the medium showed variation between PtWT and mutants under +P and –P conditions, but no clear pattern was observed (Fig. 4d).

An expression analysis was performed on *PtPSR* in PtWT and the mutant lines, using PCR primers that were not affected by the mutations. *PtPSR* expression in PtWT was quickly induced by –P conditions. After 2 h, *PtPSR* expression in PtWT was 10-fold higher in –P compared with +P cultures (Fig. 5a). *PtPSR* expression in the mutant lines in –P conditions was moderately affected compared with PtWT –P conditions; however, at 48 h *PtPSR* was downregulated in *ptpsr 2_2.3* and *ptpsr 1_3.4* compared with PtWT –P conditions (Fig. 5b). To further investigate the transcriptional effect of PtPSR inactivation, we analysed several genes belonging to the P-stress response-specific gene set that

carried the 100% conserved PtPSR core recognition motif (GAATCT) in their promoters (Figs 2, S3). As shown in Fig. 5(a), all genes displayed P limitation-induced expression in PtWT at the three time points analysed.

**Alkaline phosphatase enzyme activity and phosphomonoesterase gene expression** The survey of P starvation-induced genes containing GAATCT motifs included several phosphatases and genes of lipid metabolism, all of which were upregulated in –P conditions in wild-type (Fig. 5a). Four genes involved in phosphomonoester degradation and AP activity were analysed: *API* (*Phatr2_49678*), *atypical PhoA* (*atyPhoA*, *Phatr2_47869*) (Li *et al.*, 2018; Lin *et al.*, 2015), *phosphomonoesterase* (*PME*, *Phatr2_45757*) and *PhoD* (*Phatr2_39432*). These four genes showed a strong gradual increase in their gene expression in PtWT cells during cultivation under P limitation compared with +P conditions (Figs 5a, S7a). By contrast, expression of these genes in the mutant lines was generally downregulated in –P cultures at all time points compared with PtWT (Fig. 5c–f). *ptpsr 1_3.4* showed the lowest AP activity and highest downregulation

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 4** Growth, cellular and dissolved phosphorus of *Phaeodactylum tricornutum* wild-type (PtWT) and *ptpsr* mutants in –P and +P conditions. (a) Time analysis of cell count (cells ml⁻¹) of +P (closed symbols) and –P (open symbols) cell lines. (b) Specific growth rate µ (d⁻¹). (c) Cellular P (pg P per cell) in +P and –P conditions. (d) Dissolved P (µM) after 24 and 48 h of cultivation under +P and –P conditions. Asterisks indicate significant difference (*, P < 0.05) compared with PtWT in the same conditions. Data are expressed as means ± SD (n = 4).</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Fig. 5** Relative mRNA expression of P-stressed induced genes in *Phaeodactylum tricornutum* wild-type (PtWT) and *ptpsr* mutants under phosphate-deplete conditions. (a) The histograms show fold change (log₂ transformed) of PtWT after 2, 24 or 48 h of growth under phosphate-deplete conditions compared with 2 h growth under phosphate-replete conditions. (b–m) All PtPSR mutants (*ptpsr* 1_3.4, *ptpsr* 1_3.10, *ptpsr* 2_2.2, *ptpsr* 2_2.3) compared with PtWT cells at same time points all under phosphate-deplete conditions. Statistically significant upregulation (*) relates to the *ptpsr* mutants compared with PtWT at the same time point and is based on ANOVA test. Each data point represents the mean value (n = 4) and error bars represent 95% confidence intervals. AP1, alkaline phosphatase 1; atyPhoA, atypical PhoA; ENTPPL, phosphoethanolamine phospholyase; GDPD1/2, glycerophosphoryl diester phosphodiesterases 1/2; PhoD, alkaline phosphatase D; PME, phosphomonoesterase.</figcaption></figure>
of genes related to phosphomonoester degradation compared with PtWT under P limitation. Therefore, the most significant effects on the regulation of PME- and AP-encoding genes were observed in mutants with an impaired Myb DNA-binding domain and CC domain.

AP external (eAP) and cellular (cAP) enzyme activities were detected in the mutant lines and correlated to the expression levels of genes involved in phosphomonoester degradation. At +P conditions eAP and cAP enzyme activities were very low in PtWT and below detection limit in all mutants (Table 1). Under P limitation, however, the AP activities in PtWT increased more than 220 times, whereas *ptpsr 1_3.4*, *ptpsr 1_3.10* and *ptpsr 2_2.3* showed no detectable activities and *ptpsr 2_2.2* significantly lower activities (Table 1).

We also analysed two genes encoding a phytase (myo-inositol hexaphosphate phosphohydrolase) (*Phatr2_47612*) and a phytase-like (*Phatr2_49693*) protein, which could be involved in phytate degradation and/or phosphate scavenging. The phytase was induced at all time points in PtWT, but showed significantly lower expression in mutants except for *ptpsr 2_2.2* (Fig. 5a,g). The phytase-like gene expression was strongly induced after 24 h in PtWT in P-deplete conditions (Fig. 5a). By contrast, expression of this gene in the *ptpsr* mutants was significantly reduced compared with PtWT at 24 and 48 h in P-deplete conditions (Fig. 5h). Inorganic P is taken up by the cell via P transporters. The low affinity Pi co-transporter (*Phatr2_47667*) was induced under P stress at all time points in PtWT and significantly reduced in *ptpsr* mutants compared with PtWT except *ptpsr 2_2.2* (Fig. 5i). Expression of the low affinity Pi co-transporter, Na+/P transporter gene (*Phatr2_40433*, Fig. 5j) followed the same trend but was less consistent.

**Lipid class regulation in ptpsr mutants** Two GDPDs (GDPD1 (*Phatr2_44900*) and GDPD2 (*Phatr2_32057*)) and one phosphoethanolamine phospholipase (ENTPPL, *Phatr2_52110*) are coupled to PL degradation. In PtWT cells there was a gradual

increase of expression for genes encoding PL-degrading enzymes under both replete and deplete conditions compared with 2 h +P conditions (Fig. 5a). *ptpsr* mutant lines showed a downregulation of these genes compared with PtWT under similar conditions (Figs 5k–m, S7k–m).

Lipid class profiling showed that *ptpsr 1_3.4* and *ptpsr 1_3.10* had significantly higher PL as well as total lipid content compared with PtWT under both P-replete and P-deplete conditions (Figs 6, S8). P limitation resulted in a complete loss of PLs in PtWT, while the P-free lipid classes digitalosyliacylglycerol (DGDG), diacylglyceride (DAG) and TAG increased at 72 h. PL levels in all *ptpsr* mutant lines except *ptpsr 2_2.2* were reduced 2–3 times under P limitation compared with nonlimited conditions but did not reach zero as in PtWT. P-free lipid classes in *ptpsr 1_3.4*, *1_3.10* and *2_2.3* mutants were comparatively higher than PtWT in P-limited conditions at 72 h.

## Discussion

PtPSR, a Myb-like TF playing an essential role in the conditional adaptation of microalgae to P limitation, has been identified and functionally characterised. Downstream genes containing the PtPSR recognition motif include genes encoding proteins linked to P scavenging/acquisition, PL remodelling, transport, carbon metabolism and cellular regulation (Fig. 2). EMSA analyses verified specific binding of PtPSR to the recognition motif. *ptpsr* knockout mutants with loss of the Myb DNA-binding domain showed generally reduced expression of P-stress response genes, low cP content, highly reduced AP activity and reduced growth under P limitation (Table 2; Figs 4, 5). The helical CD2 domain, mutated in *ptpsr 2_2.2*, is also important for the P-stress response in *P. tricornutum*, as reflected in lower gene expression levels of P-stress-inducible genes (Fig. 5).

## PtPSR identification and characterisation

The Myb-related P-stress-inducible TFs PHR1 in *A. thaliana* and PSR1 in *C. reinhardtii* contain within the Myb DNA-binding domain the conserved motif SHLQKYR, which is identical to the motif found in PtPSR and its orthologues in other diatoms. Myb proteins having the SHLQKYR motif are members of the class I SHAQKYF Myb proteins and are common in plants and algae. *A. thaliana* encodes more than 30 Mybs with the SHLQKYR motif, many of them known to be involved in regulating P starvation-induced genes. Phylogenetic studies here and in Rayko *et al.* (2010) showed a taxonomic distribution of the TFs related to P-stress response in green algae (*C. reinhardtii*), plants (*A. thaliana*) and stramenopiles that points to a common lineage of green algae-like SHAQKYF-like TFs (Fig. 1c). Rayko *et al.* (2010) showed through *in silico* studies that PtPSR belongs to the Myb-related proteins (MybR1) and is closely related to *T. pseudonana* *TpMyb1R_SHAQKYF5* and *P. tricornutum* *TpMyb1R_SHAQKYF8* (*Phatr2_50328*). In transcription studies of *T. pseudonana* and *N. oceanica*, the most significant upregulated TFs under P limitation were *TpMyb1R_SHAQKYF5* and the PtPSR-related TF *NannoCCMP1779_10770*, respectively

<table><caption>**Table 1** Alkaline phosphatase activity of *ptpsr* mutants and *Phaeodactylum tricornutum* wild-type (PtWT).</caption><thead><tr><th>PtPSR cell lines and conditions</th><th>eAP activity (mM pNP per 10<sup>6</sup> cells)</th><th>cAP activity (mM pNP per 10<sup>6</sup> cells)</th></tr></thead><tbody><tr><td>+P PtWT</td><td>0.8 ± 1.5</td><td>0.3 ± 0.4</td></tr><tr><td>2_2.2</td><td>nd*</td><td>nd*</td></tr><tr><td>2_2.3</td><td>nd*</td><td>nd*</td></tr><tr><td>1_3.4</td><td>nd*</td><td>nd*</td></tr><tr><td>1_3.10</td><td>nd*</td><td>nd*</td></tr><tr><td>-P PtWT</td><td>217.5 ± 27.1</td><td>71.1 ± 13.9</td></tr><tr><td>2_2.2</td><td>38.4 ± 0*</td><td>2.5 ± 0*</td></tr><tr><td>2_2.3</td><td>nd*</td><td>nd*</td></tr><tr><td>1_3.4</td><td>nd*</td><td>nd*</td></tr><tr><td>1_3.10</td><td>1.0 ± 0*</td><td>nd*</td></tr></tbody></table>

External and cellular alkaline phosphatase (eAP and cAP, respectively) assay was taken after 48 h of cultivation under both conditions (mM pNP/ 10⁶ cells). nd, not detected; *Significantly differentially regulated (P < 0.05) compared with PtWT. ‘±’ represents SD.
<figure><img src="image_14.png" /><figcaption>**Fig. 6** Lipid class content of *Phaeodactylum tricornutum* wild-type (PtWT) and *ptpsr* mutants under +P and –P conditions at 72 h. Targeted lipid class profiling is based on linear trap quadrupole (LTQ) identification and LC-MS/MS. (a) Phospholipids. (b) Nonphosphorus glycerolipids. DAG, diacylglyceride; DGDG, digalactosyldiacylglycerol; DGTA, diacylglycerolhydroxymethyltrimethylalanine; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyl diacylglycerols; TAG, triacylglycerides. An asterisk denotes significant (*P* < 0.05) P effect on lipid classes of *ptpsr* mutants compared with PtWT either in –P or +P conditions. The mean of four biological replicates from the third independent experiment (qPCR data showed the same gene expression), when cells were grown until 72 h, ± SE are shown.</figcaption></figure>

(Dyhrman *et al*., 2012; Mühlroth *et al*., 2017, Fig. S1). These findings indicate a similar regulation mechanism in *T. pseudonana* and *N. oceanica* under P stress as we found in *P. tricornutum*.

The MEME algorithm identified a highly enriched, conserved 8-bp sequence (5'-YGAATCTH-3') present in the promoters of almost all P-stress-induced genes analysed (Figs 1d, 2), with high similarity to the binding sites of Myb-related

TFs HRS1, HHO2 and HHO6 in Arabidopsis (Fig. S4; Rubio *et al*., 2001; Medici *et al*., 2015; Nagarajan *et al*., 2016). That the GAATCT motif is indeed a PtPSR recognition motif was confirmed by EMSA analyses (Fig. S5). Large-scale studies confirm that TFs with related DNA-binding domains tended to bind very similar DNA sequences, resulting in analogous regulation in distantly related species (Weirauch *et al*., 2014). Both HHO2 and HRS1 were shown in
<table><caption>**Table 2** Characteristics of *PtPSR* mutants and *Phaeodactylum tricornutum* wild-type (PtWT) under P limitation.</caption><thead><tr><th>Mutant line vs PtWT under P limitation</th><th>PtPSR 1_3.4</th><th>PtPSR 1_3.10</th><th>PtPSR 2_2.3</th><th>PtPSR 2_2.2</th></tr></thead><tbody><tr><td>Myb DNA-binding domain</td><td>Inactive</td><td>Inactive</td><td>Intact</td><td>Intact</td></tr><tr><td>Allele 1: CD2-CD4</td><td>X</td><td>√</td><td>X</td><td>X</td></tr><tr><td>Allele2: CD2-CD4</td><td>X</td><td>X</td><td>X</td><td>√(PM in CD2)</td></tr><tr><td>Specific growth rate (% of PtWT)</td><td>80%</td><td>74%</td><td>87%</td><td>87%</td></tr><tr><td>Cellular phosphorus (% of PtWT)</td><td>22%</td><td>22%</td><td>22%</td><td>110%</td></tr><tr><td>AP activity (% of PtWT)</td><td>nd</td><td>nd</td><td>nd</td><td>4-17.5%</td></tr><tr><td>Gene expression related to AP activity (% of PtWT)</td><td>0.2-2%</td><td>0.1-2%</td><td>0.1-2%</td><td>4-13%</td></tr><tr><td>Gene expression related to phospholipid degradation (% of PtWT)</td><td>GDPDs 0.3-3%, ENTPPL 2%</td><td>GDPDs 0.3-1%, ENTPPL 4%</td><td>GDPDs 1-2%, ENTPPL 1%</td><td>GDPDs 1-13%, ENTPPL 12%</td></tr><tr><td>Gene expression of low affinity P<sub>i</sub> transporter (% of PtWT)</td><td>11%</td><td>27%</td><td>9%</td><td>67%</td></tr><tr><td>Gene expression of high affinity Na<sup>+</sup>/P<sub>i</sub> transporter (% of PtWT)</td><td>80%</td><td>106%</td><td>61%</td><td>200%</td></tr></tbody></table>

Percentage of enzymatic activity or gene expression of the mutant lines compared with the PtWT under P limitation. AP, alkaline phosphatase; CD, conserved domain; ENTPPL, phosphoethanolamine phospholyase; GBDP, glycerophosphoryldiester phosphodiesterase; nd, not detected; PM, point mutation. Presence (√) or absence (X) of conserved domains (CD2-CD4) in the encoded proteins. Gene names in italics.

microarray analyses and functional studies to be involved in the regulation of P<sub>i</sub> acquisition and signalling under P limitation (Misson *et al.*, 2005; Medici *et al.*, 2015; Nagarajan *et al.*, 2016). The *PtPSR* promoter contains four PtPSR recognition motifs; thus, *PtPSR* expression may be regulated via a positive feedback loop. The reduced *PtPSR* gene expression level observed in *ptpsr* knockout mutants compared with PtWT support this view. However, *PtPSR* expression after 2 h of P limitation is more induced in the mutant lines compared with PtWT, suggesting that other TFs are involved in the initial induction of *PtPSR* expression during P limitation.

## PtPSR mutations and their trends

The roles of the CDs of PtPSR are currently unknown. PtPSR CD1, CD2 and CD4 domains showed low similarities to CDs of other Myb-related TFs. CD3 contained a putative CC motif that was also seen in PHR1 and PSR1 and suggested to be involved in protein-protein interactions (Rayko *et al.*, 2010).

The analyses of *ptpsr* mutants indicate that PtPSR acts as a positive regulator of P-stress-inducible genes. Mutants defective in the Myb DNA-binding domain (*ptpsr 1_3.4* and *ptpsr 1_3.10*) were unable to induce these P-stress-regulated genes in a similar way as in the PtWT. Mutants where the CD2 domain was mutated (*ptpsr 2_2.2* and *ptpsr 2_2.3*, intact Myb DNA-binding domain) showed the same responses, but had a slightly reduced transcript induction for some target genes. The truncated PtPSR *1_3.4* and *1_3.10* protein, with loss of Myb DNA-binding domain function, was likely unable to enter the nucleus due to loss of the NLS and to bind to the promoter sequences, reducing activation of P-stress-inducible genes (Figs 3, 4). *ptpsr 1_3.4* had a severe reduction of growth rate and cP content compared with PtWT. All mutant lines, except *ptpsr 2_2.2*, showed lower cP content compared with PtWT under both -P and +P conditions, indicating that PtPSR is central in regulating P acquisition and scavenging both under normal and P-limited growth conditions (Figs 2, 4). In the *ptpsr 1_3.10* mutant line, which lacks the Myb DNA-binding domain but has intact CDs, most of the P-

stress-inducible genes showed significant downregulation compared with wild-type, particularly at 24 and 48 h. The *ptpsr 2_2.3* mutant line has an intact Myb DNA-binding domain and CD1, but no CD2-4. Therefore, the PtPSR protein expressed in *ptpsr 2_2.3* can bind to P-stress-inducible genes but may not be able to interact with other key regulatory proteins. Together these findings indicate that other TFs, such as PtMyb1R_SHAQKYF8, which has a CD2 domain similar to PtPSR, may partially compensate for the lack of PtPSR. The *ptpsr 2_2.2* mutant line underlined the importance of CD2, although its function is still unknown. *ptpsr 2_2.2* contains one complete copy of PtPSR with an L373F substitution in the CD2 domain and one copy with deletion of CD2-4, which led to reduction of AP activities and gene expression levels of P-stress response genes compared with PtWT under P limitation (Table 2; Fig. 5). The observed phenotypes of *ptpsr 2_2.2* indicated that the replacement of the conserved Leu with a Phe in the CD2 domain render it partially functional.

## P acquisition and scavenging in *ptpsr* mutants

Our findings showed rapid transcriptional induction of proteins involved in P acquisition, such as P<sub>i</sub> transporters, during -P cultivation. It was also seen that the complex regulation of P<sub>i</sub> transporters is partly regulated by PtPSR. Reduced cP content in *ptpsr* mutant lines indicated low P<sub>i</sub> supply, which supports a reduced intracellular and extracellular P<sub>i</sub> transport. The reduced cP may also explain the low cell division rate for some of the mutants (Fig. 4a). The low affinity P<sub>i</sub> co-transporter (*Phatr2_47667*) was induced by PtPSR under P stress. The postulated high affinity Na<sup>+</sup>/P<sub>i</sub> co-transporter (*Phatr2_40433*), related to human SLC34A1, showed variable differential regulation in the *ptpsr* mutant lines when compared with PtWT (Fig. 5i-j). In addition to PtPSR, other TFs are likely involved in regulation of P supply to the cell. Posttranscriptional regulation might also have a role, as shown for the low affinity Na<sup>+</sup>-dependent P<sub>i</sub> transporter DsSPT1 in the green alga *Dunaliella salina* (Moseley *et al.*, 2006; Li *et al.*, 2012).
We identified genes that were involved in AP activity regulated by PtPSR as an immediate response to low Pᵢ concentration. *API*, *atyPhoA*, *PME* and *PhoD*, encoding putative phosphomonoester degrading enzymes, showed strongly reduced transcript induction under P limitation in *ptpsr* mutant lines (Fig. 5c–f). In line with these results, the *ptpsr* mutant lines displayed strongly reduced or no AP activity compared with PtWT (Table 1).

AP activity is an essential survival mechanism under low Pᵢ in
several algae (Dyhrman, 2016). *In silico* studies of AP abundance
showed a wide range of algal species containing several AP
sequences per genome with similarities to identified bacterial AP
forms (Lin *et al.*, 2012). Our results show that *API*, *atyPhoA*,
*PME* and *PhoD* are regulated by PtPSR and are potentially
involved in eAP and cAP activities to degrade DOP under P limi-
tation. From plants it is known that SPX domain-containing pro-
teins control a set of processes for Pi homeostasis by fine tuning
Pi transportation and Pi sensing (Secco *et al.*, 2012; Wang,
2004). Whether the SPX proteins in diatoms have related func-
tions remains to be determined, but one gene encoding a SPX
protein (*Phatr2_47434*) may be regulated by PtPSR (Fig. S3).

Phospholipid degradation in *P. tricornutum* *ptpsr* mutants

Phospholipid degradation and betaine and TAG lipid synthesis are common P-stress responses in stramenopiles, and have been observed in *P. tricornutum* (Abida *et al.*, 2015; Cañavate *et al.*, 2017). The lipid profile of PtWT at 72 h of P limitation showed such common P-stress response as depletion of PL and increase in TAG, DAG and DGDG compared with PtWT grown under normal conditions (Fig. 6). However, the amount of DGTA, SQDG and MGDG were almost similar in -P and +P conditions, and could indicate that the lipid class remodelling was in its early stage in our study. Previous studies in *P. tricornutum* have shown a significant increase of P-free lipids classes such as SQDG and DGTA after 7–10 d of P limitation (Abida *et al.*, 2015; Cañavate *et al.*, 2017). These lipid classes are important for PL replacement and lipid droplet formation under nutrient stress (Lupette *et al.*, 2019). Our findings and previous studies have shown that the dynamics of lipid class changes in *P. tricornutum* under P stress are complex and have to be investigated in depth in future studies.

Our lipid analysis showed a higher level of PLs in the *ptpsr
1_3.4* and *1_3.10* mutants compared with PtWT under both P-
replete and P-deplete conditions, indicating reduced PL degrada-
tion. PLs are phosphodiesters and are degraded extracellularly and
intracellularly by GDPDs and lipases; not much information is
known about these enzymes in stramenopiles, but several putative
annotated lipases and GDPDs are transcriptionally induced
under P stress (Dyhrman *et al.*, 2012; Yamaguchi *et al.*, 2014;
Mühlroth *et al.*, 2017; Alipanah *et al.*, 2018). We have shown that
*ENTPPL*, *GDPD2* and *GDPD1* are under the regulation of
PtPSR (Fig. 5k–m). Extracellular GDPD2 may hydrolyse glyc-
erolphosphodiesters into alcohols and *sn*-glycerol-3-phosphates
that are further degraded to inorganic P, while GDPD1 (with
unknown compartmentalisation) could be involved in

intracellular PL degradation producing precursors for lipid accu-
mulation. ENTPPL degrades the PL biosynthesis intermediate
phosphoethanolamine to acetaldehyde and release Pᵢ. Membrane-
bound PL degradation has been suggested to be a part of the P-
stress-inducible PL-recycling scheme and P-acquiring mechanism
in stramenopiles (Mühlroth *et al.*, 2017; Alipanah *et al.*, 2018).
Our findings support the hypothesis that PL degradation is a P-
stress-inducible pathway that, in part, is regulated by PtPSR.

Previous studies have proposed that the PL-recycling scheme
includes degradation of PLs, reduction of PL biosynthesis and
synthesis of P-free lipid classes (Martin *et al.*, 2011; Cañavate
*et al.*, 2017; Mühlroth *et al.*, 2017). Whether PtPSR is involved
in the regulation of genes involved in TAG and betaine lipid
accumulation is still unclear. However, *ptpsr 1_3.4*, *1_3.10* and
*2_2.3* showed significantly increased TAG, galactolipid and
DGTA content compared with PtWT, indicating either PtPSR
regulation of genes involved in the synthesis of betaine lipid,
TAG and galactolipid classes or an indirect effect of reduced PL
degradation. Bioinformatic data showed that the betaine lipid
synthesis gene (*Phatr2_42872*) contains putative PtPSR recogni-
tion motifs (Fig. 2), supporting the idea that PtPSR may regulate
P-free lipid biosynthesis in later time points.

In summary, we have identified one of the master regulators of
specific P-stress responses in the diatom *P. tricornutum*, the Myb-
related TF PtPSR and its binding motif. Phylogenetic analysis
showed that PtPSR-like Mybs are evolutionarily conserved in
stramenopiles. Functional studies indicate a central role for
PtPSR in regulation of extra- and intracellular P acquisition,
scavenging and phospholipid remodelling during acclimation to
P-deplete conditions.

Acknowledgements

We sincerely thank Torfinn Sparstad, Trude Johansen and Kjersti
Andresen for excellent technical assistance. We would also like to
thank Valérie Gros for technical help with the lipid analysis.
Financial support from the HAVBRUK2 programme (project
267474/E40) of the Norwegian Research Council to AMB is
gratefully acknowledged. EM and JJ are supported by the French
National Research Agency (Oceanomics ANR-11-BTBR-0008,
GlycoAlps ANR-15-IDEX-02 and GRAL 10-LABX-0049).

Author contributions

PW, TB and AMB conceived the study. AKS and PW designed
target sequences and developed the mutant lines. AKS, AM and
LA performed experiments. JJ and EM performed lipid analyses.
RK and AKS performed EMSA experiments. AMB provided
resources. AKS and AM drafted the paper together with AMB,
PW and TB. All authors reviewed and approved the manuscript.

ORCID

Tore Brembu ID https://orcid.org/0000-0001-5622-8037
Per Winge ID https://orcid.org/0000-0003-4380-1951
References

Abida H, Dolch L-J, Mei C, Villanova V, Conte M, Block MA, Finazzi G, Bastien O, Tirichine L, Bowler C *et al*. 2015. Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in *Phaeodactylum tricornutum*. *Plant Physiology* 167: 118-136.

Alipanah L, Rohloff J, Winge P, Bones AM, Brembu T. 2015. Whole-cell response to nitrogen deprivation in the diatom *Phaeodactylum tricornutum*. *Journal of Experimental Botany* 66: 6281-6296.

Alipanah L, Winge P, Rohloff J, Najafi J, Brembu T, Bones AM. 2018. An integrated analysis of phosphorus depletion responses in the diatom *Phaeodactylum tricornutum*. *PLoS ONE* 13: e0193335.

Bailey TL, Williams N, Misleh C, Li WW. 2006. MEME: Discovering and analyzing DNA and protein sequence motifs. *Nucleic Acids Research* 34: W369–W373.

Baldwin DS. 2013. Organic phosphorus in the aquatic environment. *Environmental Chemistry* 10: 439–454.

Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A, Maheswari U, Martens C, Maumus F, Otillar RP *et al*. 2008. The Phaeodactylum genome reveals the dynamic nature and multi-lineage evolutionary history of diatom genomes. *Nature* 456: 239–244.

Brembu T, Jorstad M, Winge P, Valle KC, Bones AM. 2011. Genome-wide profiling of responses to cadmium in the diatom *Phaeodactylum tricornutum*. *Environmental Science and Technology* 45: 7640–7647.

Brembu T, Mühlroth A, Alipanah L, Bones AM. 2017. The effects of phosphorus limitation on carbon metabolism in diatoms. *Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences* 372: 20160406.

Cañavate JP, Armada I, Hachero-Cruzado I. 2017. Aspects of phosphorus physiology associated with phosphate-induced polar lipid remodelling in marine microalgae. *Journal of Plant Physiology* 214: 28–38.

Chiou T-J, Lin S-I. 2011. Signaling network in sensing phosphate availability in plants. *Annual Review of Plant Biology* 62: 185–206.

Cruz de Carvalho MH, Sun HX, Bowler C, Chua NH. 2016. Noncoding and coding transcriptome responses of a marine diatom to phosphate fluctuations. *New Phytologist* 210: 497–510.

Donald KM, Scanlan DJ, Carr NG, Mann NH, Joint I. 1997. Comparative phosphorus nutrition of the marine cyanobacterium *Synechococcus WH7803* and the marine diatom *Thalassiosira weissflogii*. *Journal of Plankton Research* 19: 1793–1813.

Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. 2010. MYB transcription factors in Arabidopsis. *Trends in Plant Science* 15: 573–581.

Dyhrman ST. 2016. Nutrients and their acquisition: phosphorus physiology in microalgae. In: Borowitzka M, Beardall J, Raven J, eds. *The physiology of microalgae*. Dordrecht, the Netherlands: Springer, 155–183.

Dyhrman ST, Jenkins BD, Rynearson TA, Saito MA, Mercier ML, Alexander H, Whitney LAP, Drzewianowski A, Bulygin VV, Bertrand EM *et al*. 2012. The transcriptome and proteome of the diatom *Thalassiosira pseudonana* reveal a diverse phosphorus stress response. *PLoS ONE* 7: e33768.

Elser JJ, Bracken MES, Cleland EE, Gruner DS, Harpole WS, Hillebrand H, Ngai JT, Seabloom EW, Shurin JB, Smith JE. 2007. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. *Ecology Letters* 10: 1135–1142.

Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C. 1999. Transformation of nonselectable reporter genes in marine diatoms. *Marine Biotechnology* 1: 239–251.

Feng TY, Yang ZK, Zheng JW, Xie Y, Li DW, Murugan SB, Yang WD, Liu JS, Li HY. 2015. Examination of metabolic responses to phosphorus limitation via proteomic analyses in the marine diatom *Phaeodactylum tricornutum*. *Scientific Reports* 5: 10373.

Flynn KJ, Öpik H, Syrett PJ. 1986. Localization of the alkaline phosphatase and 5'-nucleotidase activities of the diatom *Phaeodactylum tricornutum*. *Microbiology* 132: 289–298.

Hansen HP, Koroleff F. 1999. Determination of nutrients. In: Grasshoff K, Kremling K, Ehrhardt M, eds. *Methods of seawater analysis*, 3rd edn. Weinheim, Germany: Wiley-VCH, 159–228.

Hu J, Wang D, Li J, Jing G, Ning K, Xu J. 2014. Genome-wide identification of transcription factors and transcription-factor binding sites in oleaginous microalgae Nannochloropsis. *Scientific Reports* 4: 5454.

Jouhet J, Lupette J, Clerc O, Magneschi L, Bedhomme M, Collin S, Roy S, Maréchal E, Rébeillé F. 2017. LC-MS/MS versus TLC plus GC methods: consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation. *PLoS ONE* 12: e0182423.

Jyoti A, Kaushik S, Srivastava VK, Datta M, Kumar S, Yugandhar P, Kothari SL, Rai V, Jain A. 2019. The potential application of genome editing by using CRISPR/Cas9, and its engineered and ortholog variants for studying the transcription factors involved in the maintenance of phosphate homeostasis in model plants. *Seminars in Cell & Developmental Biology*. doi: 10.1016/j.semcdb.2019.03.010.

Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips APR, Noddings CM, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J *et al*. 2015. Designer diatom episomes delivered by bacterial conjugation. *Nature Communications* 6: 6925.

Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. *Molecular Biology and Evolution* 33: 1870–1874.

Kong XY, Kissen R, Bones AM. 2012. Characterization of recombinant nitrile-specifier proteins (NSPs) of *Arabidopsis thaliana*: dependency on Fe (II) ions and the effect of glucosinolate substrate and reaction conditions. *Phytochemistry* 84: 7–17.

Li SH, Xia BB, Zhang C, Cao J, Bai LH. 2012. Cloning and characterization of a phosphate transporter gene in *Dunaliella salina*. *Journal of Basic Microbiology* 52: 429–436.

Li T, Guo C, Zhang Y, Wang C, Lin X, Lin S. 2018. Identification and expression analysis of an atypical alkaline phosphatase in *Emiliania huxleyi*. *Frontiers in Microbiology* 9: 2156.

Lin X, Wang L, Shi X, Lin S. 2015. Rapidly diverging evolution of an atypical alkaline phosphatase (PhoA(aty)) in marine phytoplankton: insights from dinoflagellate alkaline phosphatases. *Frontiers in Microbiology* 6: 868.

Lin X, Zhang H, Cui Y, Lin S. 2012. High sequence variability, diverse subcellular localizations, and ecological implications of alkaline phosphatase in dinoflagellates and other eukaryotic phytoplankton. *Frontiers in Microbiology* 3: 235.

Lupas A, Van Dyke M, Stock J. 1991. Predicting coiled coils from protein sequences. *Science* 252: 1162–1164.

Lupette J, Jaussaud A, Seddiki K, Morabito C, Brugière S, Schaller H, Kuntz M, Putaux J-L, Jouneau P-H, Rébeillé F. 2019. The architecture of lipid droplets in the diatom *Phaeodactylum tricornutum*. *Algal Research* 38: 101415.

Martin P, Van Mooy BA, Heithoff A, Dyhrman ST. 2011. Phosphorus supply drives rapid turnover of membrane phospholipids in the diatom *Thalassiosira pseudonana*. *ISME Journal* 5: 1057–1060.

Matthijs M, Fabris M, Broos S, Vyverman W, Goossens A. 2016. Profiling of the early nitrogen stress response in the diatom *Phaeodactylum tricornutum* reveals a novel family of RING-domain transcription factors. *Plant Physiology* 170: 489–498.

Matthijs M, Fabris M, Obata T, Foubert I, Franco-Zorrilla JM, Solano R, Fernie AR, Vyverman W, Goossens A. 2017. The transcription factor bZIP14 regulates the TCA cycle in the diatom *Phaeodactylum tricornutum*. *EMBO Journal* 36: 1559–1576.

McDonnell AV, Jiang T, Keating AE, Berger B. 2006. Paircoil2: improved prediction of coiled coils from sequence. *Bioinformatics* 22: 356–358.

Medici A, Marshall-Colon A, Ronzier E, Szponarski W, Wang R, Gojon A, Crawford NM, Ruffel S, Coruzzi GM, Krouk G. 2015. AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip. *Nature Communications* 6: 6274.

Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R, Ortet P, Creff A, Somerville S, Rolland N *et al*. 2005. A genome-wide transcriptional analysis using *Arabidopsis thaliana* Affymetrix gene chips determined plant
responses to phosphate deprivation. *Proceedings of the National Academy of Sciences, USA* 102: 11934–11939.

Moseley JL, Chang CW, Grossman AR. 2006. Genome-based approaches to understanding phosphorus deprivation responses and PSR1 control in *Chlamydomonas reinhardtii*. *Eukaryotic Cell* 5: 26–44.

Mühlroth A, Li K, Røkke G, Winge P, Olsen Y, Hohmann-Marriott MF, Vadstein O, Bones AM. 2013. Pathways of lipid metabolism in marine algae, co-expression network, bottlenecks and candidate genes for enhanced production of EPA and DHA in species of chromista. *Marine Drugs* 11: 4662–4697.

Mühlroth A, Winge P, El Assimi A, Jouhet J, Maréchal E, Hohmann-Marriott MF, Vadstein O, Bones AM. 2017. Mechanisms of phosphorus acquisition and lipid class remodelling under P limitation in a marine microalga. *Plant Physiology* 175: 1543–1559.

Nagarajan VK, Satheesh V, Poling MD, Raghothama KG, Jain A. 2016. Arabidopsis MYB-Related HHO2 exerts a regulatory influence on a subset of root traits and genes governing phosphate homeostasis. *Plant and Cell Physiology* 57: 1142–1152.

Norwegian Standard NS-EN ISO 6878. 2004. *Water quality – determination of phosphorus – ammonium molybdate spectrometric method*. Oslo, Norway: Norwegian Standards Association.

Nakata M, Ohme-Takagi M. 2014. Electrophoresis mobility shift assay. *Bio-Protocol* 4: e1099.

Nitta KR, Jolma A, Yin Y, Morgunova E, Kivioja T, Akhtar J, Hens K, Toivonen J, Deplancke B, Furlong EEM. 2015. Conservation of transcription factor binding specificities across 600 million years of bilateria evolution. *eLife* 4: e04837.

Nymark M, Sharma A, Hafskjold M, Sparstad T, Bones A, Winge P. 2017. CRISPR/Cas9 gene editing in the marine diatom *Phaeodactylum tricornutum*. *Bio-Protocol* 7: e2442.

Nymark M, Sharma AK, Sparstad T, Bones AM, Winge P. 2016. A CRISPR/Cas9 system adapted for gene editing in marine algae. *Scientific Reports* 6: 24951.

Nymark M, Valle KC, Brembu T, Hancke K, Winge P, Andresen K, Johnsen G, Bones AM. 2009. An integrated analysis of molecular acclimation to high light in the marine diatom *Phaeodactylum tricornutum*. *PLoS ONE* 4: e7743.

Quisel JD, Wykoff DD, Grossman AR. 1996. Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived *Chlamydomonas reinhardtii*. *Plant Physiology* 111: 839–848.

Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. *Neuroscience Letters* 339: 62–66.

Rayko E, Maumus F, Maheswari U, Jabbari K, Bowler C. 2010. Transcription factor families inferred from genome sequences of photosynthetic stramenopiles. *New Phytologist* 188: 52–66.

Rubio V, Linhares F, Solano R, Martín AC, Iglesias J, Leyva A, Paz-Ares J. 2001. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. *Genes & Development* 15: 2122–2133.

Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM. 2009. Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data. *Nucleic Acids Research* 37: e45.

Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. *Molecular Biology and Evolution* 4: 406–425.

Sapriel G, Quinet M, Heijde M, Jourdren L, Tanty V, Luo G, Le Crom S, Lopez PJ. 2009. Genome-wide transcriptome analyses of silicon metabolism in *Phaeodactylum tricornutum* reveal the multilevel regulation of silicic acid transporters. *PLoS ONE* 4: e7458.

Sarthou G, Timmermans KR, Blain S, Tréguer P. 2005. Growth physiology and fate of diatoms in the ocean: a review. *Journal of Sea Research* 53: 25–42.

Secco D, Wang C, Arpat BA, Wang Z, Poirier Y, Tyerman SD, Wu P, Shou H, Whelan J. 2012. The emerging importance of the SPX domain-containing proteins in phosphate homeostasis. *New Phytologist* 193: 842–851.

Sharma AK, Nymark M, Sparstad T, Bones AM, Winge P. 2018. Transgene-free genome editing in marine algae by bacterial conjugation – comparison with biolistic CRISPR/Cas9 transformation. *Scientific Reports* 8: 14401.

Shimogawara K, Wykoff DD, Usuda H, Grossman AR. 1999. *Chlamydomonas reinhardtii* mutants abnormal in their responses to phosphorus deprivation. *Plant Physiology* 120: 685–694.

Slattery SS, Diamond A, Wang H, Therrien JA, Lant JT, Jazey T, Lee K, Klassen Z, Desgagné-Peníx I, Karas BJ *et al*. 2018. An expanded plasmid-based genetic toolbox enables Cas9 genome editing and stable maintenance of synthetic pathways in *Phaeodactylum tricornutum*. *ACS Synthetic Biology* 7: 328–338.

Sperschneider J, Catanzariti AM, Deboer K, Petre B, Gardiner DM, Singh KB, Dodds PN, Taylor JM. 2017. LOCALIZER: Subcellular localization prediction of both plant and effector proteins in the plant cell. *Scientific Reports* 7: 44598.

Theodorou ME, Elrifi IR, Turpin DH, Plaxton WC. 1991. Effects of phosphorus limitation on respiratory metabolism in the green alga *Selenastrum minutum*. *Plant Physiology* 95: 1089–1095.

Thiriet-Rupert S, Carrier G, Chénais B, Trottier C, Bougaran G, Cadoret JP, Schoefs B, Saint-Jean B. 2016. Transcription factors in microalgae: genome-wide prediction and comparative analysis. *BMC Genomics* 17: 282.

Van Mooy BAS, Fredricks HF, Pedler BE, Dyhrman ST, Karl DM, Koblížek M, Lomas MW, Mincer TJ, Moore LR, Moutin T *et al*. 2009. Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity. *Nature* 458: 69–72.

Vieler A, Wu G, Tsai C-H, Bullard B, Cornish AJ, Harvey C, Reca I-B, Thornburg C, Achawanantakun R, Buehl CJ. 2012. Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga *Nannochloropsis oceanica* CCMP1779. *PLoS Genetics* 8: e1003064.

Wang Y. 2004. Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis. *Plant Physiology* 135: 400–411.

Weirauch MT, Yang A, Albu M, Cote AG, Montenegro-Montero A, Drewe P, Najafabadi HS, Lambert SA, Mann I, Cook K *et al*. 2014. Determination and inference of eukaryotic transcription factor sequence specificity. *Cell* 158: 1431–1443.

Wykoff DD, Grossman AR, Weeks DP, Usuda H, Shimogawara K. 1999. Psr1, a nuclear localized protein that regulates phosphorus metabolism in *Chlamydomonas*. *Proceedings of the National Academy of Sciences, USA* 96: 15336–15341.

Xu J. 2006. Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances. *Molecular Ecology* 15: 1713–1731.

Yamaguchi H, Arisaka H, Otsuka N, Tomaru Y. 2014. Utilization of phosphate diesters by phosphodiesterase-producing marine diatoms. *Journal of Plankton Research* 36: 281–285.

## Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** Transcriptional induction of two PtPSR-like Myb TFs by P limitation in Nannochloropsis oceanica.

**Fig. S2** Phylogenetic relationship of diatom SHAQKYF-like motif-containing Mybs.

**Fig. S3** Regulated genes under stress conditions containing identified PtPSR recognition sites in promoter region in Phaeodactylum tricornutum.

**Fig. S4** Comparison of the PtPSR recognition motif to binding sites of Arabidopsis P-stress inducible Myb-related TFs.

**Fig. S5** EMSA analysis of PtPSR binding to the specific motif.
**Fig. S6** A schematic description of the deletions and rearrangements in ptpsr PAM1 mutants deduced from RNA-seq data.

**Fig. S7** Relative mRNA expression of P-stress induced genes in PtWT and ptpsr mutants under phosphate-replete conditions.

**Fig. S8** Total lipid content of PtWT and ptpsr mutants in +P and -P conditions at 72 h.

**Table S1** Primers for gDNA amplification of mutants and High-resolution melting (HRM) primers.

**Table S2** Primer pairs used for qRT-PCR.

**Table S3** Off-target locus for PAM1 and PAM2. Mismatch bases are underlined.

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the *New Phytologist* Central Office.

<figure><img src="image_15.png" /></figure>

## About New Phytologist

* *New Phytologist* is an electronic (online-only) journal owned by the New Phytologist Trust, a **not-for-profit organization** dedicated to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews and Tansley insights.

* Regular papers, Letters, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication 'as ready' via *Early View* – our average time to decision is <26 days. There are **no page or colour charges** and a PDF version will be provided for each article.

* The journal is available online at Wiley Online Library. Visit **www.newphytologist.com** to search the articles and register for table of contents email alerts.

* If you have any questions, do get in touch with Central Office (np-centraloffice@lancaster.ac.uk) or, if it is more convenient, our USA Office (np-usaoffice@lancaster.ac.uk)

* For submission instructions, subscription and all the latest information visit **www.newphytologist.com**