Hydrolysis of organophosphorus by diatom purple acid phosphatase and sequential regulation of cell metabolism

Abstract

Phosphorus (P) limitation affects phytoplankton growth and population size in aquatic systems, and consequently limits aquatic primary productivity. Plants have evolved a range of metabolic responses to cope with P limitation, such as accumulation of purple acid phosphatases (PAPs) to enhance acquisition of phosphates. However, it remains unknown whether algae have evolved a similar mechanism. In this study, we examined the role of PAPs in the model microalga Phaeodactylum tricornutum. Expression of PAP1 was enhanced in P. tricornutum cells grown on organophosphorus compared to inorganic phosphate. PAP1 overexpression improved cellular growth and biochemical composition in a growth-phase dependent manner. PAP1 promoted growth and photosynthesis during growth phases and reallocated carbon flux towards lipogenesis during the stationary phase. PAP1 was found to be localized in the endoplasmic reticulum and it orchestrated the expression of genes involved in key metabolic pathways and translocation of inorganic P (Pi), thereby improving energy use, reducing equivalents and antioxidant potential. RNAi of PAP1 induced expression of its homolog PAP2, thereby compensating for the Pi scavenging activity of PAP1. Our results demonstrate that PAP1 brings about sequential regulation of metabolism, and provide novel insights into algal phosphorus metabolism and aquatic primary productivity.

Phaeodactylum tricornutum, phosphate acquisition, phosphorus, Pi transport, purple acid phosphatase

Introduction

Phosphorous (P) is a key constituent of nucleic acids, membrane phospholipids, ATP, and other co-factors involved in extensive core energy metabolic pathways and its availability limits plant primary productivity. As a consequence of their direct assimilation by phytoplankton, inorganic phosphates (Pi) have been considered as the most preferred phosphate source for these organisms (Yang et al., 2014). P limitation in marine systems affects phytoplankton growth and the size of the algal population, and consequently limits ocean primary productivity. To cope with P limitation, plants have evolved a range of metabolic responses that enhance P acquisition from the environment (Ticconi et al., 2009). Among such mechanisms, the accumulation of purple acid phosphatases under P deficiency has been considered crucial.

Purple acid phosphatases (PAPs; EC 3.1.3.2), a distinct class of non-specific acid phosphatases belonging to the family of binuclear metallohydrolases, hydrolyse phosphate monoesters and anhydrates to liberate Pi (Schenk et al., 2013). PAPs possess various biological functions other than phosphorous metabolism, such as peroxidase activity (GmPAP3 and AtPAP17) (Del Pozo et al., 1999; Liao et al., 2003), cell wall regeneration (NtPAP12) (Kaida et al., 2008), and plant growth regulation and lipid production (AtPAP2) (Sun et al., 2012; Zhang et al., 2012). The enzymatic activities and substrate specificities of acid phosphatases vary significantly despite the sequence similarities among PAPs from various organisms. Because of the functional significance of PAPs in response to P deficiency, numerous studies have examined their potential for sequestering both extracellular organic and inorganic P (Tian et al., 2012). However, data on the substrate specificity of PAPs remain unclear despite their significant role in growth and metabolism under P deficiency. To date, there are few reports an PAPs in algae. In this study, we examined the role of PAPs in the model microalga species Phaeodactylum tricornutum and determined its mechanistic role in the hydrolysis of organophosphorus and subsequent sequential regulation of cellular metabolism.

Materials and methods

Algal strain and culture conditions

Phaeodactylum tricornutum Bohlin was procured from the Provasoli-Guillard National Center for Marine Algae and Microbiota, USA (No: CCMP-2561) and conserved in seawater with added f/2 medium without Na₂SiO₃.9H₂O (Guillard, 1975). The cultures were incubated at 20±0.5 °C in an artificial climate incubator under a 12/12 h photoperiod with an irradiance of 200 μmol photons m^–2 s^–1 and a 13-d cultivation cycle.

For different phosphorus conditions, the cultures were first centrifuged at 4337 g for 10 min and then subcultured in seawater with added f/2 medium (without Na₂SiO₃.9H₂O and NaH₂PO₄.H₂O) supplemented with 36.5 μM of either NaH₂PO₄.H₂O (PO₄ treatment), pyrophosphate (PPi treatment), p-nitrophenyl phosphate (pNPP treatment), or adenosine 5′-triphosphate disodium salt hydrate (ATP treatment) as the sole P source (all Sigma-Aldrich). The ATP treatment is referred to as dissolved organic P (DOP) conditions. A concentration of 36.5 μM was chosen because it represents P-sufficient conditions for growth. In addition, f/2 medium with no added phosphate was used (–P treatment).

PAP1 sequence analysis and bioinformatic predictions

The amino acid sequence of Arabidopsis thaliana AtPAP2 was retrieved from the TAIR database (http://www.arabidopsis.org/) (Lamesch et al., 2012) and used as a query to BLAST search for putative PAPs of P. tricornutum (http://genome.jgi.doe.gov/pages/blast-query.jsf?db=Phatr2). The deduced amino acid sequences of PAP1 proteins from various species were aligned using MUSCLE (Edgar, 2004), Tcoffee (Di Tommaso et al., 2011), and ESPript (Gouet et al., 1999). A phylogenetic tree based on these deduced sequences was constructed using a maximum-likelihood algorithm in MEGA7. The web-based prediction programs SMART (Letunic et al., 2006), SignalP (Almagro Armenteros et al., 2019), TargetP (Emanuelsson et al., 2007), and WoLF PSORT (Horton et al., 2007) were applied to predict the conserved domains and subcellular localization of PAP1.

Preferred substrates, co-factors, and inhibitors of recombinant P. tricornutum PAP1 from a Pichia pastoris expression system

To examine its characteristics, recombinant P. tricornutum PAP1 protein from a Pichia pastoris expression system was obtained as previously described by Hluska et al. (2017). The purified protein was characterized for its preferred substrates, potent co-factors, and inhibitors. Briefly, 100 μl of reaction mixture containing 50 mM sodium acetate buffer (pH 5.0), 5 mM MgCl₂, and 10 mM pNPP as standard substrate and 10 μg purified PAP1 protein were incubated at 37 °C for 30 min to release Pi. This released Pi was measured using the yellow vanadomolybdate method by adding 100 μl vanadate-molybdate reagent. All the samples and standards were measured at 470 nm absorbance. For the substrate assays, ATP, ADP, AMP, and PPi were chosen instead of pNPP as the substrate. For determination of co-factors, 10 mM of chloride salts with different cations (Mn, Co, Ni, Na, K) were added into the reaction mixture with pNPP as the standard substrate. For determination of inhibitors, five sodium salts with different anions (citrate, molybdate, phosphite, sulfate, carbonate) were added into the reaction mixture described above. In addition, purified proteins were detected by determination of phosphorus under different pH and temperature conditions following the yellow vanadomolybdate method.

Cloning, vector construction, and alga transformation

Total RNA from P. tricornutum harvested after 7 d cultivation was isolated using a E.Z.N.A. Plant RNA kit (Omega) and transcribed into cDNA using a PrimeScript^TM RT Reagent Kit with gDNA Eraser (Takara). For gene overexpression, a 1620-bp length cDNA of PAP1 (without the stop codon TAG) was PCR-amplified using the primers PAP1-f and PAP1-r (Supplementary Table S1). The resulting amplicon was cloned into the pHY18 expression vector (chloramphenicol-resistance expression system) containing the fucoxanthin chlorophyll a/c binding protein (fcpC) promoter, ACC motif, and fcpA terminator, which was constructed within our laboratory (Li et al., 2019). In addition, the coding sequence of c-Myc tag was fused between the target PAP1 gene and the fcpA terminator to generate PAP1 protein with c-Myc tag. For gene knockdown, RNAi vectors were constructed based on a previous study (De Riso et al., 2009). Two fragments (short, 224 bp, antisense orientation; long, 524 bp, sense orientation) of the full-length PtPAP1 cDNA were amplified and inserted into the pHY18 vector using a ClonExpress MultiS One Step Cloning Kit (Vazyme). The recombinant expression vectors were electroporated into P. tricornutum using a Gene Pulser Xcell System (Bio-Rad) as reported previously (Xue et al., 2017). The transformants were screened in f/2 liquid medium without antibiotics for at least 48 h, after which the cells were transferred on to f/2 solid medium with chloramphenicol (250 mg l⁻¹). After 1 month, surviving colonies were selected and inoculated into f/2 liquid medium with chloramphenicol (250 mg l⁻¹). Phaeodactylum tricornutum was sub-cultured once a week with chloramphenicol (250 mg l⁻¹) for preservation. Cells retrieved from preserved microalgae were firstly sub-cultured twice without antibiotics every 7 d and then further harvested at designed time-points for experimental analysis.

Verification of transformants using molecular approaches

To detect the integration of the expression cassette, putative transgenic P. tricornutum cultured under normal conditions were analysed by PCR. Genomic DNA was extracted from both the transgenic and wild-type (WT) cells using a HP Plant DNA Kit (Omega). The extracted genomic DNA was PCR-amplified to detect whether integration of the CAT gene existed in pHY18-PtPAP1 using the primers CAT-f and CAT-r (Supplementary Table S1).

qPCR was performed in PAP1-overexpression (OE) and WT lines under normal conditions to validate the expression levels following the method described by He et al. (2019). Specifically, real-time qPCR was carried out using an AceQ SYBR Green Master Mix (Vazyme) on a CFX96 Connect™ Real-Time PCR Detection System (Bio-Rad). Total RNA was prepared from transgenic and WT cells using an E.Z.N.A. Plant RNA Kit (Omega), and subsequently 20 μl of cDNA was synthesized using HiScript II Q RT SuperMix for qPCR (Vazyme) with 1 μg total RNA following the manufacturer’s instructions. qPCR was performed in eight-strip qPCR reaction tubes in a total reaction volume of 20 μl with AceQ qPCR SYBR Green Master Mix (Vazyme), distilled water, primers, and cDNA. Three programs, namely NormFinder, geNorm, and BestKeeper, were performed to explore the gene expression stability of the putative housekeeping genes β-actin (ACT), TATA box binding protein (TBP), Histone H4, 18S rRNA, translation elongation factor a subunit (EF1a), and ribosomal protein small subunit 30S (RPS). Among these, TBP and RPS were found to be the most stable and they were selected as the internal reference genes to normalize the transcriptional expression of the target genes in each sample. Standard curves were generated to check for PCR amplification efficiency over 90% and for correlation coefficients of 0.99 (Bustin et al., 2009). The relative expression of each gene was analysed according to the NRQ formula considering multi-internal reference genes and inter-run calibration algorithms (Hellemans et al., 2007). All primers used in this study are listed in Supplementary Table S1. At least three biological replicates were analysed for qPCR assay.

Protein expression in the PAP1 transgenic and WT lines was examined by western blot analysis. Briefly, P. tricornutum was collected for protein extraction using RIPA Lysis buffer (Beyotime) with phenylmethanesulfonyl fluoride. The protein concentration was determined using an Enhanced BCA Protein Assay Kit (Beyotime). The protein was then separated by SDS-PAGE and electro-transferred onto a carved polyvinylidene difluoride (PVDF) membrane. The electro-transferred PVDF was blocked using skimmed milk at 4 °C for at least 1 h. Anti-c-Myc antibody (1:3000; Abcam) and HRP-conjugated goat anti-rabbit antibody (1:5000; Cell Signaling Technology) were used as the primary and secondary antibodies, respectively, for incubation at 4 °C. The membrane was developed by using a chemiluminescent system. Endogenous β-actin protein was used as the internal reference. The APase activity of P. tricornutum under normal cultivation conditions was determined using an enzymatic assay containing 50 mM sodium acetate buffer (pH 5.0), 5 mM MgCl₂, and 10 mM pNPP as standard substrate with the released Pi being measured following the yellow vanadomolybdate method as described above.

Growth curve analysis and expression levels of PAPs in P. tricornutum

Growth curves of WT and transgenic P. tricornutum were measured at an initial cell density at 1×10⁶ cells ml^–1 by a direct-count method using a bright-line Neubauer hemocytometer under a light microscope. Measurements were taken every day and the specific growth rate of the algal cells (µ, d^–1) was calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle \mu (\mathrm{d}\mathrm{a}{\mathrm{y}}^{-1})=\frac{lnN-ln{N}_0}{t-t_0}}\end{array}}$$

where N₀ and N_t represent the initial cell number at time zero (t₀) and at time t, respectively, during the exponential (log) growth phase.

The PAP1 expression level was determined by qPCR on day 4 (mid-log phase) and on day 7 (early-stationary phase) of cultivation using RNA extracted from P. tricornutum under DOP conditions (i.e. ATP treatment) according to the qPCR method described above.

Determination of total cellular P content

For determination of total cellular P content, 500 ml of WT and transgenic P. tricornutum cultures grown under DOP conditions were centrifuged at day 4 and day 7 of cultivation. The cell samples were then transferred into ceramic crucibles and placed in a muffle furnace at 550 °C for 5 h until they had turned to ash. Then, 2 ml 2N HCl was added to the crucibles for P extraction at room temperature for at least 20 min. The suspensions were then centrifuged at 16 128 g for 5 min at 4 °C, and 100 µl of the supernatant was used to determine the P content according to the yellow vanadomolybdate method. For determination of the cellular inorganic P content, fresh samples of the P. tricornutum cultures without ashing at high temperature were used for the P extraction.

Chlorophyll contents and photosynthetic parameters

At 3-d intervals, 100 ml of WT and transgenic P. tricornutum cultures grown under DOP conditions were collected, centrifuged, and used for pigment determination. The cells were pulverized and 90% acetone was added to extract the pigments. Chlorophyl (Chl) a and c concentrations were determined spectrophotometrically using wavelengths of 630, 664, 750, and 759 nm and calculated by following equations of Jeffrey and Humphrey (1975). A PHYTO-PAM-II Multiple Excitation Wavelength Phytoplankton and Photosynthesis Analyser (Walz) was used to determine the chlorophyll fluorescence parameters of photosynthetic efficiency of PSII (F_v/F_m), electron transport rate (ETR), and non-photochemical quenching (NPQ), as described by Macedo et al. (2008).

Elemental analysis of cells

Cells of WT and transgenic P. tricornutum cultivated under organic P-sufficient conditions were harvested on day 4 and day 7 by centrifugation of 100-ml samples at 4 °C. The pelleted cells were washed three times with water and then oven-dried at 60 °C for 48 h. The cells were then ground into powder for carbon and nitrogen analysis using a FE-SEM S-4800 (Hitachi) according to the manufacturer’s instructions.

Determination of cellular metabolites

Carbohydrate content of 100-ml samples of WT and transgenic P. tricornutum was analysed by spectrophotometric assays following the method described previously by Dubois et al. (1956). Soluble proteins were extracted using RIPA Lysis Buffer (Beyotime) and quantified using an Enhanced BCA Protein Assay Kit (Beyotime). Total lipids were extracted following the method described by Bligh and Dyer (1959). The extracted lipids were dried by N₂ flow and then determined by gravimetric analysis. The relative neutral lipid content was measured by fluorometric determination using Nile Red. Samples of 1 ml of the cultures were mixed with 10 μl Nile Red solution (0.1 mg ml^–1 in acetone). Non-stained cultures and stained seawater medium were used as the auto-fluorescence and seawater-fluorescence controls, respectively. All measurements were made using a Synergy H1 Hybrid Multi-Mode Reader (Bio-Tek) at 530 nm excitation and 580 nm emission wavelengths. Final values were automatically calculated according to the cell density and reflected the relative neutral lipid content of the cells. Solid-phase extraction (SPE) through gravity fractionation was used to determine the contents of the lipid fractions (neutral lipids, phospholipids, and glycolipids) using pre-packed silica cartridges (Sep-Pak Silica 6 cc Vac Cartridge, 500 mg; Waters). ATP and NADH contents were measured using a Plant ATP and NADH ELISA kit (Shanghai BangYi Biotech) according to the manufacturer’s instructions. The NADPH content was determined using an Amplite^TM colorimetric NADPH assay kit (AAT Bioquest) according to the manufacturer’s instructions.

Expression analyses of key genes

qPCR was used to examine the effects of PAP1 overexpression of the following key genes involved in photosynthesis, triacylglycerols, galactoglycerolipids, Pi transport, energy transfer, and co-factors: POR1, POR2, ChlG, AtpC, PetJ, PetC, PsbO, PsbM, PsaA, GPAT, LPAT, PAP, DGAT, MGD1, MGD2, DGD1, DGD2, Pht1;1 like, PHF1 like, Atp1, ID, AOD, MD, ME, and G6PD. Samples of 100 ml of WT and transgenic P. tricornutum grown under DOP conditions were collected and centrifuged at 3-d intervals during the cultivation cycle for RNA extraction using E.Z.N.A. Plant RNA Kits (Omega). Then, 1 μg total RNA was transcribed into 20 μl cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme). qPCR was performed on a CFX 96 Connect™ Real-Time PCR Detection System (Bio-Rad) using AceQ qPCR SYBR Green Master Mix (Vazyme) with TBP and RPS as references genes All the primers used in this study are listed in Supplementary Table S1. At least three biological replicates were analysed for qPCR assay.

Analysis of antioxidant capacity

To evaluate the antioxidant responses of WT and transgenic P. tricornutum grown under DOP conditions, we measured the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). SOD was measured using a Total Superoxide Dismutase Assay Kit with WST-8, POD using a Cellular Glutathione Peroxidase Assay Kit with NADPH, and CAT using a Catalase Assay Kit (all Beyotime) according to the manufacturer’s instructions. The concentration of reactive oxygen species (ROS) was determined using the cell-permeable fluorogenic probe 2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime). For the assays, 1 mM DCFH-DA was added to P. tricornutum cultures at a ratio of 1:100 (v/v) and incubated at 37 °C for 30 min in darkness. The ROS concentration was determined according to the fluorescence intensity measured using a Synergy H1 Hybrid Multi-Mode Reader (Bio-Tek) at an excitation wavelength of 488 nm and an emission wavelength of 500–600 nm.

Confocal microscopy

Cells of WT and transgenic P. tricornutum after 7 d cultivation under DOP conditions were stained with Nile Red (0.1 mg ml^–1 in acetone) at ratio of 1:100 (v/v) and incubated for 20 min in the dark. Stained cells were observed under a Zeiss LSM510-META laser-scanning confocal microscope with excitation and emission wavelengths of 488 nm and 505–550 nm, respectively.

In vivo subcellular localization of PAP1

Subcellular localization of PAP1 was analysed by transiently expressing PtPAP1-EGFP in the protoplasts of Arabidopsis following the protocol described by Miao and Jiang (2007). The sequence of EGFP was fused with the 3´-terminal of PAP1 in the pBI221 vector by homologous recombination. The resultant recombinant vector was then transformed into Arabidopsis protoplasts. After incubation for 12 h, the transformed protoplasts were washed with PBS for detection of the protein using a confocal microscope.

RNAi validation of PAP functioning

The relative expression of PAP1, PAP2, and PAP3 was first determined in pap1-knockdown (KD) and WT lines grown under DOP conditions to validate the cellular PAP1 expression level using the qPCR method described above. In addition, APase activity, specific growth rate, and cell density of the KD and WT lines were determined as described above. To determine the cellular P and inorganic P contents, culture samples were centrifuged and ashed for determination using the yellow vanadomolybdate method as described above. Other parameters including contents of chlorophyll, carbohydrates, lipids, ATP, and ROS were also determined as described above.

Statistical analysis

All the experiments were performed at least three times. Statistical comparisons between the control and treatment groups were performed with Student’s t-test using GraphPad Prism 7.0. Data were also analysed using Kruskal–Wallis one-way ANOVA followed by Duncan’s test using GraphPad Prism 7.0.

Accession numbers

Sequences from genome and data used for this study can be found in GenBank data library or TAIR database by following accession numbers: XP_002180324.1, XP_002182961.1, XP_002176825.1, XP_009374738.1, XP_008387079.1, XP_009366751.1, XP_007204390.1, XP_008241255.1, XP_010101588.1, XP_002528725.1, CDP15400.1, AGL44399.1, XP_009623849.1, XP_009775250.1, XP_006341129.1, XP_004246886.1, XP_007027701.1, XP_003553594.2, KHN02236.1, XP_006650263.1, XP_011097894.1, EAY90700.1, XP_009762491.1, XP_009591701.1, XP_008867792.1, XP_008867791.1, XP_009838177.1, XP_012194718.1, XP_008604917.1, XP_009529776.1, XP_008904647.1, ETP45786.1, ETO76675.1, ETM47648.1, EWM24421.1, XP_002288709.1, NP_001313126.1, NP_001236677.1, NP_001149655.1, ACF75910.1, AAX20028.1, KMZ70543.1, AT1G52940.1, AT2G18130.1, AT3G46120.1, AT1G56360.1, AT4G36350.1, AT2G16430.1, AT2G27190.1, AT5G34850.1, AT3G07130.1, AT4G13700.1, AT2G32770.3, AT3G20500, AT3G52780.1, AT3G52810.1, AT3G52820.1, AT1G13900.1, AT2G03450.1, AT1G13750.1, AT4G24890.1, AT5G50400.1, AT3G17790.1, AT2G01880.1, AT1G25230.1, AT1G14700.1, AT2G01890.1, AT3G10150.1, AT5G57140.1, AT2G46880.1, and AT5G63140.1.

Results

Transcriptional activation of P. tricornutum PAPs is orchestrated by the type of P sources irrespective of PAP homologs

Given the existence of multiple PAP homologs in organisms, we performed a BLAST search of the predicted 29 AtPAPs (Li et al., 2002) using the amino acid sequence of AtPAP2 (AT1G13900.1) as the query against the P. tricornutum genome. Three putative PAP homologs were retrieved, and the one with the highest amino acid similarity was designated as PAP1 (Phatr2_12331, XP_002180324.1), whilst the other two were designated as PAP2 (Phatr2_2761, XP_002176825.1) and PAP3 (Phatr2_29551, XP_002182961.1). Three functional domains were predicted in PAP1, namely purple acid phosphatase, calcineurin-like phosphoesterase, and iron/zinc purple acid phosphatase-like (Supplementary Fig. S1A).

We then evaluated algal growth rates and the expression patterns of these PAPs in P. tricornutum grown with both organic (ATP and pNPP treatments) and inorganic P sources (PO₄ and PPi treatments), and without P (–P). As expected, the growth rates of cells cultivated in media with various phosphate sources was higher than in the P-deficit medium. Interestingly, inorganic P (Pi) resulted in significantly higher growth rates than that of the other P sources (Fig. 1A, B). This suggested that the different sources of P had an important impact on cellular physiological parameter, and hence we examined the expression patterns of PAPs under the different sources (Fig. 1C–E). The results revealed varying PAP expression patterns according to the different P sources. PAP1 expression significantly increased with time with the provision of P in the form of ATP and a similar pattern was seen with pNPP. A greatly reduced effect was seen with P-depletion (–P), whilst PPi and PO₄ did not influence PAP1 expression. PAP2 expression increased with time in the PO₄ and –P treatments, but was not affected by the provision of P as PPi, pNPP, or ATP. In the case of PAP3, expression greatly increased when P was supplied as pNPP and when the medium was P-deficient, whilst a moderate increase was observed for ATP and no effects were seen for PO₄ and PPi. Induction of PAP expression mediated by Pi depletion is known as a universal response to P depletion in plants (Liang et al., 2010; Tran et al., 2010b; Wang et al., 2011; Tian et al., 2012). The transcriptional responses of PAP1 under the different P sources demonstrated that P. tricornutum PAP1 exhibited a preference for organic P.

Fig. 1.

Impact of different P sources on growth and PAP expression in Phaeodactylum tricornutum. Cells were cultured in seawater with added f/2 medium (without NaH₂PO₄.H₂O) supplemented with 36.5 μM of either NaH₂PO₄.H₂O (PO₄), pyrophosphate (PPi), p-nitrophenyl phosphate (pNPP), or adenosine 5′-triphosphate disodium salt hydrate (ATP) as the sole P source; –P indicates f/2 medium without phosphorus. (A) Specific growth rate during the log phase. (B) Cell density on day 7. (c–e) Relative transcript levels during the cultivation cycle of (C) PAP1, (d) PAP2, and (E) PAP3. Expression is relative that measured on day 1. TBP and RPS served as the internal references. Data are means (±SD), n≥3. Different letters indicate significant differences as determined using Kruskal–Wallis one-way ANOVA followed by Duncan’s test (P<0.05).

Distinct enzyme properties of P. tricornutum PAP1

Typical functional motifs conserved in plants were predicted in the algal PAP1, with slight amino acid variations (Supplementary Fig. S2, Supplementary Table S2). In addition, examination of phylogenetic relationships demonstrated a high sequence similarity with the diatom Thalassiosira pseudonana (Supplementary Fig. S3A). Another phylogenetic analysis showed that the diatom PAPs belonged to Group I, which included Arabidopsis with low similarity (Supplementary Fig. S3B). Hence, although P. tricornutum PAP1 possessed typical plant-related functional motifs, it was not very close to plants, especially the model plant Arabidopsis (Supplementary Fig. S2). Our observation of significantly induced PAP1 expression by organic P (ATP) relative to P-depletion lead us to characterize the biochemical properties of PAP1, and hence we expressed and purified codon-optimized P. tricornutum PAP1 as a recombinant protein from Pichia pastoris and characterized its enzyme properties in vitro. Given the differing substrate preferences and their ability to influence PAP1 transcription (Fig. 1C), we sought to characterize PAP1 under a wide range of substrates, co-factors, inhibitors, temperatures, and pH. Although the results indicated broad organic P substrate specificity, ATP followed by pNPP were found to be the most preferred, and then ADP and AMP (Fig. 2A), which is in contrast to the reported broad organic- and inorganic-P substrate specificity of PAPs in other organisms (Wang et al., 2011). Given the regulatory role of metal co-factors in phosphatase activity and that different co-factors are required for different phosphatases (Bhadouria et al., 2017), we investigated the effects of various metals on PAP1 activity. We found that APase activity was significantly higher when Co²⁺ was provided compared with other co-factors (Fig. 2B). We then investigated the sensitivity of PAP1 to known inhibitors and found that its activity was significantly reduced by molybdate and phosphite (Fig. 2C), and intriguingly that it was comparatively less sensitive to EDTA, citrate, carbonate, and sulfate). Next, we examined the effects of temperature and pH when P was supplied as either ATP or pNPP, and found that enzymatic activity was highest at pH 4.5 and that temperature optima occurred at 20 °C and 37.5 °C (Fig. 2D, E). These resultant enzymatic analyses characterized the optimum substrate, cofactors, temperature and pH.

Fig. 2.

Biochemical characteristics of purified Phaeodactylum tricornutum PAP1 protein. (A) Relative APase activity of PAP1 with a range of different substrates. The data are relative to the value measured for pNPP, which was set as 1. (B) Relative APase activity in the presence of a range of different metal co-factors. The data are relative to the value measured for Co, which was set as 1. P was provided as 10 mM pNPP. (C) Relative APase activity in the presence of a range of different inhibitors. The data are relative to the value measured without an inhibitor, which was set as 1. P was provided as 10 mM pNPP. (D) Relative APase activity under a range of pH values with either ATP or pNPP as the sole P substrate. The data are relative to the value measured at pH 4 with pNPP. (E) Relative APase activity under a range of different temperatures with either ATP or pNPP as the sole P substrate. (F) Variation in relative APase activity over time with either ATP or pNPP as the sole P substrate. (G) Variation of total P and inorganic P concentrations in the medium over time with ATP as the sole substrate. Data are means (±SD), n≥3. Different letters indicate significant differences as determined using Kruskal–Wallis one-way ANOVA followed by Duncan’s test (P<0.05).

In order to examine long-term APase activity under the provision of ATP as the substrate, we measured enzymatic activity over a 96-h period. As expected, similar APase activity was observed between ATP and pNPP as the substrate (Fig. 2F). When ATP was provided as the P source, the concentration of inorganic P in the medium increased (Fig. 2G), implying that the organic P was degraded into inorganic P, demonstrating that PAP1 preferred ATP.

PAP1 overexpression enhances cellular physiological activity regardless of the organic P source

Previous studies have been conducted to determine the role of PAPs in P-scavenging in plants, particularly under conditions of Pi deprivation (Wang et al., 2011; Mehra et al., 2017; Kong et al., 2018); however, characterization of PAPs remains rare in microalgae. Hence, we overexpressed PAP1 under the control of the fucoxanthin chlorophyll a/c binding protein (fcpC) promoter and the fcpA terminator, with an omega leader sequence and an ACC motif fused between the promoter and the PAP1 region in order to enhance PAP1 translation in host cells (Supplementary Fig. S1B). c-Myc tag was fused to the end of the PAP1 region to verify the protein overexpression by western blotting. Several independent lines (>10) were obtained and examined for growth and physiological properties, and results from two representative overexpression lines, OE-1 and OE-2, are presented. The integration of PAP1 into the host genome was detected using genomic PCR. A 0.6-kb amplicon of the chloramphenicol resistance gene CAT region was detected in the OE lines, but was absent in the wild-type (WT) (Supplementary Fig. S4A). The relative transcript abundance of PAP1 determined by qPCR was significantly higher in the OE-1 and OE-2 lines compared to the WT (Supplementary Fig. S4B). Western blotting showed a specific cross-reactive protein band in accordance with the calculated molecular weight of PAP1 in the OE lines, but not in the WT (Supplementary Fig. S4C). Furthermore, APase activity increased significantly in the OE lines compared to the WT (Supplementary Fig. S4D). These results demonstrated the successful integration and expression of PAP1 in P. tricornutum.

Phosphatase overexpression has been shown to facilitate Pi acquisition and energy production, and to regulate cellular signaling pathways, thereby allowing cellular growth and photosynthesis to continue in plants under nutrient stress. We therefore first determined substrate preference in vivo (Supplementary Fig. S5). The WT specifically selected Pi as the P source, while the OE lines exhibited a clear shift of preference from Pi to organic P, which was in accordance with the previous studies showing that PAP prefers organic P as the sole source (Mehra et al., 2017; Kong et al., 2018).

We then investigated the impact of PAP1 overexpression on growth, photosynthesis, transcript abundance, and P acquisition under the provision of ATP, as a sole source of P in the form of dissolved organic phosphate (DOP). The specific growth rate and cell density of the OE lines were significantly greater than in the WT (Fig. 3A, b), clearly showing the crucial role of PAP1 in P-scavenging under DOP conditions. Next, we examined PAP1 expression and, as expected, the relative transcript abundance was greater in the OE lines (Fig. 3C), suggesting the high PAP1 expression triggered cell growth. The consumption rate of total P from the medium was higher in the OE lines than in the WT (Fig. 3D), suggesting that extracellular P was more efficiently absorbed. We then determined the cellular P content, and found that the acquisition of P in the OE lines was significantly higher than in the WT on both day 4 and day 7 (Fig. 3E). The inorganic phosphate (Pi) content also exhibited a similar pattern (Fig. 3F). There were reductions in the total cellular P content between day 4 and day 7 in both the OE lines and the WT, and the cellular Pi content also reduced between day 4 and day 7 in the WT; however, no such reduction in Pi occurred in the OE lines. This implied that there was effective utilization of acquired P in the late growth phase in the OE lines. These results demonstrated the potential of an organic P source (ATP, provided as the sole external source of P) to enhance Pi acquisition, similar to previously reported plant PAPs that show increased activity primarily under Pi deprivation (Liang et al., 2010).

Fig. 3.

Growth, PAP1 expression, and P contents in wild-type (WT) and PAP1-overexpression (-OE) lines of Phaeodactylum tricornutum with ATP as the sole P substrate. (A) Specific growth rate during day 4. (B) Cell density on day 7. (C) Expression of PAP1 on day 4 and day 7, relative to the value measured in the WT on day 4. TBP and RPS served as the internal references. (D) Total P content in the growth medium during the course of the cultivation cycle. (E) Total cellular P content on day 4 and day 7. (F) Cellular inorganic P content on day 4 and day 7. Data are means (±SD), n≥3. Individual data points are also shown in (E, F). Significant differences compared with the WT were determined using Student’s t-test: **P<0.01.

PAP1 overexpression enhances growth and photosynthetic activity

Given the indispensable role of P in photosynthesis and respiration (Sun et al., 2012), we next examined the impact of PAP1 overexpression on photosynthetic parameters over time. Chlorophyll a and c contents, non-photochemical quenching, the electron transport rate, and F_v/F_m all indicated enhanced photosynthetic efficiency in the OE lines (Fig. 4, Supplementary Fig. S6). Notably, photosynthetic efficiency gradually decreased in the WT, whereas it remained stable in the OE lines throughout the cultivation cycle. Previous studies have correlated the expression levels of genes related to photosynthesis with photosynthetic efficiency (Fujimoto et al., 2012), and phosphate availability has also been shown to significantly influence the expression of such genes, and hence photosynthetic efficiency (Bettini et al., 2016). We therefore examined key genes during the course of cultivation, and found that the expression levels of POR1, POR2, ChlG, AtpC, PetJ, PetC, PsbO, PsbM, and PsbA were significantly higher in the OE lines than in the WT (Supplementary Fig. S7). Consistent with a previous report (Wu et al., 2003), our results demonstrated that photosynthetic performance was enhanced a PAP1-mediated increase in acquisition of Pi from an organic P source , and this in turn elevated growth.

Fig. 4.

Photosynthetic properties of wild-type (WT) and PAP1-overexpression (-OE) lines of Phaeodactylum tricornutum during the cultivation cycle with ATP as the sole P substrate. (A) Electron transport rate (ETR), (B) non-photochemical quenching (NPQ), and (C) The maximal quantum efficiency of PSII (F_v/F_m). Data are means (±SD), n≥3. Significant differences compared with the WT were determined using Student’s t-test: **P<0.01.

PAP1 overexpression alters cellular biochemical content in a growth phase-dependent manner

Given that several cellular properties were altered in a growth phase-dependent manner, we sought to examine this aspect of PAP1 overexpression in more detail. Total carbon and nitrogen contents were determined on day 4 and day 7 of cultivation (Supplementary Fig. S8A, B). Cellular nitrogen was not affected in either the OE lines or the WT, indicating that cellular growth was not limited by nitrogen availability as the cells were grown in regular cultivation media (Cho et al., 2016). In contrast, the carbon content of the OE lines was greater than that of the WT on day 7, whereas no differences were observed on day 4. Interestingly, we observed a marked decrease in the Chl/C ratio from day 4 to day 7, which was more pronounced in the OE lines (Supplementary Fig. S8C). We further determined the content of primary metabolites such as protein, carbohydrates and lipids at 3 days interval. In terms of primary metabolites, there were no differences in the protein content over time (Fig. 5A), which was in accordance with the results for nitrogen content. The total carbohydrate content was significantly higher in the OE lines than in the WT during the log-phase of growth (days 1–4 of cultivation). Intriguingly, however, by day 7 the carbohydrate content had decreased in the OE lines to become significantly lower than in the WT (Fig. 5B). These results implied that assimilated photosynthetic carbon was converted into metabolites during the growth phase, and also indicated a need to explore the mechanisms underlying the effective utilization of photosynthetic carbon.

Fig. 5.

Primary metabolites in wild-type (WT) and PAP1-overexpression (-OE) lines of Phaeodactylum tricornutum during the cultivation cycle with ATP as the sole P substrate. (A) Total soluble proteins per cell, (B) Total carbohydrate content per cell, and (C) total lipid content. Data are means (±SD), n≥3. Significant differences compared with the WT were determined using Student’s t-test: *P<0.05, **P<0.01.

PAP1 redirects carbon flux towards lipogenesis, specifically to neutral and glycolipids

Given the increased total carbon and decreased carbohydrate contents that we observed, we next examined lipids. Lipids and carbohydrates compete for metabolic precursors, energy, and reducing power, and hence decreases in carbohydrates are correlated with increases in lipids and vice versa (Xue et al., 2017). We determined the lipid content using both fluorometric and gravimetric analyses, and found that it gradually increased in both the OE lines and the WT during cultivation (Fig. 5C). As expected, it showed the opposite pattern to carbohydrates, being initially lower in the OE lines and then significantly higher than in the WT from day 7. The results were corroborated by staining with Nile Red, which showed that the volume and number of lipid droplets were concomitantly increased in the OE lines compared with the WT (Fig. 6A, Supplementary Fig. S9). We further fractionated the total lipids into neutral lipids, phospholipids, and glycolipids, and found that the phospholipid content was similar in the OE lines to the WT, whereas the glycolipid content was significantly increased in the OE lines (Fig. 6). The neutral lipid content was initially lower in the OE lines and then became significantly higher from day 7. The pattern of the phospholipid content indicated that the algal cells had not undergone P-related stress conditions, and hence the cells did not utilize membrane phospholipids to generate intracellular P metabolites, as would be expected under P deprivation (Nakamura, 2013). The results indicated that PAP1 mediated the accumulation of photosynthetic sugars during the log-phase of growth and the reallocation of carbon flux towards de novo lipogenesis during the stationary-phase of growth, resulting in enhanced accumulation of neutral and glycolipids. This was in good agreement with the observed increase in photosynthetic efficiency and increased expression of genes associated with photosynthesis.

Fig. 6.

Analysis of lipids in wild-type (WT) and PAP1-overexpression (-OE) lines of Phaeodactylum tricornutum during the cultivation cycle with ATP as the sole P substrate. (A) Relative neutral lipid content, as measured by relative fluorescence units (RFUs) of cells stained by Nile Red. (B–D) Cellular contents of (B) neutral lipids, (C) phospholipids, and (D) glycolipids. Data are means (±SD), n≥3. Significant differences compared with the WT were determined using Student’s t-test: *P<0.05, **P<0.01.

To further explore the molecular mechanisms underlying the enhancement of neutral and glycolipids, we examined the expression patterns of key genes involved in the triacylglycerol (TAG) and glycolipid biosynthetic pathways. Interestingly, we found that the relative expression of GPAT3, LPAT3, PAP and DGAT1 was significantly higher in OE lines than in the WT, particularly from day 7 onwards when high and steady levels were reached (Supplementary Fig. S10). We also examined the expression levels of key genes involved in the glycolipid biosynthetic pathway, namely MGD1, MGD2, DGD1, and DGD2 and found that they were all consistently higher in the OE lines from day 1 (Supplementary Fig. S11). Expression of these genes would eventually result in accumulation of neutral and glycolipids (Martin et al., 2014).

PAP1 liberates inorganic P and orchestrates Pi transport

In order to determine the subcellular localization of PAP1, we transiently expressed the PtPAP1-GFP fusion protein (Supplementary Fig. S1C) in Arabidopsis protoplasts, and the resulting fluorescent signal was detected in the endoplasmic reticulum (ER; Supplementary Fig. S12). Thereafter, we sought to elucidate the mechanistic role of Pi accumulation in TAG biogenesis. Previous reports have demonstrated the impact of Pi content on P mobilization between organelles, which alters the subcellular and total lipid contents (Kobayashi et al., 2009; Nakamura et al., 2009). We found that increased Pi level resulted in the activation of Pi transporters (Supplementary Fig. S13), which in turn would remobilize Pi in plant cells (Ticconi and Abel, 2004). Another study has demonstrated that loss of function of PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 in Arabidopsis impairs Pi uptake and mobilization, which leads to the retention of Pi in the ER and reduces the abundance of membrane transporters (González et al., 2005). Previous reports have also correlated the impact of Pi translocation and abundance on governing the generation of energy and reducing equivalents (Lau et al., 2000; González et al., 2005), and we therefore examined the levels of ATP, NADH, and NADPH. We found that the contents of ATP and NADH were significantly higher in OE lines compared with the WT, and there was significant up-regulation of transcripts of associated genes (Fig. 7, Supplementary Fig. S14A–D). The patterns of NADPH content in the OE lines and the WT corresponded to those observed for total lipids (Fig. 7C), with the OE lines showing greater content during the stationary phase, indicating the role of PAP1 in the generation of lipogenic NADPH. The genes encoding glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme, which are involved in NADPH synthesis (Xue et al., 2017), showed increasing and relatively stable expression, respectively, indicating the primary role of G6PD rather than ME in providing NADPH, and also corroborating the lipogenic role of PAP1 (Supplementary Fig. S14E, F). We also assessed antioxidant potentials and found that ROS levels were significantly lower in the OE lines than the WT, and that the activities of SOD, POD, and CAT were also lower (Fig. 8). The increased antioxidant potential might be due to the increased NADH content, thereby obviating the impact of ROS (Yu et al., 2016).

Fig. 7.

Changes in contents of ATP and reducing equivalents in wild-type (WT) and PAP1-overexpression (-OE) lines of Phaeodactylum tricornutum during the cultivation cycle with ATP as the sole P substrate. Relative fold-change compared with the WT on day 1 of (a) ATP, (b) NADH, and (c) NADPH. Data are means (±SD), n≥3.

Fig. 8.

Antioxidant content and activity of antioxidant genes in wild-type (WT) and PAP1-overexpression (-OE) lines of Phaeodactylum tricornutum during the cultivation cycle with ATP as the sole P substrate. (A) Relative content of reactive oxygen species (ROS) as determined using a cell-permeable fluorogenic probe. RFU, relative fluorescent units, with the WT value on day 4 set as 100%. Activity of (B) peroxidase (POD), (C) superoxide dismutase (SOD), and (D) catalase (CAT). Data are means (±SD), n≥3. Significant differences compared with the WT were determined using Student’s t-test: *P<0.05, **P<0.01.

Our results indicated the crucial role of ER-localized PAP1 in generating energy and reducing equivalents, and also revealed the role of ER-associated transporters in mobilizing Pi across the membrane. The translocated Pi could be instrumental in generating NADPH, possibly by orchestrating dehydrogenases (Bose et al., 2003). In addition, the expression of the key genes involved in non-phospholipid synthesis DGDG and MGDG was enhanced in the OE lines (Supplementary Fig. S11), implying a role of ER-localized PAP1 in Pi acquisition and remobilization (Okazaki et al., 2013). Collectively, our data demonstrated the role of ER-localized PAP1 in Pi acquisition and lipogenesis; however, further studies are required to elucidate the detailed mechanisms of Pi mobilization. The results uncovered the crucial role of PAP1 in generating redox pairs and ATP, which in turn will maintain the redox state of the cell (Hirrlinger and Dringen, 2010) and various metabolic circuits such as energy metabolic pathways and reductive biosynthesis (Collins et al., 2012; Nakazawa et al., 2016).

PAP1 RNAi reveals that PAP2 compensates to maintain cellular physiological processes

We used RNAi to knockdown PAP1 expression in order to confirm the function of PAP1 in liberating Pi (Supplementary Fig. S1D). The relative transcript abundance of PAP1 was significantly decreased in the RNAi lines (designated as KD1 and KD2; Fig. 9A), but the activity of APase was affected (Fig. 9B). We also examined the impact of PAP1 RNAi on cellular physiological characteristics (Fig. 9C–J).Contrary to our expectations, we found that cell growth rate was not impaired in the RNAi lines and it remained at the same level as the WT, which was in contrast to previous findings that RNAi of PAP in rice results in significant physiological impairments under both P sufficient and deficient conditions (Mehra et al., 2017). we also observed no significant differences in total cellular P and inorganic P contents between the RNAi lines and the WT (Fig. 9E, F). To examine the mechanisms underpinning the maintenance of the growth rate of the transgenic lines, we determined the transcript abundance of other PAP homologs and found that the relative expression of PAP2 was significantly higher in the RNAi lines than in the WT on both day 4 and day 7 of cultivation, whilst PAP3 expression was unaffected (Fig. 9K, L). These results demonstrated functional redundancy between PAP genes in P. tricornutum, thereby allowing cell growth to be maintained under P deprivation.

Fig. 9.

Effects of PAP1-knockdown on growth and physiology of Phaeodactylum tricornutum with ATP as the sole P substrate. (A) Relative expression of PAP1 on day 4 of cultivation in the wild-type (WT) and PAP1-knockdown (-KD) lines. Expression is relative to that in the WT, and TBP and RPS served as the internal references. (B) Relative APase activity on day 4. (C) Specific growth rate during the log phase (day 4). (D) Cell density on day 7. (E) Total cellular P content on day 4 and day 7. (F) Cellular inorganic P content on day 4 and day 7. (G) Chlorophyll to carbon ratio on day 4 and day 7. (H) Total lipid contents on day 4 and day 7. (I) Relative fold-change of ATP on day 4 and day 7, compared with the WT on day 4. (J) Relative content of reactive oxygen species (ROS) as determined using a cell-permeable fluorogenic probe. RFU, relative fluorescent units, with the WT value on day 4 set as 100%. (K, L) Expression of (K) PAP2 and (L) PAP3 on day 4 and day 7 relative to the value measured in the WT on day 4. TBP and RPS served as the internal references. Data are means (±SD), n≥3. Individual data points are also shown in (E, F). Significant differences compared with the WT were determined using Student’s t-test: *P<0.05, **P<0.01.

Discussion

PAPs catalyse the liberation of inorganic phosphate (Pi) from a wide array of phosphate monoesters and anhydrates, thereby helping to facilitate growth under phosphate deficiency, and various studies have demonstrated the role of plant PAPs in releasing Pi and promoting cellular biogenesis (Liang et al., 2010; Tran et al., 2010b; Sun et al., 2012).However, detailed examination of their roles in crucial metabolic systems and molecular mechanisms remain lacking, particularly in microalgae. In this study, we have demonstrated the mechanistic role of PAP1, which maintains physiological and biochemical functioning in the microalga Phaeodactylum tricornutum in a substrate- and growth-phase dependent manner. Despite sequence similarities among various PAPs, we found that their expression patterns and substrate specificities were unique and are probably governed by their organic P preference. Expression of PAP1 was highly induced by organic phosphate (ATP) rather than by P depletion, whereas expression of the homologs PAP2 and PAP3 exhibited entirely different patterns (Fig. 1C–E). Interestingly, purified PAP1 exhibited strict specificity towards organic P in vitro, which was in contrast to the results of a previous study of plant PAPs, which found that they exhibited broad organic and inorganic P substrate specificity (Wang et al., 2011). In addition, the data uncovered the distinct characteristics of PAP1 towards specific cofactors and inhibitors when compared to other PAPs which were in contrast to the previous findings (Bhadouria et al., 2017) Our results were further confirmed by finding that organic phosphate mediated enhancements of growth and acquisition of Pi in transgenic PAP1-overexpression (OE) lines (Fig. 3A, B, D–F). These results provide novel insights in the biochemical characteristics of PAP1, and highlight its potential to perform a regulatory role when organic P (i.e. ATP) is supplied, and they also reveal the functional potential of PAP1 in hydrolysing organophosphates, which are known to be the primary P source under deficiency conditions (Tran et al., 2010a).

Although the role of PAPs in enhancing growth has been well-documented in plants (Sun et al., 2012; Tian et al., 2012; Kong et al., 2014), it remains largely unexplored in microalgae. We found that the cellular contents of Pi and total P were higher in the OE lines compared to the wild-type (WT) during both the log- and late-growth phases (Fig. 3E–F); however, the phosphate content was lower in the late-growth phase compared to the log phase, during which cells require adequate provision of a P source for growth (Tran et al., 2010a; Yang et al., 2014), and this is consistent with previous findings that support the role of PAP1 in P metabolism (Veljanovski et al., 2006; Mehra et al., 2017). In addition, photosynthetic parameters and expression of key photosynthetic genes were significantly enhanced in the OE lines ( Fig. 4, Supplementary Figs S6, S7), which supports a crucial role of Pi in enhancing growth and photosynthesis (Veljanovski et al., 2006; Zhang et al., 2012). Taken together, these findings provide evidence for continued utilization of the elevated P content in the cells during the late growth phase. It appears that the enhanced P content was effectively utilized for growth during the log phase, and thereafter it was accumulated as energy metabolites, as demonstrated by the altered carbon content and chlorophyll/carbon ratio (Supplementary Fig. S8A, C; Wu et al., 2015). Hence, a complete understanding of how PAP1 affects associated biochemical composition is required to elucidate the molecular mechanisms.

Biochemical analyses of the OE lines revealed an increased carbohydrate content relative to the WT during the growth phase and a decreased content thereafter, whilst the opposite trend was observed for lipid content (Fig. 5). Consistent with a previous report (Papanikolaou and Aggelis, 2011), the enhanced carbohydrate content during the growth phase was shown to play a central role in carbon assimilation and in providing the carbon source for various cellular processes. The reduction in sugar content and increase in lipid content in the stationary phase in the OE lines indicated that the synthesized sugar might be converted to lipids via metabolite remodeling. The fluctuations in the lipid content during the different growth phases demonstrated that PAP1-overexpression redirected the carbon flux towards lipogenesis during the stationary phase, which was further corroborated by the elevated expression of key genes involved in NADPH generation and in the lipogenic pathway (Supplementary Figs S10, S14).

In summary, our findings uncover the mechanistic role of PAP1 in enhancing phosphate assimilation, thereby regulating key metabolic pathways (Fig. 10). PAP1 plays a role in enhancing growth and photosynthesis during the growth period, possibly via enhanced phosphate assimilation, whilst enhancing the lipid content during the late growth phase. Hence, its function in microalgal homeostasis acts in a growth phase-dependent manner. PAP1 plays a crucial role in potentiating reducing equivalents and also in Pi translocation through orchestrating the expression of Pi transporters. Knockdown of PAP1 selectively induces the expression of PAP2 to replenish the activity of PAP1 and maintain cellular growth.

Fig. 10.

Proposed model of the mechanistic role of PAP1 in Phaeodactylum tricornutum. PAP1 facilitates ATP uptake and subsequently orchestrates crucial cellular metabolic pathways, possibly located in the endoplasmic reticulum (ER), thereby governing cellular growth and lipogenesis in a growth-phase dependent manner. DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; DGD, digalactosyldiacylglycerol synthase; FASII, fatty acid synthetase II; FFA, free fatty acid; LD, lipid droplet; MGD, monogalactosyldiacylglycerol synthase; PHF1, PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1; Pht1;1, inorganic phosphate transporter; Pi, inorganic phosphate; PPP, pentose phosphate pathway; TAG, triacylglycerol.

Supplementary data

The following supplementary data are available at JXB online.

Table S1. List of primers used in this study.

Table S2. Signature motifs of PAP proteins from a range of plant species.

Fig. S1. Conserved domains and schematic maps of the transformation constructs.

Fig. S2. Multiple sequence alignment of PAP1 with homologous sequences from various plant species.

Fig. S3. Phylogenetic trees of PAP family proteins.

Fig. S4. Molecular validation of transgenic PAP1-overexpression lines.

Fig. S5. P content of growth medium during the cultivation period for the wild-type and a PAP1-overexpression line with ATP as the P source.

Fig. S6. Chlorophyll contents of the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S7. Relative transcript abundance of genes related to photosynthesis in the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S8. Carbon and nitrogen contents and chlorophyll/ carbon ratios of the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S9. Location of lipid droplets as shown by staining with Nile Red in the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S10. Relative transcript abundance during the course of cultivation of genes related to triacylglycerol biosynthesis in the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S11. Relative transcript abundance during the course of cultivation of key genes related to galactoglycerolipid biosynthesis in the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S12. Location of PtPAP1-EGFP expression in Arabidopsis protoplasts.

Fig. S13. Relative transcript abundance during the course of genes related to Pi-transporters in the wild-type and PAP1-overexpressing lines with ATP as the P source.

Fig. S14. Relative transcript abundance during the course of genes for energy-related small molecules and co-factors in the wild-type and PAP1-overexpressing lines with ATP as the P source.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (51908244, 31870027), and China Postdoctoral Science Foundation (2018M643363, 2019T120789).

Author contributions

XW was responsible for conceptualization, investigation, data curation, formal analysis, visualization, and writing the original draft of the paper; SB was responsible for investigation, formal analysis, and visualization; S-FL, C-YJi, and Y-HL were responsible for investigation and formal analysis; W-DY was responsible for project administration and resources; LJ was responsible for writing and editing the revised paper; H-YL was responsible for conceptualization, project administration, supervision, validation, and writing and editing the revised paper.

Data availability

All data supporting the findings of this study are available within the paper and within its supplementary data published online.