SPX-related genes regulate phosphorus homeostasis in the marine phytoplankton, Phaeodactylum tricornutum

SPX gene and CRISPR/Cas9 knockout

Using the SPX domain as a query to search in the P. tricornutum genome, we found a total of six genes that harbor an SPX domain (Supplementary Table 1), including Vpt1 and Vtc4 that were recently described by Dell’Aquila et al.²¹. Pfam analysis of the six identified sequences in P. tricornutum resulted in the identification of the SPX domain in these proteins, which shared several conserved sites with land plants’ SPX domains (Fig. 1a, b). Phylogenetic analyses further verified the high similarity of these sequences with other known SPX domain proteins (Supplementary Fig. 1). One of these genes possesses a SPX domain as the sole functional domain (named SPX, and its encoding gene named SPX gene, from here on) while the other five (including Vpt1 and Vtc4) contain at least one other domain. From our recently published transcriptome dataset²⁷, we found that SPX, Vpt1, and Vtc4 genes were differentially expressed (log₂ Fold Change > 1 and adjusted p value < 0.05). (Supplementary Table 1). To focus on determining the function of the SPX domain, only the SPX gene was chosen for mutagenesis, and the target site was located in the SPX domain but away from (upstream of) the conserved motif (Fig. 1c).

Five mutant strains of SPX (mSPX) were obtained. The mutation efficiency was 9.3%, and the mutation included various insertions and deletions (indels) at the target site (Fig. 1d–f). The deletion or insertion ranged from single nucleotide (nt), various short multi-nt, to a long tract (Fig. 1e). These indels are indicative of the non-homologous end joining (NHEJ) DNA repair mechanism operating in P. tricornutum. The four mutant clones shown in Fig. 1e had a deletion of 1 nt (clone 1-4 or mSPX1-4), 23 nt (clone 65-2), or 195 nt (clone 15-3 or mSPX15-3), or an insertion of 6 nt (clone 1-1), all but one causing a frameshift translation (mSPX1-4) or deletion of translation (mSPX15-3), and hence presumably functional loss or reduction.

The expression of SPX and its related genes in the mutants and WT grown under P+ were analyzed and compared using qPCR. When using primers located near the mutation site (F1/R1; Fig. 1c), qPCR results showed that SPX expression in mSPX1-4 and mSPX15-3 decreased by 4.4 folds and 6.2 folds, respectively (Fig. 1g). When using another pair of primers located in the 3′end region (F2/R2; Fig. 1c), far downstream of the mutation site, the expression of SPX in mSPX1-4 and mSPX15-3 exhibited no significant change compared to WT (Fig. 1g). The discrepancy between the two pairs of primers was most likely due to the influence of the mutation on F1/R1 priming efficiency in PCR. In addition, the other two P-stress inducible SPX domain-containing genes (Vpt1 and Vtc4) were significantly upregulated in both mSPX1-4 and mSPX15-3 compared to WT (Fig. 1g). To more systematically investigate the effects of SPX inactivation, a series of physiological and transcriptomic analyses were performed on the mSPX15-3 strain.

Decreased growth rate, increased pigment contents and photosynthetic rate in SPX mutant

Starting from the same initial cell concentration (400,000 cells ml⁻¹), the growth of P. tricornutum under different P conditions started to diverge one day later. P deficiency suppressed growth in both WT and mSPX15-3, whereas rapid growth occurred in both strains under P+ condition. Surprisingly, however, the mSPX15-3 grew slower than the WT under P+ condition (Fig. 2a). The average growth rate of mSPX15-3_P+ was 0.40 ± 0.03 day⁻¹ during the exponential growth phase (from day 1 to day 7), lower than that of the WT_P+ culture group, which was 0.47 ± 0.02 day⁻¹ (Fig. 2a).

Remarkable increases were observed in cellular pigment contents in the mSPX15-3 cells under both P + and P− conditions (Fig. 2b–d). After SPX mutation, chlorophyll a (Chl a) content increased by 14% (ANOVA, F_1,4 = 24.535, p = 0.008) in P+ and 18% in P− culture groups (ANOVA, F_1,4 = 36.125, p = 0.004), and carotenoid content rose by 22% (ANOVA, F_1,4 = 65.283, p = 0.001) in P+ and 39% (ANOVA, F_1,4 = 50.548, p = 0.002) in P− groups, respectively (Fig. 2b, d). The elevation was also noted in chlorophyll c (Chl c), albeit without statistical significance (ANOVA, F_1,4 = 2.219, p = 0.211 in P+; ANOVA, F_1,4 = 2.079, p = 0.223 in P−) (Fig. 2c). Furthermore, photosynthetic rate, as determined as the rate of oxygen evolution, increased by 89% (ANOVA, F_1,4 = 58.638, p = 0.002) in mSPX15-3 relative to WT under P− condition, although the effect was not detected under P+ condition (ANOVA, F_1,4 = 2.753, p = 0.172) (Fig. 2e). Clearly, SPX mutation led to significant upregulation of photosynthetic capacity under P− condition.

Increased cellular carbon content, nitrogen content, and neutral lipid content in SPX mutant

Carbon content in mSPX15-3 cells increased by 22% (ANOVA, F_1,4 = 15.853, p = 0.016) under P+ condition and 39% (ANOVA, F_1,4 = 146.728, p < 0.001) under P− condition, respectively, relative to WT cells (Fig. 2f). Nitrogen content was higher by 24% (ANOVA, F_1,4 = 26.546, p = 0.007) in mSPX15-3 cells than in WT cells under P+ condition, but no significant difference was observed under P− condition (ANOVA, F_1,4 = 1.212, p = 0.333) (Fig. 2g). Consequently, the C: N ratio was not affected by SPX mutation under P+ condition (ANOVA, F_1,4 = 2.344, p = 0.200) but increased (by 31%) under P− condition (ANOVA, F_1,4 = 63.385, p = 0.001) (Fig. 2h).

The neutral lipid content per cell, as measured as fluorescence intensity of BODIPY 505/515 stain, increased by 5.83-fold (ANOVA, F_1,4 = 167.279, p < 0.001) and 4.72-fold (ANOVA, F_1,4 = 2856.099, p < 0.001) in WT and mSPX15-3, respectively comparing P− with P+ conditions (Fig. 2i), indicating that neutral lipid synthesis was markedly promoted by P deficiency. More notably, this lipid increase was magnified by SPX mutation, as the neutral lipid content in mSPX15-3 was 49% (ANOVA, F_1,4 = 47.547, p = 0.002) and 21% (ANOVA, F_1,4 = 11.448, p = 0.028) higher than in WT under P+ and P− conditions, respectively (Fig. 2i).

Transcriptomic alteration caused by SPX mutation

A total of 12 transcriptome libraries were constructed from four combinations of genotype and P conditions (mSPX15-3_P+, mSPX15-3_P−, WT_P+, and WT_P−, triplicated cultures for each) and sequenced to saturation. After removing low-quality sequences and adaptor sequences, on average 84.66% reads were uniquely mapped onto the reference genome, covering 93.24–94.44% of the genome-predicted proteome (Supplementary Table 2). Comparing mSPX15-3 with WT under both P conditions revealed 885 differentially expressed unigenes (Supplementary Data 1), indicating a broad impact of SPX mutation on the transcriptomic landscape. When mSPX15-3 was compared with WT separately for P+ and P− conditions, we found 528 differentially expressed genes (DEGs) for the P+ condition, 250 upregulated and 278 downregulated, and 560 DEGs for the P− condition, 306 upregulated and 254 downregulated (Supplementary Data 1). Of these, 203 DEGs appeared in both the P+ and P− conditions, indicating metabolic functions were influenced by SPX regardless of P nutrient condition. The 885 non-redundant DEGs represent metabolic processes regulated by SPX under P− or P+ conditions.

Increased alkaline phosphatase (AP) activity and gene expression in SPX mutant

SPX disruption led to significant increases in AP enzyme activity (Fig. 3a, b) and gene expression (Supplementary Table 3) under both P+ and P− conditions. The overall upregulated gene expression pattern was reflected in RNA-seq (Supplementary Table 3) and RT-qPCR, with similar magnitudes of elevation in the two different mutants examined (mSPX1-4 and mSPX15-3) (Fig. 3c). These results indicate that SPX is an upstream negative regulator of AP genes.

Upregulation of Pi transporter genes in SPX mutant

SPX mutation led to significant upregulation of P-responsive phosphate transporters (PTs) under P+ as well as P− conditions (Supplementary Table 4). Remarkably, the Nap_i3 (Phatr3_J47239) gene showed an 81.6-fold upregulation in mSPX15-3 under the P+ condition in our transcriptomic data (Supplementary Table 4), a trend also verified by RT-qPCR results in both mutants examined (Fig. 3c). In addition, one mitochondrial Pi transporter (PtMPT) was upregulated in mSPX15-3 under both P+ and P− conditions (Supplementary Table 4).

Upregulation of phospholipid degradation genes in SPX mutant

Eleven phospholipid degradation-related genes were found to be upregulated in mSPX15-3 compared to WT under either P+ or P− conditions (Supplementary Table 5). Two glycerophosphoryl diester phosphodiesterases (GDPDs, Phatr3_J32057 and Phatr3_J49693) that participate in glycerophospholipid metabolism displayed strongly increased expression in both mSPX1-4 and mSPX15-3 mutants under P+ condition (Fig. 3c). In addition, one phosphoethanolamine phospholipase (Phatr3_J52110) showed upregulation in RNA-seq (Supplementary Table 5), which was not substantiated by RT-qPCR probably due to low expression levels overall (Fig. 3c). These results demonstrate that the SPX mutation causes the same effect as expected of P stress on WT, providing evidence that SPX is a negative regulator of phospholipid degradation, which is likely the first responder to P-nutrient dynamics.

Relationship between SPX and PHR

As a negative regulator for PT and AP as shown above, SPX is expected to be downregulated under P− condition. Surprisingly, SPX was upregulated instead in WT_ P− relative to WT_P+ (Supplementary Table 1). This suggests that AP and PT are not immediate targets of SPX; rather, an intermediate control mechanism may be at play between SPX and effectors AP and PT (Fig. 3d). We looked to Myb TFs because they are known to function as a central regulator of Pi starvation signaling in plants and algae¹⁶. We examined the nine Myb TFs that contain a single Myb domain (Myb1R) described by Rayko et al.^20,28. Among these, we found that only Myb1R_5 (PHR) exhibited a significant response (upregulation) to both P-stress condition and SPX mutation (Supplementary Table 6), but not to PhoA or PhoD mutations^27,29, indicating that it functions downstream of SPX and upstream of APs. This is evidence that SPX-PHR is a coupled regulatory cascade of APs and PTs.

Distribution and expression of SPX in all major phytoplankton phyla and across global oceans

Based on a signature domain search on 306 pelagic and endosymbiotic marine eukaryotic species transcriptomes in the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) dataset²⁴, we found SPX domain-containing genes in 206 phytoplankton species that cover all major phytoplankton phyla (Supplementary Data 2). This indicates that the SPX gene is widespread in the phytoplankton tree of life, even though some of the detected genes might contain additional functional domains. Furthermore, we investigated the expression patterns and geographical distribution of SPX genes across Tara Oceans stations. The SPX gene was found to exist and be expressed widely across the global oceans, in size fractions from pico-, nano-, micro-, to small meso-plankton (Fig. 4a). Totally, 1131 SPX domain-containing genes were detected in the Marine Atlas of Tara Ocean Unigenes (MATOU-v1 catalog), of which 1091 belong to eukaryotes. They were identified at all of the 66 Tara Oceans stations (Fig. 4a) and were mainly (34%) distributed in the pico-eukaryote size fraction (0.8–5 µm), which were dominated by Chlorophyta in the subsurface layer (SRF) and Fungi in the deep chlorophyll maximum layer (DCM), respectively (Supplementary Fig. 2). In contrast, the other size fractions were dominated by Bacillariophyta (5–20 μm) and Metazoa (20–180 and 180–2000 μm) in both the SRF layer and DCM layer, respectively. Furthermore, the majority (96%) of the expressed SPX domains were concentrated in six lineages: Opisthokonta (Metazoa and Fungi), Stramenopiles, Haptophyceae, Viridiplantae (Chlorophyta), Rhizaria, and Alveolata, in decreasing order (Fig. 4b). In addition, 226 SPX domain-containing genes distributed in 58 species, were detected in 91 different Metagenomics-based Transcriptomes (MGTs), two of which (MGT-15 and MGT-50) account for 34%. These expressed SPX domain-containing genes were most greatly contributed by the pelagophyte Aureococcus anophagefferens, the phaeophyte Ectocarpus siliculosus, and the chlorophyte Chlamydomonas eustigma (Supplementary Data 3). These results demonstrate the prevalence and hence importance of the SPX in global marine phytoplankton communities.

Source: Ecology - nature.com