Introduction

Iron (Fe) is a crucial micronutrient that serves as an essential cofactor in various biological processes, including photosynthesis, nucleotide biosynthesis, electron transport chains, and nitrogen fixation. It is often a limiting factor for photosynthetic productivity, biomass accumulation, and the community structure of phytoplankton1,2. However, the growth of phytoplankton can be significantly hindered by the low bioavailability of iron, particularly in one-third of the open ocean3,4. In surface seawater, the majority of iron exists as insoluble organic complex, while dissolved unchelated inorganic iron is at exceedingly low concentration5.

In the marine diatom Phaeodactylum tricornutum, there are three proposed pathways for iron acquisition from ocean6,7. First, un-chelated ferric irons can be concentrated at the cell surface and subsequently transported via endocytosis after binding to the phototransferrin iron starvation-induced protein 2 A (ISIP2A)8,9. ISIP2A, which possessed carboxylate iron-binding domains, was initially identified in P.tricornutum10, and is localized to both the outer membrane and internal vesicles. A deficiency in ISIP2A impedes high-affinity iron uptake in P.tricornutum, though uptake can be restored through complementation with human transferrin9. Second, ferric irons complexed with hydroxamate siderophores can bind to the cell surface protein FBP1 (ferrichrome-binding protein) and are transported via endocytosis in a process depended on ISIP1 into the cytosol11,12. Third, ferrous iron may be directly taken up by divalent metal transporters belonging to the ZRT/IRT-like protein (ZIP) family, which were identified in P.tricornutum genome through homologous genes related to yeast and plant ferrous ion transporters (ZIP family), however, experimental validation for this pathway is still lacking7,10,13.

In vivo, ferritin and vacuoles serving as the two primary sites for long-term iron storage in most plants14,15. Overexpression of ferritin in P. tricornutum has been shown to induce a morphological transformation from fusiform to ovoid forms, exhibiting characteristics typical of resting cells and benthic adaption, including reduced growth rates, limited light adaption ability, and silicified valves16. Additionally, the vacuole can function as heavy metal repositories for ion homeostasis, necessitating a variety of metal transporters, such as vacuole iron transporter (VIT) and VIT-like (VTL) gene family, as well as Na+/H+ antiporters, Mn2+ transporters, Copper transporter COPT5, metal transporter proteins (MTPs), cation exchangers (CAXs), heavy metal ATPases (HMA), natural resistance-associated macrophage proteins (NRAMPs), ABC transporter, etc17,18,19. However, it remains undocumented whether specific iron transporters exist in P. tricornutum for vacuole sequestration to regulate the iron homeostasis.

In the present study, we identified four genes encoding for vacuolar iron transporters of the VIT, the vacuolar iron transporter gene family, firstly in the P. tricornutum genome using in silico approaches. We explored the phylogeny, chromosome distribution, conserved domains, subcellular localization, with a particular focus on the functional analysis of PtVTL3. The findings from this research will enhance our understanding of the regulation mechanism of VIT in P. tricornutum.

Methods

Identification and characterization of vacuolar iron transporter (VIT) genes from the Phaeodactylum tricornutum genome

To identify potential VIT genes in the P. tricornutum genome, we downloaded the Hidden Markov Model (HMM) profile of the VIT ___domain (PF01988) from the Pfam database (http://pfam.xfam.org/). This profile was used as a query to perform HMMsearch20 on the P. tricornutum genome obtained from the Ensembl Protists database (E value < 1.0 E-5 ) (http://protists.ensembl.org/). Redundant sequences were eliminated and verified by three protein structure databases: CDD (https://www.ncbi.nlm.nih.gov/cdd), HMMER (https://www.ebi.ac.uk/Tools/hmmer/) and SMART (http://smart.embl.de/).

The Expasy ProtParam tool (https://web.expasy.org/cgi-bin/protparam/protparam) was utilized to analyze the basic physicochemical property of the PtVIT genes, including the number of amino acids, molecular weight, and the isoelectric point. Transmembrane ___domain predictions were conducted using the TMHMM − 2.0 online program (https://services.healthtech.dtu.dk/services/TMHMM-2.0/)21.

The signal peptides of PtVTLs were predicted by SignalP-4.1 online program (https://services.healthtech.dtu.dk/services/SignalP-4.1/), and the subcellular localization of PtVTLs were predicted by TargetP-2.0 online program (https://services.healthtech.dtu.dk/services/TargetP-2.0/).

Phylogenetic analysis of PtVITs

To investigate the phylogenetic relationship between PtVITs and other plant VITs, we collected 93 VIT amino acid sequences from 12 species, including those from P. tricornutum (4), Oryza sativa (7), Arabidopsis thaliana (6), Chlamydomonas reinhardtii (4), Selaginella moellendorffii (10), Zea mays (7), Populus trichocarpa (8), Physcomitrella patens (5), Vitis vinifera (7), Glycine max (21), Medicago truncatula (13) and Saccharomyces cerevisiae (1). These sequences were sourced from following databases: JGI, Ensembl, RGAP, TAIR and UniPort, and extracted the conserved ___domain sequences to construct a phylogenetic tree using the maximum likelihood (ML) method in MEGA 7.022, with a bootstrap value set at 1000. The Saccharomyces cerevisiae CCC1 sequence (P47818)23,24,25 served as the outgroup for rooting the tree. The resulting phylogenetic tree was visualized using the iTOL online tool (https://itol.embl.de/)26.

Conserved motifs, domains, and gene structure of PtVITs

The phylogenetic tree of PtVITs conserved ___domain amino acid sequences was constructed using the ML method in MEGA 7.0. Conserved motifs and domains were predicted using the MEME online program (suite 5.5.2) (https://meme-suite.org/meme/tools/meme) with a p value < 0.0527, as well as the CDD protein structure database with a similar p value threshold28. The genome annotation file for P. tricornutum was downloaded from the Ensembl database and visualized using TBtools29.

Chromosome distribution, and gene duplication pattern of PtVIT genes

Chromosome distribution data of PtVIT genes were extracted from the P. tricornutum genome annotation file and visualized using the MG2C v2.1 online program (http://mg2c.iask.in/mg2c_v2.1/)30. The Simple Ka/Ks Calculator in TBtools software was utilized for analyzing the gene duplication pattern.

Cis-acting regulatory elements and expression profile of PtVIT genes

The 500 bp DNA sequences upstream of the initiation codon of PtVIT gene were extracted from the P. tricornutum genome using TBtools software29, subsequently, cis-acting regulatory elements of the promoters were identified using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)31, and visualized with GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, California, USA, www.graphpad.com).

Based on the transcriptome data provided in the literature32, the expression profile of PtVTL genes at diel light cycling under iron limitation was studied. The values of FPKM (fragments per kilobase of exon per million mapped reads) were calculated as log2 values, and the expression data were normalized and viewed using the TBtools software29.

Functional analysis of PtVITs

To investigate the functions of PtVITs, we cloned the genes from P. tricornutum into yeast expression vectors for the complementary assay and the subcellular ___location study.

Total RNA was extracted from the P. tricornutum strain CCMA 106, sourced from the State Key Laboratory of Marine Environmental Science of Xiamen University, using the HiPure Plant RNA Mini Kit (Magen, Guangzhou, China). Reverse transcription and RT-PCR were performed using the corresponding primers (Supplementary Table 1), employing a reverse transcription kit and FastPfu DNA polymerase (TransGen Biotech, Beijing, China). The PtVTLs genes were inserted into the yeast expression vector pYES2 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China).

The recombinant plasmids were transferred into the yeast wild type yeast strain DY150 and into the mutant strains △ccc1, △smf1, △Zrt1Zrt2 using the PEG/LiAc method. Positive clones were selected on solid SD medium lacking uracil (SD-Ura) (WEIDI, Shanghai, China), and cultured in glucose-containing SD-Ura liquid medium until the logarithmic growth phase. After centrifugation at 2000 g for 1 min, cell pellets were washed twice with sterile ultrapure water, resuspended in sterile ultrapure water to an OD600 of approximately 1, and then two microliters of the cell suspension were spotted on SGR medium (SD-Ura plates containing 2% galactose and 1% raffinose) (WEIDI, Shanghai, China) in four serial 10-fold dilutions. Plates were incubated at 30 ℃ for 2–4 days and subsequently imaged.

The eGFP (enhanced Green Fluorescent Protein) gene was synthesized by Sangon Biotech Co., Ltd (Shanghai, China) (no stop codon) and fused with PtVTL3 gene into the pYES2 vector using the ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing, China). The recombinant vector was transformed into yeast mutant strain △ccc1 via the PEG/LiAc method. Cultures were grown in SD-Ura liquid medium containing 2% galactose and 1% raffinose (WEIDI, Shanghai, China), and incubated with 10 µM of the vacuolar membrane fluorescence dye FM4-64 (Coolaber Technology Co., Ltd., Beijing, China) for 0.5 h at room temperature. The recombinant yeast was imaged using a laser confocal microscope (Leica, Germany).

Results

Identification and characterization of the VIT family in Phaeodactylum tricornutum

Using the Hidden Markov model (HMM) profile of the VIT1 (PF01988) via HMMsearch, we identified four putative VIT family genes in the P. tricornutum genome. These findings were subsequently validated through three protein structure databases: CDD, SMART and HMMER. The different physiochemical properties of these genes are detailed in Table 1. The results indicated that the four genes are distributed across chromosomes: 2, 4, and 13, and the lengths of the deduced amino acid sequences varied from 280 to 318 residues, with molecular weights ranging from 30 to 34 kD. The number of transmembrane domains (TMD) ranged from 3 to 5.

Table 1 Basic information of VITs in Phaeodactylum tricornutum.
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The signal peptides were predicted by SignalP-4.1 online program, and the results showed that there are not any signal peptides in PtVITs. The subcellular localization of PtVITs were predicted by TargetP-2.0 online program and the results showed that they didn’t exist at mitochondria, chloroplast or thylakoid luminal. Therefore, they should be localized at other subcellular organ (Supplementary Table 2).

Phylogenetic analysis of PtVITs

We collected a total of 93 VIT protein sequences (Supplementary Table 3) and extracted the ___domain sequences to perform multiple sequences alignment in Mega 7.0 and construct a phylogenetic tree by the ML method. The tree revealed two distinct groups: the VIT group and the VTL group (Fig. 1). The VIT group showed a closer relationship to Saccharomyces cerevisiae CCC1 and included characterized ferrous iron transporters such as OsVIT1, OsVIT233, AtVIT134. In contrast, the VTL group was larger and comprised many uncharacterized members of the protein family, along with various ferrous iron transporters, including AtVTL1, AtVTL2, AtVTL535, MtVTL4, MtVTL836, GmVTL1a, and GmVTL1b37. Notably, all the four members of VIT family from P. tricornutum clustered within the VTL clade. Consequently, based on the chromosomal distribution and phylogenetic relationship, we designated Phatr3_J43313, Phatr3_J43314, Phatr3_J26157 and Phatr3_J37632 as PtVTL1, PtVTL2, PtVTL3 and PtVTL4 respectively. Moreover, the phylogenetic analysis indicated that the evolutionary pathway of PtVTL3 diverges from the other three PtVTLs, as it was not clustered with them in the VTL group.

Fig. 1
figure 1

The phylogenetic tree of PtVTLs basis on their protein ___domain sequences by ML method. ScCCC1 is used as outgroup. The red clade means the VTL group, and the blue clade represents the VIT group. Red asterisk represented the PtVTLs.

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Conserved motifs, domains, and gene structure of PtVTLs

To investigate the diversity of PtVTLs, we analyzed their conserved motifs. Our findings revealed five motifs associated with the VIT family (PF01988) in the PtVTLs (Supplementary Table 4). These motifs were arranged in the same order in PtVTL1, PtVTL2 and PtVTL4, whereas PtVTL3 only contained motif 2, 3 and 4. The characteristics of PtVTL3 were closely aligned with its phylogenetic relationships (Fig. 2A,B).

All PtVTLs contained the VIT family ___domain, which was localized on the C-terminus (Fig. 2C). The gene structure analysis indicated that only PtVTL4 contained one intron, while other three PtVTL genes were intronless (Fig. 2D).

Fig. 2
figure 2

Phylogenetic tree, conserved motifs, domains and gene structure of PtVTLs. (A) the ML phylogenetic tree of PtVTLs based on the protein sequences. (B) The conserved motifs of PtVTLs. (C) The conserved ___domain of PtVTLs. (D) the gene structure of PtVTL genes.

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Chromosomal distribution and gene duplication of PtVTL genes

Using the annotation file of the P. tricornutum genome, we employed the MG2C online program to visualize the distribution of PtVTL genes. The PtVTL genes were found on chromosomes: 2, 4, and 13, with PtVTL1 and PtVTL2 tightly linked on the same chromosome (Fig. 3). The distance between these two genes was only 772 bp (Table 1). A BLAST analysis revealed an identification rate of 88.49%, and 75% coverage between the two genes. This strongly suggests that the gene pair of PtVTL1 and PtVTL2 resulted from a gene tandem duplication event. We calculated the ratio of nonsynonymous substitutions per nonsynonymous site (Ka) to synonymous substitutions per synonymous site (Ks) of the PtVTL1 and PtVTL2 gene pair, finding that the Ka/Ks ratio was less than 1 (Table 2), indicating that this gene duplication event underwent negative selection38.

Table 2 Ka/Ks ratio and duplicated date calculation for PtVIT genes.
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Fig. 3
figure 3

Chromosome distribution of PtVTL genes. Red font represents the linked gene pair.

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Cis-acting regulatory elements and expression profile of PtVTL genes

Cis-acting elements in the promoter region can influence the precise and efficient expression of downstream genes. Given the gene density in the P. tricornutum genome39, we extracted the 500 bp upstream sequence from the start codon of each gene for promoter analysis. In total, we identified 124 regulatory elements associated with PtVTL genes (Fig. 4). These elements were categorized into seven classes (included function unknown), and three classes occupy the vast majority: 54 elements for transcriptional regulation (Fig. 4, shown in red), 30 elements for abiotic stress responses (such as drought, light, and anoxic) (Fig. 4, shown in orange), and 28 elements related to phytohormone response (including abscisic acid, MeJA, jasmonic acid, auxin, and gibberellin) (Fig. 4, shown in yellow). Among these, CAAT-box (27) and TATA-box (7) were the most abundant regulatory elements for transcriptional regulation, while ABRE (7), as-1 (5), CGTCA-motif (5) and TGACG-motif (5) were the richest elements for phytohormone response. The G-Box (7), MYB (5), and MYC (4) elements were the most prevalent for abiotic stress. Notably, cis-acting elements related to transcription regulation, environmental stress, phytohormone response and morphological development were present in all PtVTL genes promoter regions, while elements associated with metabolism and biotic stress appeared only in some promoter regions. These findings suggest that the expression of PtVTL genes is predominantly regulated by environmental factors (e.g. light) and phytohormone. In addition, we try to seek some iron-responsive cis-elements, such as C(A/G)C(A/G)C(G/T)40, A(A/C)G(G/C)C(G/-)C(A/G)TG, or CACGTG(T/C)C41. But regrettably, these iron-responsive cis-elements are not found.

Fig. 4
figure 4

Cis-acting regulatory elements analysis of PtVTL gene family. Red represents transcriptional regulation elements, orange represents abiotic stress responses elements, yellow represents phytohormone responses elements, green represents morphological development elements, blue represents biotic stress responses elements, purple represents zein metabolism responses elements, and gray represents function unknown responses elements.

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To understand the roles of specific PtVTL genes at diel light cycling and different iron concentration, expression data for the four PtVTL genes were downloaded from the literature32, and TBtools software was used to create HeatMap (Fig. 5). The results revealed that the expression of PtVTL genes were induced at different light condition. PtVTL3 was highly expressed under light, and others were highly expressed under dark condition, which might be associated with the cis-acting elements in the promoters. Specifically, PtVTL2 was highly expressed at low iron conditions (20 pM Fe’), and PtVTL3 exhibited high expression at high iron condition (400 pM Fe’).

Fig. 5
figure 5

The expression profile of PtVTL genes to diel light cycling (12:12) under iron limitation (standardized RPKM across Fe conditions). L: 20 pM Fe’, M: 40 pM Fe’, H: 400 pM Fe’. Fe’: sum of all Fe species not complexed to EDTA. (data resource32: ).

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Function analysis of PtVTLs

We amplified the PtVTL genes from the P. tricornutum strain CCMA 106 using RT-PCR, and the results indicated a premature stop code (TAA) at positions 100–102 bp in the PtVTL4 gene, leading to early termination of translation. Therefore, we considered that PtVTL4 has not function in heavy metal transportation, and adopted PtVTL1, PtVTL2 and PtVTL3 to analysis the function of PtVTLs.

To assess the metal transport activity of PtVTLs, we conducted a yeast complementary assay. The PtVTLs (PtVTL1, PtVTL2 and PtVTL3) were expressed in a yeast mutant strain, △ccc1 (Ca2+-sensitive cross-complementer 1 mutant), which is particularly sensitive to high extracellular Fe levels and fails to thrive in Fe enriched media24. The result demonstrated that PtVTL3 successfully rescued the Fe2+ sensitivity in mutant △ccc1, indicating its role as an iron transporter (Fig. 6). But PtVTL1 and 2 was not able to complement the Fe uptake mutant. PtVTL1 and 2 rescue mutants did not exhibit any growth differences after 48 h of growth on SGR medium containing 5 and 10 mM FeSO4 compared with the mutant △ccc1 cells. Interestingly, heterologous expression of PtVTL3 in the Zn2+ uptake-defect mutant yeast △Zrt1Zrt2 exhibited increased sensibility to 5 mM ZnSO4 compared to the control. Conversely, the expression of PtVTL3 in Mn2+ uptake-defective mutant yeast △smf1 did not exhibit any growth differences compared to the control across various metal ion-enriched medium (Supplementary Fig. 1). And PtVTL1 and 2 still appear to be unaffected by increased Zn2+ and Mn2+ concentrations.

Fig. 6
figure 6

The heterologous expression of PtVTL genes in yeast. Yeast wild type (WT) strain DY150 and mutant △ccc1 cells containing pYES2 (EV), pYES2-PtVTL1, pYES2-PtVTL2 and pYES2-PtVTL3 grown on SGR medium without or with 5 and 10 mM FeSO4 for 48hs and photographed. Each spot represents a 1:10 dilution of the culture starting with an OD of 1 on the far left (10-, 100-, and 100-fold dilutions).

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The subcellular ___location of PtVTL3 in yeast was observed using confocal microscope, and the images displayed that the eGFP-PtVTL3 fluorescence signal overlapped with the tonoplast fluorescence dye FM4-64 (Fig. 7), suggesting that the PtVTL3 is primarily localized at the tonoplast.

Fig. 7
figure 7

Subcellular ___location of PtVTL3 in yeast mutant △ccc1 cells. (A) eGFP-PtVIT3; (B) tonoplast fluorochrome FM4-64; (C) Bright Field; D: merged image. The green and red signal obtained with confocal microscopy indicated fusion protein eGFP-PtVIT3 and tonoplast fluorochrome FM4-64, respectively. The overlap image eGFP-PtVIT3 and FM4-64 signal is indicated in merged image. Bar = 5 μm.

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Discussion

Saccharomyces cerevisiae CCC1, the first identified member of the VIT family, is located at the tonoplast, where it plays a crucial role in regulating the accumulation of iron and manganese within this organelle24. The overexpression of the CCC1 protein leads to a reduction in cytosolic iron concentration while increasing iron content in the vacuole. The first VIT protein discovered in plants was Arabidopsis thaliana AtVIT1, which shared 62% amino acid similarity with the yeast CCC1 protein. AtVIT1 is responsible for transporting iron into the vacuole under high-iron conditions, thereby supporting the normal development of A. thaliana seedling34. Similarly, OsVIT1 and OsVIT2 are located in the tonoplast and participate in the transport of Fe2+ and Zn2+ in rice33. OsVIT2, in particular, is expressed in the parenchyma cell bridges of nodes and play a significant role in distributing iron to grains by sequestering it into the vacuole within the mestome sheath, node, and aleurone layer42. In Tulipa gesneriana, the TgVIT1 protein is linked to iron accumulation in the blue-colored inner segments of petals, contributing to the blue pigmentation in purple cells43,44,45. Likewise, Centaurea cyanus CcVIT is involved in the blue coloration of cornflower petals46.

In contrast to VIT proteins, VTL proteins lack a cytosolic loop in their tertiary structure, which serves as a metal-binding ___domain17,36,47,48. VTL proteins have been identified in both monocots and dicots, as well as in Chlamydomonas and Physcomitrella35. Overexpression of A.thaliana AtVTL1, AtVTL2 or AtVTL5 significantly increased seed iron concentration in nramp3/nramp4 or vit1-1 mutants35. In Medicago truncatula, MtVTL4 and MtVTL8 were exclusively expressed in nodule, with MtVTL8 serving as the primary route for delivering iron to symbiotic rhizobia36. Similarly, in soybean (Glycine max), GmVTL1a and GmVTL1b are localized into the symbiosome membrane, with GmVTL1a facilitating iron transport across the membrane to bacteroids and playing a crucial role in the nitrogen-fixing symbiosis37.

In this study, four members of the VIT family were identified in the genome of the marine diatom Phaeodactylum tricornutum. A phylogenetic tree constructed from different plant VIT family members revealed two distinct groups: the VIT group and the VTL group (Fig. 1), aligning with the phylogenetic trees created by Brear et al.37 and Sharma et al.49. All four identified members belong to the VTL group.

Similar to findings in tomato and soybean50, a gene duplication event was observed in the VIT family of P. tricornutum. The gene pair of PtVTL1 and PtVTL2 arose from tandem duplication, which is a key factor contributing to the expansion of this gens family in P.tricornutum (Fig. 3; Table 2).

The VIT family is involved in the transport of Fe2+, Mn2+ and Zn2+, regulating the homeostasis of these heavy metals within cells19. Like AtVTL135, GmVTL1a51, MtVTL4 and MtVTL836, the heterogeneous expression of PtVTL3 in yeast mutant △ccc1 was able to rescue growth under high Fe concentrations, indicating that PtVTL3 functions to transport Fe2+ and regulate iron homeostasis (Fig. 6). Additionally, we observed that in medium containing 5 mM ZnSO4, the expression of PtVTL3 in Zn2+-sensitive yeast mutant △Zrt1Zrt2 exhibited increased Zn2+ sensitivity compared to the control (Supplementary Fig. 1), suggesting that PtVTL3 may also function as a bi-functional protein.

Like AtVTL1 and AtVTL234 and GmVTL1a51, the eGFP-PtVTL3 fusion protein localized to the tonoplast in yeast (Fig. 7), which indicated that PtVTL3 may sequester Fe2+ into the vacuole for detoxification under high Fe2+ concentration conditions, thereby contributing to the regulation of iron homeostasis within cells.

Conclusions

In the genome of the marine diatom Phaeodactylum tricornutum, four members of the VIT gene family were identified, all of which belong to the VTL group. Each of the PtVTLs contains a VIT family ___domain, indicating a shared evolutionary origin. This gene family appears to have expansion through tandem duplication events. Based on the analysis of cis-acting regulatory elements, it is evident that PtVTLs play a role in responding to environmental stress and phytohormones, and the expression profile verified that PtVTL3 gene was induced to express highly under light, and others were induced to express highly under dark condition. Specially, PtVTL2 and PtVTL3 genes were induced to express highly by low Fe and high Fe condition respectively. Additionally, the heterologous expression of PtVTL3 in yeast mutant strain △ccc1 demonstrated that it is predominantly localized at the tonoplast, and is effective in rescuing yeast growth under the elevated iron concentrations.