Molecular chaperones are the guardians of protein homeostasis [1]. They widely exist in eubacteria archaebacteria and eukaryotic cells [2]. Molecular chaperones could interact with another protein to form its functional active conformation [3]. Previous studies have already shown that molecular chaperones are essential for cellular protein folding, refolding, and the ubiquitin-proteasome system, playing an important role in removing the misfolded and aggregated proteins timely under stresses [4]. Additionally, molecular chaperones could promote cell survival in a non-physiological state and stressful conditions by preventing aggregation and promoting refolding of proteins. It was reported that these functions are ubiquitous and conserved in all organisms [1].

Protein disulfide isomerases (PDIs), a kind of 55 kDa proteins, are important molecular chaperones in the Endoplasmic Reticulum quality control (ERQC). ERQC is known as a calreticulin/calnexin cycle and plays a key role in glycoprotein folding, secretion, and maturity [5,6]. The ERQC recognizes the misfolded/unfolded proteins on the way to their secretory pathway [7]. In the ERQC, together with the ER lectins calnexin (CNX) and/or calreticulin (CRT), molecular chaperones (e.g., PDIs) combine to the N-linked mono-glucosylated glycan of glycoproteins and aid the folding of glycoproteins [5]. The structure of PDIs was widely studied in plants. The PDIs are composed of five domains, including a, b, b’, a’, and c domains. There are two active sites in the a and a’ domains, both have two cysteine residues [8]. The a-type domain carrying the “Cys-Gly-His-Cys” motif contains catalytic active sites and plays a role in the reduction-oxidation cycle. The b-type domain is enriched in hydrophobic residues, involving substrate binding. It was reported that PDIs are a kind of thiol oxidoreductase chaperones located in the ER [9]. It was shown that PDIs are divided into three classes in microalgae. The first class contains a central Trx module with a canonical YAPWCGHC active site sequence. The second class harbors an N-terminal Trx domain with a variable active site sequence. The third class has two domains, a N-terminal Trx domain and a long α-helix domain at the C-terminus [10]. For example, Rb60 in Chlamydomonas reinhardtii, the third class of PDI protein, contains two thioredoxin-like domains with a putative catalytic site of “-Cys-Gly-His-Cys-” [11]. Although characteristics of PDIs were compared between plants and chromalveolates [12], however, the structure of PDIs and their characteristics in microalgae, including the conserved motifs and domains, classification, the number of introns-exons, and phylogenetic relationship are still unknown so far.

Additionally, PDIs display different functions in plants. The classical function of PDI proteins is responsible for the introduction or isomerization of disulfide bonds in nascent ER proteins, aiding the refolding of misfolded/unfolded proteins, and finally retaining the protein homeostasis [8,11,13]. For example, AtPDIs located in chloroplasts of leaves are associated with starch granule biosynthesis in Arabidopsis thaliana [14]. In Soybean DG330, the upregulated expression of the GmPDIL7 gene helps the formation of disulfide bonds in nascent proteins under ER stress [15]. SlPDI (Protein disulfide isomerases of Solanum Lycopersicum) promotes the tomato resistance to TYLCV virus by reducing hydrogen peroxide (H₂O₂) levels, increasing the activity of superoxide dismutase (SOD) and peroxidase (POD), and enhancing antioxidant activity. It was indicated that SlPDI plays an essential role in enhancing the protein folding function of ER and promoting the synthesis and conformation of antioxidant-related proteins [16]. In addition to plants, the function of PDIs in microalgae was also studied. In C. reinhardtii, Rb60 regulates the translation of proteins and can maintain photosynthesis. CrPDI2 is a redox-active protein in C. reinhardtii. It was found that CrPDI2 participates in the regulation of the circadian signaling pathway [17]. So far, a lot of PDI studies have been focused on Chlorophyta, such as C. reinhardtii. However, the expression characteristics and function of the PDI gene family in diatom remain unknown.

Phaeodactylum tricornutum, a diatom, is one of the most important primary producers in the ocean [18]. In this study, P. tricornutum was used to study the conserved motifs, gene structures and the distribution of genes on the chromosomes, evolutionary relationship, and putative function of PtPDIs via bioinformatic analysis and molecular biotechnology. Tunicamycin is an effective inhibitor of protein N-glycosylation to disrupt the N-glycosylation modification of proteins in organisms [19]. N-glycosylation is an important co- and post-translational event in the modification of proteins in eukaryotes. N-glycosylation plays a vital role in the function of protein. In P. tricornutum, the pathway of protein N-glycosylation mainly takes place in the ER and Golgi apparatus [14]. The stress of tunicamycin will disrupt protein homeostasis. Therefore, it is valuable to study the expression characteristics of PDIs and propose their potential functions under tunicamycin stress. The results obtained in this study will provide an important foundation for clarifying the mechanism of PDIs proteins in protein homeostasis [20].

In this study, 42 PtPDI candidates were identified from P. tricornutum. However, only 8 CrPDIs were identified from the genome of C. reinhardtii, and 10 OsPDIs were observed in Ostreococcus lucimarinus [21]. The different number of PDIs might be dependent on the algal species. Many more PtPDI in P. tricornutum were identified here, indicating that Diatom might need more PDIs to regulate the complex ERQC pathway. In high plants, PDI proteins were classified into nine classes, including PDI-A, PDI-S, PDI-E, PDI-B, PDI-C, PDI-M, PDI-D, and PDI-F. The difference between these 9 PDI classes was the number and the position of the Trx domain and the presence of additional domains [14]. Previous reports showed that PDI-A in Arabidopsis thaliana could bind an iron-sulfur (Fe−S) cluster. It was certificated that this binding scavenged the free iron to prevent the production of reactive oxygen [22]. PDI-M from plant Populus trichocarpa x Populus deltoides had the ability of oxidation-reduction regulation to activate the NADP-MDH [23]. This indicated that different kinds of PDIs played diverse roles. It was reported that PDI-F was found in microalgae, such as C. reinhardtii, Thalassiosira pseudonana, and Emiliania huxleyi [14,21,24]. Previous papers showed that three PDI-F proteins were found in diatom T. pseudonana [14,21]. Differently, 27 PtPDI-F subfamily was found in P. tricornutum in this study. These results enriched the PDI-F subfamily, and it was indicated that the number of PDIs was species-specific. At the same time, it was found that PDIs were divided into different groups. For example, in Brassica rapa ssp. Pekinensis, 32 PDIs were clustered into PDI-A (15.6%, 5/32), PDI-L (34.3%, 11/32), PDI-C (18.7%, 6/32), PDI-S (6.25%, 2/32), PDI-M (6.25%, 2/32), PDI-D (6.25%, 2/32), and PDI-E (12.5%, 4/32) [25]. In Arabidopsis thaliana, 21 PDIs were grouped into PDI-A (19%, 4/21), PDI-L (33.3%, 7/21), PDI-C (14.28%, 3/21), PDI-S (4.7%, 1/21), PDI-M (9.5%, 2/21), PDI-D (9.5%, 2/21) and PDI-E (9.5%, 2/21) [25]. 17 PDIs from Medicago truncatula belonged to PDI-L (29.4%, 5/17), PDI-S (23.5%, 4/17), PDI-C (17.6%, 3/17), PDI-B (5.8%, 1/17), PDI-A (5.8%, 1/17), PDI-E (5.8%, 3/17) [25,26]. 8 PDIs identified from C. reinhardtii were clustered into PDI-E (12.5%, 1/8), PDI-C (12.5%, 1/8), PDI-L (12.5%, 1/8), PDI-S (12.5%, 1/8), PDI-M (12.5%, 1/8), PDI-D (12.5%, 1/8) and PDI-F (25%, 2/8) [21]. In a previous paper, 7 PtPDIs were identified by blast search in the genome, including 1 PtPDI-B, 2 PtPDI-C, 1 PtPDI-E, and 4 PtPDI-F [14]. Many more PtPDIs (4 PtPDI-B, 4 PtPDI-C, 2 PtPDI-E, 27 PtPDI-F, 5 PtPDI-L) were identified via the Pfam number in this study, indicating a further method was important for the genome wild research. It was also proposed that more PtPDIs in P. tricornutum were needed to adapt to complex environments during secondary endosymbiosis.

Except PtPDI1, all the other PtPDIs contained motif 1 and motif 2 in P. tricornutum in this study, indicating that these two motifs were critical for the function of PtPDIs. Similarly, motif 1 and motif 2 were also highly conserved in rice [27]. Additionally, it was reported that motif 1 and motif 2 were responsible for the resistance of plant Panax ginseng to environmental stresses, such as drought [27,28]. Therefore, it was speculated that these two motifs in P. tricornutum might have a similar function under environmental pressures. In this study, 42 PtPDIs were located in different subcellular organelles, including cytoplasm, endoplasmic reticulum, Golgi apparatus, chloroplast, and nucleus. Consistent with the previous reports [14,29], ER-localized PtPDIs also contained ER retention signal, for example, PtPDI17 contained KEEL at the C-terminus, and PtPDI19 had EDEL motif. The subcellular localization of PtPDIs in C. reinhardtii was similar to that in P. tricornutum [30]. This suggested that PtPDIs were widely distributed into different subcellular organelles and played different functions in these diverse structures. However, all these putative localizations needed experiments to certify, such as via the observation of PtPDIs-eGFP fluorescence in confocal laser scanning microscopy. It was also found that PtPDIs played a vital role in forming disulfide bonds and modifying misfolded proteins in ER [31]. Moreover, it has been shown that PDIs could regulate thiol-disulfide switches and help repair the cytoskeleton in cell surface and cytosol [32]. Hence, ER-localized PtPDIs might also participate in the formation of disulfide bonds in proteins and regulate the correct folding of misfolded/unfolded proteins to maintain protein homeostasis in the cell. However, the exact function of these PtPDIs needs more experiments to clarify, such as by the overexpression, knock-out, and/or knockdown of genes in P. tricornutum.

A previous study reported that gene-duplicated events played an important role in the rapid expansion and evolution of gene families [33]. Gene-duplicated events usually happen among most plants and algae [34]. A pair of tandem duplicated genes (PtPDI15 and PtPDI18 genes) was observed in the genome of P. tricornutum. At the same time, it was observed that the gene structure, motifs, and homology of these two genes were highly conserved, indicating that these two PtPDIs might have similar functions in P. tricornutum. Compared to the reported homologous gene PDI-L5-1 (At1g04980) with two CXXC motifs in Arabidopsis thaliana [14,35], both PtPDI15 and PtPDI18 contained three CXXC motifs, indicating the structure specificity among different organisms. WP1 in yeast Rhodotorula gramini, a homologous gene of PtPDI15 and PtPDI18, could increase resistance and tolerance against a wide range of biotic and abiotic stresses, such as oxidative stress and heavy metals [34]. The homologous genes of PtPDI15 and PtPDI18 in the fungus Wallemia played an important role in osmoregulation under the hyperosmotic environment [36]. These studies suggested that PtPDI15 and PtPDI18 genes might also be important for the responses of P. tricornutum under the biotic and abiotic stresses, such as tunicamycin stress in this study.

It was known that PDIs play a crucial role in aiding the correct folding and assembly of nascent proteins in ERQC to maintain protein and cellular homeostasis. ERQC is an important pathway in ER (Fig. 5) [7]. Two ER-located PDI proteins were reported to be related to ER stress in Soybeans [15,37]. In this paper, PtPDI6, 17, 19, 20, 22, 35, and 37 with ER retention motif were predicted to be located in ER, therefore, it was reasonable to propose that these 7 PtPDIs might be involved in the ERQC pathway to resist ER stress and maintain cellular protein and cellular homeostasis. Moreover, it was found that the expression of these PtPDI genes was different under the stress of tunicamycin. Tunicamycin is an effective protein inhibitor, affecting the N-glycosylation modification of proteins and causing the accumulation of unfolded and/or misfolded proteins in the ER lumen [20,38]. PtPDI genes were differentially expressed to respond to the tunicamycin stress. Similar results were observed in tomato and Chlamydomonas [39]. The differential expression of PDI genes was dependent on the activation or repression of transcription factors. It was reported that bZIPs played a key role in the regulation of expression of PDI genes [29], therefore, it was speculated that the differentially expressed PtPDIs were regulated by different bZIP transcription factors. In this paper, it was suggested that the up-regulated PtPDIs (PtPDI1, PtPDI4-5, PtPDI7, PtPDI11-12, PtPDI15, PtPDI17, PtPDI22, PtPDI29, PtPDI35-37, and PtPDI42) might be involved in maintaining cellular protein homeostasis as positive regulatory factors. This suggestion was verified by that the homologous genes of PtPDI15 played important roles in cellular homeostasis, especially under oxidative, heavy metal, and hyperosmotic stresses [40]. The homologous gene Rb60/PDI1A of PtPDI22 in C. reinhardtii was also strongly upregulated in response to tunicamycin treatment, and it was also found that both Rb60/PDI1A and PtPDI22 were putatively targeted to ER [39,41]. The second homologous gene PDI6 in C. reinhardtii of PtPDI1 was also dramatically upregulated in tunicamycin-treated cells, indicating that this gene was involved in the cellular response to this stress [39]. The homologous relationship is shown in Supplementary Fig. 1. However, the downregulated PtPDIs (PtPDI6, PtPDI13, PtPDI16, PtPDI31, PtPDI33, PtPDI40 and PtPDI41) might act as negative regulatory factors. However, the negative regulatory mechanism of PtPDIs in P. tricornutum was still unknown. The homologous genes AtPDI2/5 of PtPDI6/16 showed the same differentially expressed profiles, indicating that these two PtPDIs might have synergistically functioned as AtPDI2/5 on the formation of a disulfide bond [42]. The differentially expressed PtPDIs could also be explained by that tunicamycin induced the unfolded protein response (UPR), and then UPR affected the transcription of PtPDIs [29]. In our recent paper, it was concluded that the differentially expressed PDI genes in C. reinhardtii might also play an important role in the response to Pb stress [21]. Anyway, it was proposed that these PtPDI genes might act as a catalyst of disulfide bond formation, helping stabilize protein folding’s tertiary and quaternary structures and maintaining cellular homeostasis during various stresses.