In aquaculture, changes in temperature influence the growth, development, reproductive ability, and survival of organisms (Viergutz et al., 2012; Aagesen and Hase, 2014). The fluctuating temperature could compromise and alter the immune function and cause bacteria to proliferate and accumulate in tissues, leading to diseases and stress (Yu et al., 2009). Pearl oysters as warm-water shellfish species were cultured in natural seas and exposed in diverse temperature ranges and their corresponding effects (Adzigbli et al., 2020b). Pinctada fucata martensii, as the main marine pearl called “South China sea pearls” production in China (Yang et al., 2019; Zhang et al., 2021), hold a low enduring capacity to extreme temperatures and just hold an optimal range of 23–28°C (Deng et al., 2010). During pearl culture, the juvenile of pearl oyster is transferred to a culturing site in the sea and thus exposed to various environmental perturbations that affect its survival and growth in the natural sea (Hao et al., 2019; Adzigbli et al., 2020b). The sensitivity of pearl oysters to temperature change has limited their culture area, which in turn affects the pearling industry. Therefore, understanding the response mechanism of the pearl oyster to environmental stress and exploring the genetic mechanism of cold tolerance in pearl oyster are essential for the breeding production.

Recently, multiple researches were performed to explore the mechanism of temperature stress such as Mytilus galloprovincialis, Mytilus trossulus, and Crassostrea gigas, and plenty of genes were identified especially genes like HSPs or HSP-related genes (Ioannou et al., 2009; Fields et al., 2012; Zhu et al., 2016). Through the analysis of high-throughput data, cellular senescence, homeostatic flexibility, inhibition of apoptosis, lysosome protein-encoding genes, and immune-related genes participated in the process of temperature tolerance (Ibarz et al., 2010; Zhu et al., 2016; Wang et al., 2018). Liu et al. (2018) showed that stearoyl-CoA desaturase showed a significantly higher expression in the low temperature tolerance, which showed the potential function of monounsaturated fatty acid in the response of temperature (Liu et al., 2018). Although many genes and related metabolic pathway have been reported to be involved in temperature stress, small genetic markers that are important for breeding were screened.

Pearl oysters rely on their innate immunity in response to diverse stress including cold tolerance and diseases (Adzigbli et al., 2020a). The protein inhibitor of activated signal transducer and activator of transcription (PIAS) is a key small ubiquitin-related modifier protein (SUMO) E3 ligase, which participates not only in cytokines but also in various immune signaling pathways (Shuai and Liu, 2005) and may be involved in diverse immune response to stimuli. In addition, extreme temperatures have been known to influence apoptosis, with PIAS protein performing this function in various organisms. PIAS proteins could modulate the function and localization of many proteins, including many components of two important pathways, the NF-κB and JAK-STAT signaling pathway (Schmidt and Müller, 2002; Shuai and Liu, 2003). Furthermore, diverse functions have been ascribed to PIAS in vertebrates and invertebrates including immune response, cell apoptosis, cell differentiation, and proliferation (Duval et al., 2003; Myllymäki and Rämet, 2014). Duck PIAS2 could interact with duck interferon regulatory factor 7 and inhibits IFN-β promoter activation induced by the duck IRF7 (Zu et al., 2020). Amphioxus PIAS inhibits NF-κB by binding with upstream signaling adaptor TICAM-like and MyD88 (Wang et al., 2017). Although most of the PIAS research has focused on model species like Drosophila and mammals, some information is available for aquatic organisms like shrimp Litopenaeus vannamei (Zhang et al., 2019). In the present study, the E3 SUMO-protein ligase PIAS in the pearl oyster P. f. martensii, a bivalve mollusk, was characterized, and its exon region SNPs related to temperature stress were also investigated to explore its potential in the breeding program for the cold tolerance.

As a member of the negative regulators of the JAK/STAT signaling pathway, PIAS participates in the regulation of immune responses, which showed its potential in response to cold tolerance. However, in the bivalve, limited reports have been researched about PIAS proteins. In the present study, a PIAS gene from P. f. martensii was cloned and SNPs in the exon region of PmPIAS were obtained to explore the genetic information for breeding.

Researches have reported that the PIAS protein family exhibit high similarity in conserved domains including the conserved RLD, PINIT motif, C-terminal Ser/Thr amino acids enriched region (S/T), AD, and LXXLL signature motif in the SAP domain (Duval et al., 2003; Suzuki et al., 2009). Among them, SAP domain aids the function of the PIAS gene, which is associated with sequence- or structure-specific DNA binding (Aravind and Koonin, 2000). The LXXLL signature motif was found to mediate the interactions between nuclear receptors and their coregulators (Glass and Rosenfeld, 2000). RLD domain is required for the SUMO-E3-ligase activity of PIAS proteins and may be involved in the interaction with other proteins (Kotaja et al., 2002). Amphioxus PIAS inhibited NF-κB activation by co-localizing and binding with TRAF6, and the interaction relied on the N-terminal SAP and PINIT domains of PIAS (Fu et al., 2020). PIASy binds to MafA through the SAP domain and negatively regulates the insulin gene promoter through a novel SIM1-dependent mechanism (Onishi and Kataoka, 2019). In the present study, the protein sequence feature of PmPIAS also contained all conserved domains and motifs, which were consistent with PIAS proteins from other species like Crassostrea virginica, Mizuhopecten yessoensis, Octopus vulgaris, Scylla paramamosain, and Lingula anatina (Shuai, 2006; Huang et al., 2015). Advanced structure analysis of PIAS proteins from H. sapiens and bivalve also showed the conserved structure of PINIT and RLD domains in the PmPIAS. Therefore, PmPIAS may be a member of the PIAS protein family.

PIAS protein participates in regulating various immune signaling pathways and immune response to stimuli (Shuai and Liu, 2005). In L. vannamei, LvPIAS exhibited an immune response function after bacteria and virus stimulation with a significant expression pattern within 48 h post-stimulation and inhibited the transcriptional activity of LvSTAT, which indicated that there was a feedback loop between LvSTAT and LvPIAS (Zhang et al., 2019). PIAS of Scylla paramamosain was involved in the pathogen-resistant activities of mud scab (Huang et al., 2015). Expression pattern of PmPIAS in the different tissues and developmental stages indicated that this gene may play an important role in the life process of P. f. martensii. Xenopus PIAS plays important roles in mesodermal induction and patterning during early frog development (Burn et al., 2011). After cold tolerance, PmPIAS expressed significantly highly at the early time points to respond to the stress, which indicated its important function in the cold tolerance stress response. In the Arabidopsis, the ectopic expression of IZ1 (a SIZ/PIAS-Type SUMO E3 Ligase) could also improve the cold tolerance (Li et al., 2020). Researches have reported that, after low temperature tolerance, Pm-SCD, Pm-HK, and PmHSP70 genes showed a significantly higher expression at different time points compared with the control group, which means the pearl oysters utilized different biological processes at different times to respond to the low temperature tolerance (Liu et al., 2018, 2019a,b). Temperature tolerance has been reported to induce the immune system, and transcriptome analysis of low water temperature stress showed that several immune-related genes and pathways were presented in response to exposure to low temperatures in pearl oysters (Wang et al., 2018). Immune-related genes were also upregulated after cold exposure of Drosophila melanogaster and suggested that immunomodulation plays an important role in response to cold stress (Zhang et al., 2011; Tusong et al., 2017). Previous studies reported that PIAS could bind to the transcription factor STAT in the cytoplasm and then inhibit the DNA-binding activity with downstream genes especially immune-related genes and pathways in mammals, jawless fishes, and some crustaceans (Kotaja et al., 2002; Niu et al., 2018; Li et al., 2020). Therefore, PmPIAS may participate in the cold tolerance through involvement in the immune activity.

In order to explore the potential of PmPIAS in the breeding program for the cold tolerance selection, exon region SNPs of PmPIAS were identified. Various studies have reported that particular environmental stresses increase SNPs. Different cold tolerances for Nile tilapia strains from Ghana, Egypt, and Ivory Coast suggest the influence of geographic location and natural selection on cold tolerance in tilapia (Sifa et al., 2002). The further the geographic location from the equator, the more cold-tolerant the strain of Nile tilapia. The two sequenced natural populations of bay scallop Argopecten irradians presented a substantial difference in T allele frequency implying that the SNP all-53308-760 T/C may have been subjected to natural selection for temperature adaptation, and the higher frequency of T allele in the southern subspecies is the consequence of local adaptation (Du et al., 2014). In the present study, 11 SNP sites were found in the exon region of PmPIAS, and they demonstrated significant differences between the R and W in genotype and allele. Accumulating evidence suggested that genetic polymorphisms in the coding regions could affect protein activity or effect of translation (Gottler et al., 2008; Frydenberg et al., 2010). The SNPs detected in the present study may provide a potential site for the cold tolerance selection in the future.

The full-length characterization of PmPIAS showed its conserved primary and advanced structures in the protein sequence. Expression analysis demonstrated the wide distribution of PmPIAS in pearl oyster and showed a significant increase after cold tolerance. Eighteen SNPs were identified in the exon region of PmPIAS, and 11 SNPs showed the potential in the cold tolerance breeding of pearl oysters. This study provided a potential molecular marker for the selective breeding of cold tolerance of P. f. martensii.

The sequence information of PmPIAS presented in the study are deposited in the GenBank repository, accession number MW326754. The SNP data are deposited in the European Variation Archive, accession number PRJEB43188.

RH and QW: conceptualization. ZL: formal analysis and writing–original draft preparation. LA and QC: resources. RH, QW, YL, and YD: writing–review and editing. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.