Identification,of,a,uroporphyrinogen,III,synthetase,gene,and,characterization,of,its,role,in,pearl,sac,formation,in,Hyriopsis,cumingii

Jiexun Shen, Dnn Hung, Jile Li,b,c, Weixin Ye, Zheng Wng, Zhiyi Bi,b,c,＊

a Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, Ministry of Agriculture, Shanghai, 201306, China

b Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China

c Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai, 201306, China

d Zhexing Pearl Trading Co. Ltd, Zhejiang, 321001, China

Keywords:Fresh water pearl mussel Hyriopsis cumingii Uroporphyrinogen III synthetase Nacre color

ABSTRACT Uroporphyrinogen III synthetase (UROS) is an important enzyme involved in the structural formation of porphyrin, which is responsible for color intensity. A UROS homolog was isolated and characterized from the pearl mussel, Hyriopsis cumingii (HcUros). The HcUros cDNA was 858 bp in length with an open reading frame that encoded a 285 amino acid protein, which contained a conserved HemD domain. HcUros exhibited widespread tissue distribution, displaying particular enrichment in the mantle and haemolymph of purple mussels,compared to white mussels. In situ hybridization of HcUros in mantle tissue demonstrated that it was specifically expressed in the dorsal epithelial cells of the mantle pallia. Additionally, HcUros expression was upregulated following implantation of the pearl sac to produce pearls. The data suggest that HcUros contributes to shell pigmentation, and nacre formation and coloring in H. cumingii.

Hyriopsis cumingiiis an important and economically valuable freshwater mussel species in China. However, the quality of pearls cannot be maintained with the current increase in production; thus, hindering the development of pearl breeding and processing industries. Improving pearl quality is a primary challenge facing the mussel and pearl industry.To culture pearls, allografts from the mantle tissue of donor mussels are inserted into the accepter mussels and this results in the formation of a thin layer of epithelial cells, from which the nacre is secreted. (Kishore &Southgate, 2016).

Pearl color is an inherited characteristic. Studies of pearl color, have mainly focused on breeding and much less attention has been paid to genetic aspects. In one study pearl color was shown to be regulated by multiple genes (Newkirk, 1980). The influence of gene expression on pearl production has been partially described forH. cumingii,and the molecular mechanisms that regulate pearl formation and pearl color are of increasing interest (Chen et al., 2016; Li et al., 2014).

Animal pigments, including melanin, carotenoids, and terapyrroles(Mcgraw, 2005) generate a variety of colors. Color variation in bird eggshells is based on tetrapyrrole pigments, and in particular, biliverdin IXα and protoporphyrin IX (Cassey et al., 2012; Gorchein et al., 2010;Igic et al., 2010; Kennedy & Vevers, 1976). Studies of shellfish pigments are restricted to pyrrole-based pigments, such as porphyrin. Porphyrins are pigments that are widely present in many organisms and play an important role in the metabolism and catabolic activity of living organisms. Porphyrin or porphyrin-like compounds are present in mammals, fish, and bivalves. Uroporphyrinogen III synthetase (UROS) is an enzyme involved in the formation of porphyrin. UROS catalyzes the intramolecular rearrangement of hydroxymethylcholine to uroporphyrinogen III, and through a series of catalytic events, forms chlorophyll in plants, hemocyanin in some mollusks, and heme in vertebrates(Battersby & Leeper, 1990; Crockett et al., 1991). UROS has been mainly studied in mammals (human and mouse) and bacteria, and defects in UROS activity causes congenital erythropoietic porphyria (CEP) (Xu et al., 1992). There have been few investigations of the role of UROS in mollusks.

It is well known that the shell of pearl oysters consists of two calcareous layers, the nacreous layer and the prismatic layer (Wilt et al.,2010). Different areas of the mantle are responsible for the expression and secretion of the matrix proteins and regulators that are involved in the formation of the shell layers. For example, prismalin-14 directs thestructure of the prismatic layer (Suzuki & Nagasawa, 2010). In the present study, the spatial localization of theH. cumingiiUROS gene(HcUros)was evaluated in mantle tissue to investigate its potential functions in shell formation and coloring.

Table 1 List of the primers used in this study.

The aim of this study was to clone and identifyHcUrosfromH. cumingiiand to measure its transcript level in purple- and white-line mussels.In situhybridization was performed to investigate the precise position ofHcUrosin mantle tissue. The spatial transcript abundance ofHcUrosafter implantation was also examined. The results of this study suggest thatHcUrosmay contribute to shell pigmentation, nacre formation and coloring inH. cumingii.

2.1. Mussels

Forty healthy mussels (20 white mussels and 20 purple mussels) with an average shell length of 10–11 cm were collected from Xuancheng Farm of Zhexing Pearl Trading Co. Ltd., Anhui Province, China. Mussels were cultured separately in two tanks (60 cm length, 40 cm width, 30 cm height, 30 L of water) at room temperature for 7 days before experimentation.

2.2. Total RNA extraction and cloning of HcUros

2.3. Bioinformatics analyses

The cDNA and amino acid sequence ofHcUroswas deduced using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/). The NCBIprotein BLAST tool was used to align theHcUrosamino acid sequence to UROS sequences from other organisms. ExPASy Compute pI/Mw(http://web.expasy.org/compute_pi/) was used to predict the molecular mass and theoretical isoelectric point ofHcUros, and SignalP 4.0(http://www.cbs.dtu.dk/services/SignalP/) was used to predict the signal peptides. Multiple alignments and phylogenetic trees were constructed using Clustal X2.0 and MEGA 5.2 software with the neighborjoining (NJ) algorithm, respectively.

2.4. Analysis of HcUros tissue distribution

Quantitative RT-PCR analysis was used to detect the expression ofHcUrosin different tissues. Nine purple mussels and nine white mussels were randomly collected, and total RNA was isolated from mantle,haemolymph, kidney, gill, intestine, foot, and adductor muscle. Total RNA extraction was performed as described above. cDNA for quantitative PCR analysis was synthesized using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara) to eliminate potential genomic DNA contamination. The qPCR reaction contained 1.6 μl of cDNA, 10 μl of 2SYBR Green PCR Master Mix (Takara), 6.8 μl of ddH2O, and 0.8 μl of each primer in a final reaction volume of 20 μl. The qPCR reaction protocols were: 95C for 3 min; 40 cycles at 95C for 5 s, followed by 58.5C for 30 s. Specific primers (Table 1) were designed using Primer Premier 5.0 software and the housekeeping gene,EF1α(Table 1), was used as the internal reference (Bai et al., 2014). Each sample (n ¼9) was analyzed in triplicate and relative expression was calculated using themethod (Livak & Schmittgen, 2001).

2.5. In situ hybridization of HcUros in mantle tissue

Digoxigenin-labeled RNA probes were prepared forHcUrosusing the DIG RNA Labeling Kit (Roche Molecular Biochemicals, Germany), according to the manufacturer’s instructions. In situhybridization was performed to evaluate the location ofHcUrosin mantle tissue from P-line mussels. Mantle samples were fixed in 4% paraformaldehyde for 6 h and then transferred to 20% sucrose at 4for 16 h. Tissues were sliced to a 12-μm thickness using a freezing microtome (Leica CM 1950; Wetzlar,Germany), andin situhybridization was performed following the manufacturer’s instructions (Enhanced Sensitive ISH Detection Kit, Boster,Switzerland).

2.6. Spatial expression of HcUros after implantation

To detect the temporal expression ofHcUrosduring early pearl formation, 90 healthy P-line mussels were randomly selected. Twenty-four small pieces of mantle tissue were implanted into each mussel, and pearl sac samples (if present) were randomly collected at 0, 3, 6, 12, 24, 48,and 96 h, and 7, 14, 28, and 35 d after operation (N ¼6 individuals for each time point). Pearl sacs at 0 h were collected as the non-implanted control. Total RNA extraction, qPCR, and data analyses were performed as described above.

2.7. Statistical analysis

All data were expressed as the meanstandard deviation (SD) and were analyzed in One-way ANOVA using SPSS 17.0 software. Statistical significance was accepted at P <0.05.

3.1. Sequence features of HcUros

Fig. 1. Multiple sequence alignment of the deduced amino acid sequence of HcUros with UROS proteins from other invertebrates. The UROS amino acid sequences are from Hyriopsis cumingii (MK599473), Meretrix petechialis (OWF51001.1), Crassostrea gigas (XP_011438551.1), Pomacea canaliculata (PVD29876.1), and Octopus bimaculoides (KOF93249.1). The conserved amino acids are shown with a black background, while similar amino acids were shaded gray.

Fig. 2. Phylogenetic analysis of HcUros from Hyriopsis cumingii and related species. The phylogenetic tree was produced using the neighbor-joining algorithm in Mega5.0. The bar (0.1) indicates the genetic distance. GenBank accession numbers are indicated next to the species names.

The amplicon forHcUroscDNA (GenBank accession no. MK599473)consisted of an open reading frame (ORF) of 858 bp, a 1391-bp 3ʹ-untranslated region (UTR), a 57-bp 5ʹ-UTR, and a canonical polyadenylation signal sequence (AATAAA) upstream of the poly-A tail. TheHcUrosgene was predicted to encode 285 amino acids with a molecular weight of 31.69 kDa and a theoretical isoelectric point of 5.1. It also contained a HemD domain (residues 34–261) and the protein belonged to the HemD superfamily. No signal peptide was predicted, and multiple sequence alignment (Fig. 1) and phylogenetic analyses (Fig. 2) revealed thatHcUroshad the closest relationship with UROS fromPomacea canaliculata.

3.2. Different expression of HcUros between purple and white mussels

The expression ofHcUroswas analyzed in different tissues using qPCR.HcUroswas detected in all tissues in the P- and W-lines (Fig. 3.HcUrosexpression levels were higher in the P-line than in the W-line,and significant differences were detected in mantle and haemolymph (P<0.01). The results suggest thatHcUrosexpression may be related to the intensity of the inner shell color.

3.3. In situ hybridization

In situhybridization revealed the presence ofHcUrosin the dorsal epithelial cells of the mantle pallia. No expression was observed in negative control cells(Fig. 4).

3.4. Temporal expression pattern of HcUros during pearl formation

To test the role ofHcUrosduring early pearl formation, qPCR was performed after implantation of donner mantle in the host. At 3 h,HcUroswas down-regulated in pearl sac tissue, but increased at 48 h and 96 h.HcUrosexpression returned to baseline levels by 7 days postimplantation, and rose sharply at 28 d when the pearl performed initially(Fig. 5).

Fig. 3. Tissue expression profile of HcUros in P- and W-line mussels, detected by quantitative RT-PCR. Indicate sample number and the meaning of ＊, ＊＊ etc. Explain here how relative expression was calculated.

Fig. 4. In situ hybridization analysis of HcUros in mantle tissue. A: ISH of HcUros expression in mantle tissue of P-line mussels; B: higher magnification image of A; C: background staining. OF: outer fold; MF: middle fold; IF: inner fold; DM: dorsal mantle; VM: ventral mantle.

In this study, the uroporphyrinogen III synthetase gene (HcUros) was identified fromH. cumingii. It was predicted thatHcUroscontained a conserved HemD domain and was closely related to theP. canaliculatahomolog. Therefore,HcUroswas predicted to belong to the HemD superfamily.

Fig. 5. Expression levels of HcUros in the pearl sac of P-line mussels after implantation.

Mollusk shell color formation is determined by porphyrin compounds. Porphyrins can be detected by fluorescence in mollusks (Comfort, 2010), and protoporphyrin can be found in mollusk shells (Verdes et al., 2015). Genes related to porphyrin and chlorophyll biosynthesis, as well as other metabolic pathways were identified by comparative analyses of gene expression profiles in individualRuditapes philippinarumwith different shell colors (Jiang, 2018). HcUrosin P- and W-line mussels was broadly expressed in all of the tissues evaluated, revealing thatHcUrosis involved in diverse physiological processes.

The expression ofHcUrosin haemolymph and the mantle in P-line mussels was significantly higher than that in W-line mussels. Based on the higher expression ofHcUrosin the haemolymph of P-line mussels,suggests it may participate in the synthesis of hemocyanin inH. cumingii.

Porphyrins usually exist as chromoproteins, and it is difficult to distinguish them from melanin. However, it has been found that color formation in bivalve shells is related to the mantles (Crockett et al.,1991). Therefore, the relationship between porphyrin compounds and shell color can be studied indirectly by evaluating mantle tissue. Additionally, the differential expression ofHcUrosin the mantle of P- and W-line mussels indicates that the differential accumulation of porphyrin compounds can lead to different shell colors. The distal mantle is further characterized by the enlargement of the sheath at the shell margin into three terminal folds: the outer fold, the middle fold, and the inner fold(Gardner, 2011). The outer fold is closest to the shell, while the inner fold is furthest from the shell.In situhybridization showed thatHcUroswas present in the dorsal epithelial cells of the mantle pallia, which is close to the nacre layer. Thus, those data suggest thatHcUrosmay be involved in nacre secretion.

The development of the pearl sac after implantation is an important step in the pearl production process, and directly affects pearl quality.According to previous research, there are many genes involved in the development of pearl sacs at different stages, including immune genes and mineralization genes (Huang et al., 2019; Jin et al., 2019a; Li et al.,2016; Wang et al., 2019). The secretion of pearls begins on the seventh day after implantation, at which time pearls and color begin to form, and continues until the thirty-fifth day (Bai et al., 2016; Jin, Zhao, & Liu,2019; Lin et al., 2013). Our data revealed a reduction inHcUrosexpression in pearl sacs 3 h after implantation, and that expression was increased at 48 h and 96 h after implantation.HcUrosexpression returned to baseline levels by 7 d post-implantation. Huang used comparative transcriptomic analysis to identify key immune-related genes. The results showed that there were remarkable changes of key immune-related genes from 0 h to 7 d (Huang et al., 2019). Based on those results, we inferred thatHcUrosis involved in an early immune response. The remarkable expression ofHcUrosat 28 d post--implantation also suggested thatHcUroswas involved in pearl formation, and its expression likely relates to pearl color.

In conclusion, we cloned the UROS gene fromH. cumingii. Higher levels ofHcUrosexpression were observed in the mantle of P-line mussels compared to W-line mussels, suggesting thatHcUrosplays a key role in mussel pigmentation.In situhybridization investigations ofHcUrosin the mantle revealed thatHcUrosmay be involved in nacre formation.Changes inHcUrostranscript levels after mantle implantation also indicated thatHcUrosmay play a role in the early stages of pearl formation. Thus, our collective findings suggest thatHcUroscontributes to shell pigmentation, and nacre formation and coloring inH. cumingii.

CRediT authorship contribution statement

Jiexuan Shen: Writing - original draft. Dandan Huang: Data curation, Methodology. Jiale Li: Supervision. Weixin Ye: Investigation.Zheng Wang: Investigation. Zhiyi Bai: Conceptualization.

Declaration of competing interest

We report no conflicts of interest.

Acknowledgements

This work was supported by the National Key R&D Program of China(2018YFD0901406), the National Natural Science Foundation of China(31872565), and sponsored by the Program of the Shanghai Academic Research Leader (19XD1421500).