Transcriptomic analysis of formation and release of monospores in Pyropia yezoensis (Bangiales, Rhodophyta)

Certain red algae (Pyropia yezoensis) of the genus Pyropia can be asexually reproduced by producing monospores, which provides a large number of secondary seeds for marine production. However, the molecular biological mechanism of how algal regulates its own monospores’ formation and release is still unclear, which brings great difficulties and challenges for regulating the yield of seaweed and discovering new strains. In this study, we compared and analyzed the data between PY26W' which release a large number of monospores and PY26W which release few monospores by Illumina sequencing platform. The number of DEGs produced by the PY26W' was much higher than that of PY26W, and upregulated genes dominantly in PY26W'. A total of 415 common DEGs were produced between comparison of PY26W and PY26W', which may be involved in formation and release of monospores. All the DEGs were highly enriched in the translation process, ribosome assembly, RNA methylation, assembly of actin and vesicles, intracellular localization of organelles, endocytosis and other biological processes and MAPK signaling pathways. Four DEGs (Contig-21827, Contig-15542, Contig-13390, Trinity-DN39215) were selected for real-time PCR, and the results were highly consistent with the transcriptome sequencing results in both strains, and their expression levels were significant. Further expression analysis indicated that Contig-21827 was abundantly expressed in Pyropia. suborbiculata, Pyropia. chauhanii and PY26W' but not in PY26W and Porphyra haitanensis (WT-8), indicating it plays a key regulatory role in the formation and release of monospores. This gene was further confirmed to be involved in the formation of monospores by analogous conidia formation pathway and was named PyMFG. This is the first time to investigate the formation and release of monospores in P. yezoensis from the RNA level. We successfully screened the monospores’ formation gene (PyMFG) of P. yezoensis and the chitinase

release, regulation of seaweed yield and discovery of new seaweed strains.

Background
Pyropia yezoensis [1] is mainly distributed in the Yellow Sea, Bohai Sea and the northern coast of the East China Sea as well as the coast of Japan and the Korean Peninsula, and its production plays an important role in the seaweed industry worldwide. Studies have shown that P. yezoensis uses filamentous sporophyte and leafy gametophyte to complete generational alternation. Besides, asexual reproduction in P. yezoensis provides a large number of secondary seeds for cultivation in the sea [2]. In the process of conversion into monospores, the vegetative cells of the algae change from red to golden yellow, and then they are released by monosporangia or germinated in situ [3]. With observation on the life history of different seaweeds, individual biomass is increased by producing monospores in Pyropia tenera, Pyropia suborbiculata, Pyropia chauhanii and so on [4][5][6]. Existing evidence suggests that the formation and release of monospores in Pyropia affect the size and quantity of algae to a certain extent, and determine its yield ultimately [7].
Current studies have confirmed that changes in culture conditions, such as light, temperature, seawater density and nutrients, can affect the formation and release of monospores of P. yezoensis [8][9][10][11]. Furthermore, the addition of exogenous substances, such as allantoin and ammonium sulfate, also has a significant effect on the release of monospores [10,12,13]. Early studies have indicated that P. yezoensis can produce mutants, which release monospores heavily by induction of methyl nitrosoguanidine [2]. Researchers have used 60 Coγ-rays to induce and separate strain of PC-Y1 that does not release monospores in recent years. Above-mentioned evidence indicates that the biological traits of formation and release of monospores are controlled by genes and can be stably inherited after several generations of cultivation [14].
At present, with the continuous improvement of the individual monospores, it has become the trend of current research to explore the formation and release of monospores at the molecular level.
Studies have shown that the amount of released monospores is closely related to the interspecific genetic distance of algae. The closer the genetic distance between the varieties of algae that can release monospores, the smaller the difference in the amount of released monospores [15]. In addition, some studies have pointed out that the molecular weight of cDNA is significantly reduced during the process of vegetative cells specializing in monospores, indicating that the expression of genetic information has been changed [16]. However, the significant expressions of genes involved in the Calvin cycle and the influence of allantoin on the release of monospores also illustrate that the mass formation and release of monospores are closely related to energy and purine metabolic pathways [13,17].
With the maturity and popularity of next-generation sequencing technology, transcriptomics is used to identify key genes controlling specificity in non-reference genomic organisms, to analyze the trend of gene expression during reproductive development, and to explain the complex molecules of specific traits. The mechanism has become a hot topic of current research. Therefore, we, in the present study, aimed to explore the key genes involved in the regulation of sporangium formation and release at the transcriptome level. Our findings provided valuable insights into the molecular mechanism underlying the formation and release of monospores in P. yezoensis.

Methods
Seaweed materials and monospore release PY26W (few monospores) and PY26W' (large-scale monospores) were used in the present study, and both of them were obtained by separating and purifying from the spottedly variegated foliose thalli produced by the hybridization between red mutant (fre-1,♀) which releases monospores heavily and wild type (U-511,♂) which releases few monospores, referred to as W and W' as follows. Separation and purification were performed as previously reported [18]. The genetic backgrounds of these two strains were similar, while their biological characteristics of monospores were significantly different, which are reliable materials for investigating the formation and release of monospores. In addition, P. suborbiculata (PS-WT), P. chauhanii (PC-WT) and P. haitanensis (WT-8) were also used in this experiment. Gametophytes of three variety were cultured at 23 ± 1 ° C under 50 μmol photons / (m 2 · s) with 10 L: 14 D light cycle. All of the above-mentioned algal varieties were saved in the laboratory in the form of free-living conchocelis filaments, and the preservation was conducted as Liu described [19]. Culture solution used for gametophytes was sterilized seawater, to which MES medium was supplemented [20].
On the 18th day of culture, 500 gametophytes with the same size and growth potential were randomly selected, and then the proportion of gametophytes releasing the monospores within 7 days was counted to confirm the starting age of the transcriptome sequencing. In addition, 30 gametophytes were randomly selected to count the total numbers of monospores per geametophyte during 18-35 days between W and W'. Three biological replicates were set for each group of experiments. First, each group of gametophytes was placed in a white plastic cup for aeration culture.
The gametophytes were removed with sterile scorpion on the next day, placed in a new plastic cup containing fresh culture medium and continued to culture. The previously released monospores were brushed down and placed under suitable conditions, and the culture was counted until the gametophyte of each strain was visible to the naked eyes. The culture conditions were as follows: 19 ± 1 °C, 40 μmol photons / (m 2 · s), 10 L: 14 D. The total amount of monospores released was obtained after 15 days of continuous monitoring, by which the ability of both strains to disperse monospores and the cut-off age of the sequencing samples were confirmed.
Transcriptome sequencing was performed in two comparisons. First, a comparison of samples in each strain was performed to obtain DEGs between day 25 and day 35, and then samples of the same age group were compared to obtain DEGs between W and W'.
RNA extraction, transcriptome de novo assembly, and annotation The gametophytes were washed with distilled water and blotted dry with absorbent paper.
Subsequently, the gametophytes with a wet weight of 0.1-0.3 g were placed in liquid nitrogen and rapidly ground, and their total RNA was extracted with Trizol reagent. The absorbance at wavelengths of 260 nm and 280 nm (A260/A280) was determined by NanoDrop 2000, respectively, and the integrity was examined by 1% agarose gel electrophoresis and Agilent 2200. After the RIN was confirmed to be greater than 6.0 in all sequencing samples, 2 μg or more RNA was used to construct a cDNA library. RNA sequencing was performed by the Illumina HiSeqXTen sequencing platform (NovelBio Bio-Pharm Technology Co. Ltd, Shanghai). Sequencing data were analyzed using the following processes. Firstly, the Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to perform an overall quality assessment of the raw sequencing data, and then the Trinity software was employed to perform de novo clean reads on a single sample. Furthermore, Cap3 was used to merge the assembly results to eliminate redundancy. The spliced sequences were then separately aligned to Arabidopsis thaliana, Aspergillus nidulans, Saccharomyces cerevisiae, Bacillus subtilis, Chlamydomonas and Porphyra purpurea to identify the homologous sequence between P. yezoensis and model species in order to determine the genetic annotation and functional prediction. Finally, mapping was performed using BWA software and BWA-MEM strategy, FPKM values were calculated, and the gene expression was reflected based on the FPKM values.

Differential expression analysis
In order to obtain the expression pattern of a certain differential gene, DEGs were screened by comparison between W or W' using the EBSeq algorithm by setting the criteria for differential screening at P-value<0.05, FDR≤0.05 and log 2 |fold change| ≥1. FDR and P-value were used to control the accuracy of comparison among multiple samples.
To further verify the gene expression at the mRNA level, four DEGs were selected for qRT-PCR verification according to the FPKM value ≥1 in W' 35d, the FPKM values of the other three samples were < 1, and the primers were designed in the open reading frame region using NCBI Primer Blast (Table 1). Firstly, RNA of each sample was reversely transcribed into cDNA using Takara reverse transcription kit (Takara, Japan). Secondly, qRT-PCR was performed with Takara's SYBR Green fluorescent dye and Bio-Rad CFX-96 real-time PCR system using cDNA as a template and PH18S as a reference gene. Three biological replicates were set for each gene, and three technical replicates were set for each biological replicate. The relative expression level of each gene was calculated according to the 2 -ΔΔCt method [13]. The program setting of qRT-PCR system was the same as previously described [21]. The experimental data were statistically analyzed using Origin 8.5 software, and the differences between the different data groups were compared by one-way ANOVA and LSD test. P < 0.05 indicates significant difference.
Gene expression pattern analysis and functional enrichment of DEGs

Statistical analysis
Relevant statistical analysis was carried out using R language-related packages.

Release of monospores
We found that the proportion of the gametophytes releasing monospores was continuously increased after the W' strain was cultured for 18 days, while such proportion at the 25th day did not exceed 30%, and the overall proportion was relatively small (Fig. 1a). Within 18-35 days, the total amount of monospores in each gametophyte of the W' strain could be as high as 1,100, and the amount of scatter was relatively large (Fig. 1b). However, only two monospores were found in the W, which released less monospores (Fig. 1a, Fig. 1b) higher than that at 35 days, but the proportion of up-regulated DEGs at 35 days was larger (Fig. 2).
Moreover, the number of DGEs in W during the 25-35 days of culture was significantly less than that in W'. All FPKM values in different samples were shown in Additional file 1: Table S1.
In addition, the consensus DEG analysis showed that there were 415 shared DEGs in the two comparison groups between W and W', while only 64 DEGs were found in the comparison group in the same strain (Fig. 3). Original specific statistics was shown in Additional file 2: Table S2.

Analysis of GO and pathway enrichment of DEGs
There were 415 DEGs between the W and W' comparison groups at the same age, and these genes might be involved in the formation and release of monospores. GO enrichment analysis showed that its function was highly enriched in biological processes, such as translation, RNA activity, actin and vesicle assembly, intracellular localization of Golgi and mitochondria, and peroxisome activity. Among them, the number of DEGs involved in translation, RNA methylation and ribosome assembly was high (Fig. 4). Moreover, KEGG analysis revealed that the above-mentioned DEGs were mainly enriched in ribosome assembly, endocytosis, amino acids (Gly/Ser/Arg/Val), carbon and nitrogen metabolic pathways, photooxidation, as well as AGE-RAGE and MAPK signaling pathways (Fig. 5). Raw data of GO and Pathway analysis can be seen in Additional file 3: Table S3 and Additional file 4: Table S4.

Analysis of expression patterns of candidate DEGs
Comprehensive genome annotation, GO enrichment (Fig. 4) and pathway signaling pathway analysis were generally consistent with RNA-Seq data. Besides, the relative expressions of these four genes in W' were significantly different from those of the W (Fig. 7). The expression of Contig-21827 in different speciesof Pyropia and strains was further analyzed. The results showed that it was abundantly expressed in P. yezoensis (W'), P. suborbiculata and P. chauhanii, and the difference was significant among species. However, it was not expressed in the P. haitanensis (WT-8) and P.
yezoensis (W) (Fig. 8). These results suggested that the function of this gene was related to the formation and release of monospores. nidulans, and the qRT-PCR results confirmed that the expression of Contig-21827 was highly consistent with the macroscopic statistical results (Fig. 1, Fig. 7, Fig. 8). Therefore, the genes involved in the traits of monospore could be found in the pathway of maturity and release of fungal conidia.
Furthermore, the molecular mechanism of the interaction could be elucidated. AbaA and WetA [22][23][24]. Previous studies have confirmed that the specific expression of abaA promotes the production of conidial head and maintains conidial production [25][26][27]. BrlA is located upstream of abaA, which can induce the specific expressions of abaA and wetA without the involvement of upstream activators to produce fluffy conidial heads. WetA is located downstream of abaA, which regulates the conidium maturation and synthesizes trehalose to maintain normal conidial walls [27][28][29]. According to the conidial formation pathway and the expression of Contig-21827 in different species of Pyropia, it was preliminarily believed that the gene was involved in the formation of monospores, and it was named PyMFG. However, based on the current annotation information, the homologous sequences of the brlA and wetA genes were not found in P. yezoensis, indicating that the formation process of monospores in P. yezoensis might not be exactly the same as the regulation of the formation of A. nidulans.

Vesicle transport
Recent studies have found that GTPase-binding proteins involved in A. thaliana secreted protein trafficking are highly homologous to the Ypt protein family in S. cerevisiae [30]. Among them, the Rab, Arf and Ras GTP protein kinase subfamilies mediate intracellular vesicle trafficking through interaction with GEFs [31]. However, the intracellular vesicle trafficking process is mainly mediated by Sec and Ypt proteins in yeast. Ypt1p regulates the secretion and fusion of secreted proteins of S.
cerevisiae, Ypt31p and Ypt32p are responsible for the transport of secretory vesicles inside the Golgi.
Sec4p mediates Golgi vesicle transport to the cytoplasmic membrane. However, Ypt7p mediates vacuolar fusion during the in vitro ligation of homozygous yeasts [32][33][34][35][36][37]. In the present study, the Rab/Ypt homologous protein family genes were found in comparison with the genomes of A. nidulans and S. cerevisiae. These genes were abundantly expressed in W' (Fig. 6). Otherwise, the expressions of DEGs generated by the comparison between W and W' were significantly enhanced in translation, RNA activity, assembly of actin and vesicles, and biological processes, such as Golgi and mitochondria, peroxisome activity, and endocytosis (Fig. 4, Fig. 5), which was consistent with the study at the cellular level [16]. Therefore, we speculated that P. yezoensis transported functional proteins required for various life activities in the form of vesicles, thereby achieving the effect by rapidly coping with its own stress about the formation and release of monospores.

System of signaling pathway
The Ca 2+ signaling pathway mainly relies on Ca 2+ /CaM and calcineurin to initiate intracellular responses and participate in a variety of biological processes in eukaryotes [38,39]. Collectotrichum.
gloeosprioides and C. trifolii begin to express the cam early in sporulation [40]. In addition, the endogenous Ca 2+ signaling pathway regulates the germination and fusion of Neurospora crassa germ tube, which is closely associated with its intact colony morphology [41]. We found that there were a large number of up-regulated calmodulin homologous genes in W' (Fig. 6), which fully demonstrated that the formation and release of monospores activated Ca 2+ signaling pathways in cells, thereby stimulating the transmission of CaM to extracellular signals. Previous study has indicated that under normal irradiance, the monosporangia of P. yezoensis can influx low-level Ca 2+ and induce intracellular signaling pathway to produce a large amount of IP 3 , which promotes the formation and release of a large amount of monospores, resulting in serious disintegration in algae [42]. However, the absence of midA leads to the imbalance of mannan and chitin components in the cell wall according to the study of midAmutant of A. nidulans, by which conidia are forced to flow out [43].
We suspected that the Ca 2+ influx involved in the initial germination of monospores activated the Ca 2+ signaling pathway, which eventually caused changes in cell wall components and cytoskeleton, thereby achieving monospores.
As a Ser/Thr protein kinase, MAPK plays a key role in gene expression and cytoplasmic activity.
Sequencing data showed that the MAPKK kinase gene was highly enriched in the sample W' 35d ( Fig.   6), and the MAPK signaling pathway was significantly enriched in the top 20 significant signaling pathways (Fig. 5). At present, the amplification reaction of MAPK cascade in animal and yeast systems can produce a variety of stress responses by regulating the expressions of related genes [44]. In addition, many biological processes, such as spore formation pathway, cell wall integration pathway In addition, the cytoplasmic flow of cells is also severely blocked due to the lack of dynein and multiple kinesin family genes [48]. However, algae in the Pyropia genus capable of dispersing monospores can achieve cytoplasmic flow and cell morphological changes in the formation of monospores. We also found that some actin and myosin homologous genes were highly expressed in W', suggesting that the intracellular components and assembly complexity of actin and myosin in this type of algae were higher than those of Porphyra genus. Therefore, it could regulate the morphological changes of vegetative cells and cause the separation of protoplasts.

Hydrolase of cell wall
The current research has confirmed that the conchase and agarase isolated from marine animals and microorganisms, such as conch, sarcophagus and Pseudomonas, can effectively decompose the cell wall of vegetative cells of algae [49,50]. However, it remains unclear whether the algae itself can produce enzymes to degrade cell wall. We successfully screened the chitinase homologous gene based on the genome of A. nidulans in P. yezoensis. Besides, the expression of this gene was significantly increased in W' 35d samples with a large number of monospores (Fig. 6). Chitinase belongs to the family of glycosyl hydrolases, which can effectively decompose the conidial wall of fungi with chitin and polysaccharide as main components to achieve spore development and cell autolysis [51][52][53][54][55][56]. In addition, the FluG-BrlA feedback pathway of A. nidulans auxotrophic strains can also produce chitinase to decompose cell walls and organelles to autolyze cells, leading to further release of conidia [28,56]. Rac-1 gene in N. crassa breaks down plant cell walls by synthesizing polysaccharide hydrolases, such as lignocellulase [57][58][59][60][61]. All of above-mentioned evidence indicated that the chitinase homologous gene in P. yezoensis was very likely to be involved in the degradation of the cell wall of monospoorangia, thus accelerating the release of monospores.

Conclusions
The transcriptome data suggested that more DEGs were obtained in W' than W in comparison group of different age, and these DEGs were mainly up-regulated. A comparison between W' and W strains revealed that more DEGs were identified in 25-day samples, the proportion of up-regulated DEGs in 35-day samples was higher, exhibiting obvious trait of monospore (Fig. 2). Besides, the consensus DEGs in the comparison group between W and W' (Fig. 3) were involved in biological processes, such as translation, actin and vesicle assembly, ribosome activity, endocytosis, phagosomes and peroxisomes, and these DEGs were significantly enriched in pathways, such as MAPK (Fig. 4, Fig. 5 Table 2 Assessment of transcriptome sequencing data quality and gene alignment rate in P. yezoensis. Figure 1 Comparison of age point and ability of monospores' release between gametophytes of W and W'. a Percentage of gametophytes that release monospores between W and W'. b Total numbers of monospores that release in per blade between W and W'.      Relative expression analysis about selected DEGs by qRT-PCR between W and W'. "*" represents significant difference with control (P<0.05).