miR8181 is Involved in the Cell Growth and Development Regulation of Saccharina Japonica

Background: Aureochrome, a blue-light receptor found in photosynthetic stramenopiles, plays an important role in brown algal growth and development. Aureochrome preserves the reversed effector-sensor domain for blue light reception and acts as the candidate optogenetic tool for light induced post-transcriptional regulation, but the inner rapid regulation of aureochrome remains to be studied. MicroRNA (miRNAs) of plant can cleavage the specic base-pairing site of mRNA by RNA interference mechanism, and such post-transcriptional regulation of miRNAs to photoreceptor has received attention due to the exible regulation pathway. However, the targeting relationship between aureochrome and miRNA is unclear. Results: In this study, the potential regulatory network between miRNAs and aureochrome were explored by transcriptome and sRNA sequencing in Saccharina japonica. Our results found that 18 miRNAs perfectly paired with aureochrome. Among the screened miRNAs, miR8181-x was negatively correlated with aureochrome5 with high credibility and exhibited tissue-specic expression in S. japonica. Degradome sequencing detected the exact cleavage site of miR8181-x on aureochrome5, conrming their targeting regulation relationship. Among the 54 target genes of miR8181-x, nine genes of ABC transporters, E3 ubiquitin-protein protein ligase, Hsp90, Mx1, PetC, EF2, GSA, HAD-superfamily hydrolase and SET2 that exhibited similar expression with aureochrome5 competed with the same binding site, thus constructing the competing endogenous RNA network. Functional analysis of miR8181-x target genes revealed that regulation of cell differentiation and development was enriched, indicating the potential role of miR8181-x in the regulation of growth and development. Conclusion: Our study found that miR8181-x negatively regulated the expression of aureochrome5. The exact cleavage site in aureochrome5 were veried by degradome sequencing, conrming the targeting relationships. Functional enrichment of miR8181-x target genes revealed that miR8181-x involved in the cell growth and development regulation


Background
Light serves as a biological stimulus to trigger signal transduction pathways via speci c photoreceptors. The responses of photoreceptors to light wavelength, direction and duration of light illumination are important for marine plant growth and development [1,2,3]. Similar to green algae, photosynthetic stramenopiles acquire the blue light responses of phototropism, chloroplast photorelocation and photomorphogenesis from secondary symbiosis [4]. Aureochrome is classi ed as new type of blue light receptor in photosynthetic stramenopiles [5], and it consists of a basic region/leucine zipper (bZIP) domain at the N-terminus and a light-oxygen-voltage (LOV) domain at the C-terminus. The DNA-binding region in the bZIP domain includes a region that is rich in basic amino acids and a heptad leucine repeat zipper [6]. The LOV domain is composed of 11 well-conserved amino acids that are responsible for FMN binding and receiving the environmental signal [7,8]. SjAUREO from the brown alga Saccharina japonica exhibits 40%-92% similarities with other photosynthetic stramenopiles [9]. Yeast two-hybrid screening has demonstrated a strong interaction between SjAUREO and the 40S ribosomal protein S6, which might be involved in blue light-mediated cellular division and photomorphogenesis [10]. In the single cellular diatom Phaeodactylum tricornutum, PtAUREO1a and PtAUREO1b mutants exhibit signi cantly decreased photoacclimation to blue light [11,12]. AUREO1 in Heterosigma akashiwois may act as a blue light-sensitive negative gene regulator, which is driven by dark-to-light transitional cues [13]. Although the function of aureochrome has been veri ed in various species, further details regarding the transcription regulation of aureochrome remain to be further explored.
MicroRNAs (miRNAs) play an important role in post-transcriptional regulation [14,15], and plant microRNAs can trigger endonucleolytic cleavage of base-pairing mRNA targets via a RNA interference mechanism [16]. Animal-like mechanisms that repress mRNA translation with the assistance of both agronaute proteins and a microtubulesevering enzyme also exist in plants [17,18]. Additionally, competing endogenous RNAs (ceRNAs) that share the common miRNA response element reduce miRNA activity, leading to the depression of speci c mRNAs [19].
MiRNAs from different lineages are nonhomologous but have strongly conserved structures, such as the presence of uracil at the rst residue. The developmental plasticity mediated by miRNAs allows plants to e ciently cope with environmental stress [20]. A previous study has revealed that mature miRNAs are involved in the red light signalling pathway, which is involved in plant photomorphogenesis [21]. The majority of miRNA target genes have been identi ed as transcription factors and may function in plant development, boundary formation and organ polarity [22].
For low tidal zone inhabitants with blue light irradiance, light reception and subsequent signal ampli cation are crucial for the S. japonica development and growth. Although miRNAs from the lineage of S. japonica have been identi ed [23,24], the interaction between miRNAs and target genes have received less attention. With integrative analysis of the transcriptome, sRNA and degradome sequencing data, the miRNAs that target to aureochrome were screened, and the exact cleavage sites were veri ed in this study. By functional enrichment of targeting genes at genome-wide levek, miRNA functions were also further investigated. Our aim was to verify the candidate miRNAs that target aureochrome and to explore the regulatory role of aureochrome in brown algae.

Results
Identi cation of miRNAs targeting aureochromes By searching the whole genome data of S. japonica, ve homologous sequences encoding aureochrome were screened. Apart from aureochrome2 and aureochrome3, the remaining three homologs, namely aureochrome1, aureochrome4 and aureochrome5, were signi cantly upregulated in response to blue light (Tukey's tests, p < 0.05; Fig. 1A), indicating their sensitivity in response to bliue light. Based on the complementarity interactions between miRNAs and target genes, we performed homology predictions for the miRNA target genes with Patmatch (v1.2) software. In total, We obtained 18 miRNAs that may target aureochromes (Table S1), and constructed a miRNAaureochrome network (Fig. 1B).
The transcription level of miR818-x was downregulated in response to blue light, which was opposite to the aureochrome5. qRT-PCR analysis veri ed the opposite expression tendency between aureochrome5 and miR8181x (Fig. 2, 3), indicating the negative regulation relationship. Moreover, the transcription levels of miR8181-x in the kelp blade was signi cantly higher than that in the holdfast (Tukey's tests, p < 0.05; Fig. 3), suggesting that miR8181-x exhibits tissue-speci c expression patterns in S. japonica.
Cluster analysis of miR8181-x target genes By integrating high-throughput sequencing of mRNAs and sRNAs, Patmatch software predicted 1915 mRNAs as potential targets of miR8181-x (Fig. 4A). According to their transcription patterns, we separately grouped these targets by hierarchical clustering. These target genes were classi ed into six clusters based on their degrees of transcription. In cluster 4, 311 genes were upregulated in blue light compared to dark and white light conditions ( Fig. 4B), exhibiting similar expression patterns with the aureochrome5 transcript, thus screening as the candidate ceRNAs.

Target gene identi cation for miR8181-x by degradome sequencing
To validate the target cleavage relationship between miR8181-x and mRNAs, we constructed three degradome libraries (BL, DR, and WL) and performed sequencing using the Illumina Hiseq 2500. After removing low-quality sequences, 6019083, 4907605, and 6578179 clean reads were obtained for the BL, DR, and WL libraries, respectively ( Table 1). The obtained sequences were mapped to the reference genome of S. japonica registered in the NCBI database (accession: MEHQ00000000). Finally, 2614536, 2018907, and 2387139 sequences were mapped to the reference genome (Table 1). These mapped sequences were analyzed to identify target genes of miRNAs. In total, 55 mRNAs that included aureochrome5 were identi ed to be cleaved by miR8181-x ( Table 2). DAVID was used to determine the functional analysis of miR8181-x target genes. The results showed that miR8181-x might be involved in the cellular component (CC) category with the following enriched terms: "organelle", "cell part", "macromolecular complex" and "membrane". The following terms in the molecular function (MF) category were signi cantly enriched: "binding", "catalytic activity", and "transporter activity" (Fig. 5). In the biological process (BP) category, the following terms were signi cantly enriched: "growth", "reproduction", "singleorganism process", "localization", "response to stimulus" and "developmental process" (Fig. 5). Together, these results indicated that miR8181-x plays important roles in growth and development of brown alga. Moreover, ABC transporters, E3 ubiquitin-protein protein ligase, Hsp90, Mx1, PetC, EF2, GSA, HAD-superfamily hydrolase and SET2, which were previously classi ed in cluster 4 ( Fig. 4B), were veri ed as the target mRNAs ( Fig. 6, 7A). The scores ranged from 2 to 5, and their functions were various, including defense, energy production and conversion, coenzyme transport and post-translational modi cation (Fig. 7B). These nine mRNAs may serve as ceRNAs of aureochrome5 as they share common miRNA response elements and inhibit normal miR8181-x-targeting activity.

Discussion
By integration analysis of the miRNA and mRNA high-throughput sequencing data, we found 18 candidate miRNAs targeting aureochrome orthologs. Among the candidate miRNAs, the level of novel miRNAs was signi cantly higher than that of known miRNAs. The high levels of novel miRNAs in brown algae are attributed to the rapid evolution process [24], which had signi cant consequences for miRNA function. Among the 18 candidate miRNAs, miR8181-x was identi ed to target the blue light receptor, aureochrome5,with high reliability. Moreover, the cleavage site of miR8181-x on aureochrome5 was identi ed by degradome sequencing, verifying their targeting relationship. Plant miRNAs cleave the RNA strand at the binding site by a RNA-induced silencing complex with negative correlation between the expression of miRNA and target gene [25]. In our study, miR8181-x showed a negative correlation with aureochrome5 in response to light irradiation, and this negative correlation was also found in different tissues of S. japonica. The tissue-speci c expression pattern of miR8181-x suggested that its fundamental roles include maintaining tissue development.
A previous study has reported that ceRNAs play important roles in miRNA-mediated posttranscriptional regulation of gene expression [26]. Here, based on targeting relationship and transcriptional expression pro les, we constructed the ceRNA regulation network of miR8181-mRNA, which may regulate the aureochrome5 transcript. The mRNAs in the ceRNA network were involved in various pathways, indicating the intricate regulatory relationship. In the ceRNA network, one nuclear gene encoding petC had competing roles with aureochrome5 for light sensing in kelp. PetC plays a regulatory role with other cytochrome b6f subunits [27], and overexpression of petC enhances the light conversion e ciency and CO 2 assimilation rate [28]. The competing endogenous relationship between aureochrome5 and petC might act as bridge to connect the light sensing and energy production in kelp.
In green plants, the miR8181 family is involved in anthocyanin biosynthesis [29], but the function of miR8181 in algae remains unclear. Integrated analyses of target veri cation and functional enrichment provided clues for elucidating the precise underlying mechanisms of miR8181-x. We found that the miR8181-x-mediated regulatory pathway involved glycolysis/gluconeogenesis for energy supply, and the most common processes included cell differentiation, cell cycle and cell development in the kelp. Therefore, we speculated that miR8181-x might regulate cell growth and development in S. japonica. After removing reads containing adapters or low-quality bases, all clean tags were aligned with small RNAs in the Rfam and GenBank databases (Release 209.0) with our previous S. japonica genome data registered in the NCBI database (accession: MEHQ00000000). The rRNA, scRNA, snoRNA, snRNA and tRNA sequences were ltered out.
All clean tags were validated using the miRBase database with known miRNAs. We selected the following prediction criteria for novel miRNAs: length, 18-25 nt; maximal free energy allowed for a miRNA precursor, 18 kcal/mol; space between miRNA and miRNA*, 14-35 nt; maximal asymmetry of the miRNA/miRNA* duplex, 5 nt; and ank sequence length of miRNA precursor, 10 nt. Finally, the identi ed miRNAs were predicted by Patmatch (v1.2) software. The minimum free energy of the miRNA/target duplex was set at ≥ 74% and there were no more than two adjacent mismatches in the miRNA/target duplex and no mismatches at positions 10-11 of the miRNA/target duplex.

Target gene function enrichment analysis
Blast2Go was employed for exploring the Gene Ontology (GO) annotation terms. Database for Annotation, Visualization and Integrated Discovery (DAVID) was adopted for pathway analysis with the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database [30,31]. P values less than 0.05 indicated enriched gene sets.  Table S2. Actin and U6 were adopted as internal control markers, and the relative expression of the miRNAs was calculated by the 2 −ΔΔCt method. All qRT-PCR tests were performed with three biological replicates.

Degradome library construction and data analysis
Three degradome libraries (DR, BL and WL) were constructed using the juvenile sporophytes of S. japonica. Following the enrichment of mRNA, the obtained poly(A)-enriched RNA was ligated to oligonucleotide adaptors harbouring an MmeI recognition site. First-strand cDNA was generated from the ligated sequence via reverse transcription. After PCR ampli cation, the additional DNA products were yielded. After puri cation, digestion and ligation, the cDNA library was subjected for sequencing with Illumina Hiseq 2500.
Raw data obtained from HiSeq sequencing were processed to lter out the low-quality tags. The 5′ adapters, 3′ adapter contaminants, insert tags, and reads shorter than 18 nts were removed to obtain clean data, which were further mapped to the reference genome of S. japonica. By performing Blastn searches against the Rfam and National Centre for Biotechnology Information (NCBI) databases with an E-value cutoff of 10 − 2 , the full-length sRNA tags were annotated to non-coding RNAs, and all of which were discarded. Additionally, t-plots were constructed according to the category of sites to analyse the miRNA targets and RNA degradation patterns.

Statistical analysis
Statistical differences were examined using one-way analysis of variance (ANOVA) by SPSS 22.0. P-values less than 0.05 were considered to be signi cant. Availability of data and materials The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Competing Interests
All the authors have approved the manuscript and agree with submission to your esteemed journal. There are no con icts of interest to declare.      Targets plots of the targets cleaved by the miR8181-x. The T-plots showed the distribution of the degradome tags along the full-length of the target mRNA sequence.