Novel-m2866-5p and miR8181-x are involved in the regulation of aureochrome and photosynthesis in Saccharina japonica

Background: Aureochrome, a blue-light receptor only in photosynthetic stramenopiles, plays an important role in algal growth and development. Previous researches have reported that aureochrome is involved in the photoacclimation, cell division and sex organ development. It holds reversed effector-sensor topology for the reception of blue light, acting as the candidate of optogenetic tools in transcription regulation. However, the inner regulatory mechanism of aureochrome is still unclear. In this study, we explored the expression proles of microRNAs (miRNAs) and mRNAs, aiming to construct the regulatory network between miRNAs and aureochrome. Results: Our results showed that 18 identied miRNAs have potential aureochrome regulatory function. Among the screened microRNAs, both novel-m2866-5p and miR8181-x exhibited a negative correlation with Aureochrome5. The transcription of novel-m2866-5p and miR8181-x exhibited tissue specicity, indicating that these two microRNAs might function in different tissues. Moreover, miR8181-x could regulate kelp growth, while novel-m2866-5p was possibly involved in the arginine metabolic pathway. Aureochrome5 and photosynthetic genes exhibited similar expression trends, suggesting coordination between photosynthesis and blue light receptors. Moreover, the proton gradient regulation 5 (PGR5), which functions in the induction of energy-dependent quenching, exhibited a co-expression pattern with Aureochrome5. These patterns were further observed upon incubation with N,N’-dicyclohexylcarbodiimide (DCCD), which exhibited up-regulation of both PGR5 and Aureochrome5 and conrmed their endogenous competition. Conclusion: Our study revealed that both novel-m2866-5p and miR8181-x were involved in the regulation of aureochrome, and that miR8181-x could act as a bridge between blue light receptors and photosynthesis in kelp.


Background
Light serves as a biological stimulus that triggers signal transduction pathways via a speci c photoreceptor. The responses of photoreceptors to dynamic changes in the wavelength, direction and duration of light illumination are important for seaweed growth and development (Jiao et al. 2007;Masuda et al. 2008;Jaubert et al. 2017). Following the absorption of light energy, photosynthetic antenna pigments act as a driving force for photosynthesis, which triggers the splitting of H 2 O and regeneration of NADPH by the electron transport chain (Nixon and Mullineaux, 2001). The association between photoreceptor and photosynthesis has been elucidated in the green alga Chlamydomonas reinhardtii (Petroutsos et al. 2016;Allorent et al. 2017). Blue light receptors (phototropins) control energy-dependent non-photochemical quenching (qE) by inducing light-harvesting complex stress-related protein 3 (LHCSR3) expression under high light intensity (Petroutsos et al. 2016). Induction of the expression of a UV photoreceptor (ultra-violet resistance locus 8, UVR8) on LHCSR1 and photosystem II subunit S (PsbS) has been documented (Heijde and Ulm, 2013;Allorent et al. 2017). These studies revealed that both phototropins and UVR8 can protect green algae from over-reduction in electron transport chain.
Similar to the primary symbiosis exhibited by green algae, photosynthetic stramenopiles acquire the blue light responses of phototropism, chloroplast photorelocation, and photomorphogenesis from secondary symbiosis (Matiiv and Chekunova, 2018).
Aureochrome is new type of blue light receptor in this clade, belonging to the species Vaucheria frigida (Takahashi et al. 2007). Aureochrome consists of a basic region/leucine zipper (bZIP) domain at the N-terminus and a light-oxygen-voltage (LOV) domain at the C-terminus. SjAUREO from the brown alga Saccharina japonica was shown to share 40%-92% sequence similarity with other photosynthetic stramenopiles (Deng et al. 2014). Yeast two-hybrid screening further proved the strong interaction between SjAUREO and the light-harvesting complex (LHC) in S. japonica (Luan et al. 2019), indicating that photosynthesis and aureochrome are associated at the protein level. Moreover,PtAUREO1a and PtAUREO1b mutants exhibit signi cantly decreased photosynthetic potential in Phaeodactylum tricornutum (Coasta et al. 2013;Mann et al. 2017). However, details regarding the association between aureochrome and photosynthesis remain to be further explored.
MicroRNAs (miRNAs) play an important role in post-transcriptional gene regulation via base paring with their complementary sequences (Bartel, 2004;Voinnet, 2009). Tarver et al. (2015) noted that the miRNAs in brown algae have evolved independently with ve other eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges and slime moulds. The developmental plasticity mediated by miRNAs allows plants to e ciently cope with environmental stress (Song et al. 2019). A previous study reported that mature miRNAs participate in the red light signalling pathway to regulate plant photomorphogenesis (Sun et al. 2018).
Although miRNAs from the lineage of brown algae have been identi ed (Cock et al. 2010), the interaction of miRNAs with algal photoreceptors is unclear. Additionally, competing endogenous RNAs (ceRNAs) that share the common miRNA response element can reduce miRNA activity, leading to the derepression of speci c mRNAs (Gupta, 2014). Therefore, it is important to explore the in uence of the miRNA regulatory network on aureochrome in brown algae.
Incubation in low tidal zone inhabitants with blue light irradiance, light reception and subsequent signal ampli cation are crucial for the development and growth of S. japonica. Through analysis of mRNA and miRNA data, we aimed to explore the relationship between miRNA and aureochrome, to verify the associated miRNAs and to examine the function of these miRNAs in the regulation of the blue light receptor followed by photosynthesis. Our study will be aid the understanding of the role of miRNAs in the regulation of kelp growth and development.

Results
Identi cation of miRNAs that targeted aureochromes By conducting a high-throughput mRNA array to test the response of S. japonica to light quality, we screened ve homologous sequences that encoded aureochrome. In addition to Aureochrome3, the remaining four homologs were upregulated in response to blue light (BL) (Fig. 1). Based on the complementarity interactions between miRNAs and target genes, we made homology predictions for the miRNA target genes with Patmatch (v1.2) software. In turn, we obtained 18 miRNA sequences targeting aureochromes (Table. 1), constructing one miRNA-aureochrome network (Fig. 2). The two candidate miRNAs, namely, miR8181-x and novel-m2866-5p, targeted Aureochrome5 with high delity (Fig. 3). The conserved miRNA miR818-x, 18 nt in length, was classi ed into the miR8181 family. The newly identi ed novel-m2866-5p with 20 nt exhibited stem loop structures (Fig. S1), and both miR8181-x and novel-m2866-5p were down-regulated in response to BL, which was the opposite to the result obtained for aureochrome5 (Table. 2).
qRT-PCR analysis of miR8181-x and novel-m2866-5p showed that these two miRNAs were negatively correlated with aureochrome5 ( Fig. 4), and the transcription levels of these miRNAs were signi cantly higher in the kelp blade than in the holdfast, whereas the Aureochrome5 transcription patterns were the opposite.
Cluster analysis of the miRNA target genes In total, 1915 sequences and 31 mRNAs were screened as potential targets of miR8181-x and novel-m2866-5p, respectively. According to these transcription patterns, we separately grouped these genes by hierarchical clustering. The target genes of novel-m2866-5p were grouped into 4 clusters, and those from cluster 1 was signi cantly up-regulated in response to BL (Fig. 7). In cluster 1, Kif6 exhibited a similar trend as Aureochrome5. To further screen the related target sequences of miR818-x, a total of 1915 sequences were classi ed into 6 clusters based on their degrees of transcription using TCseq. There were 311 genes (including PsbO, cluster 4) that were signi cantly up-regulated in response to BL than dark and WL conditions (Fig. 8). Moreover, we conducted GO and KEGG analysis to target sequences in cluster 4, and functional analysis indicated high transcription levels of sequences associated with kelp growth, establishment of localization, cytoplasmic transport, photosynthesis, ubiquitin-mediated proteolysis, RNA transport, and carbon xation ( Fig. 9).

Functional prediction of objective miRNAs
We used DAVID to determine the KEGG and GO pathway for biological analysis of target genes. The results showed that miR8181-x was possibly involved in the cellular component category, with the enriched terms "organelle", "cell part", "macromolecular complex" and "membrane". In the molecular function (MF) category, "binding" and "catalytic activity", and "molecular function regulator" were signi cantly enriched (Fig. 10a). In the biological process (BF) category, "metabolic process", "cellular process", "single-organism process", "localization", "response to stimulus", and "developmental process" were signi cantly enriched. KEGG pathway analysis revealed that the top three pathways were glycolysis, cyanoamino acid metabolism, and biosynthesis of amino acids (Fig. 10b). The number of target sequences of novel-m2866-5p was lower than that of miR8181-x. GO analysis results showed that "metabolic process", "cellular process", and "single-organism process" were the three most common BP terms and the most highly enriched MF terms were "binding" and "catalytic activity" (Fig. 11).
ceRNA network construction and functional analysis Based on the target analysis, we further screened the negative-correlation pairs between the miRNAs and mRNAs. A total of 311 sequences and 4 mRNAs were screened as the potential ceRNAs of miR8181-x and novel-m2866-5p,respectively (Fig. 12a). Functional analysis of these ceRNAs indicated that they were involved in metabolic pathways, biosynthesis of secondary metabolites and carbon metabolism (Fig. 12b). Moreover, the proton gradient regulation 5 (PGR5), which played an important role in metabolic pathways, was also screened and can be regarded as one of the potential ceRNAs of Aureochrome5.

Dynamic transcript regulation of photosynthetic electron transport
In response to BL, PsaA and PsaD in the photosystem I, PetA and PetD in the cytochrome b6/f complex, and ATP synthase were upregulated, but PsbA and PsbB in photosystem II were down-regulated (Fig. S2a). In addition to PsbA and PetA, the transcription patterns under WL coincided with those under BL (Fig. S2b).
Effect of DCCD on the photosynthetic ability of S. japonica During cultivation under WL and BL, the non-photochemical quenching (NPQ) index was signi cantly increased. After adding N,-N'dicyclohexylcarbodiimide (DCCD), NPQ was down-regulated under BL, which was accompanied by a decrease in qE (Fig. 13, 14). The decrease in NPQ was also consistent with the decrease of Fv/Fm and Rfd, which indicated the decrease of photosynthetic capacity. Additionally, the transcription of PGR5 was up-regulated under BL, and Aureochrome5 exhibited a co-expression pattern with PGR5 ( Fig. 15).

Discussion
By integration analysis of the miRNA and mRNA data, we found that there were 18 candidate miRNAs that targeted aureochrome orthologs. Both Aureochrome1 and Aureochrome5 had different miRNA-binding sites, indicating their complex regulatory network. Among the candidate miRNAs, the level of novel miRNAs was signi cantly higher than that of known miRNAs. The high amount of novel miRNAs in brown algae was attributed to the rapid evolution process (Cock et al. 2017).
Usually, miRNA can splice or cleave the RNA strand at the binding site by guiding the RNA-induced silencing complex and, thus, the transcription of miRNA is negatively correlated with targeted gene transcription (Denzler et al. 2014). In our study, the conserved miRNA miR8181-x and novel miRNA novel-m2866-5p were negatively associated with aureochrome5 in response to light irradiation. The differential expression of these two miRNAs in the blade and holdfast indicated their complex and independent roles in kelp tissue. Due to the vertical cultivation of kelp, the holdfast is closer to the sea surface than the other part of the blade. The high transcription level of aureochrome5 in the holdfast indicated the rapid response to increased light irradiation.
In addition to Aureochrome5, novel-m2866-5p and miR8181-x were presumed to target 30 and 1914 gene sequences, respectively. A small number of target genes of novel-m2866-5p revealed signi cant enrichment of KEGG pathways associated with arginine metabolic processes. In green plants, the miR8181 family is involved in anthocyanin biosynthesis (Sun et al. 2017). KEGG pathway analysis of the miR8181-x target genes revealed that the genes were mainly involved in glycolysis/gluconeogenesis for energy supply. GO enrichment analysis indicated that the most common processes are cell differentiation, cell cycle and cell development, indicating the involvement of miR8181-x in the regulation of growth and development in S. japonica.
BL promotes the growth of S. japonica sporophytes through photosynthetic processes (Wang et al. 2010;Deng et al. 2012). Among the photosynthetic electron transport components, PSII is the most sensitive site of the photosynthetic electron transport chain in the response to environment interference (Chen et al. 2016). In our study, both the PsbA and PsbB PSII subunits were down-regulated, indicating the inhibition of PSII activity. These results are also consistent with the reversible decrease in PSII quantum yield, which may an effective approach to increase the photoprotection capacity via a rapid increase in NPQ. Therefore,the kelpcan su ciently cope with BL and resist the damage caused by excess light. Moreover, the manganese-stabilizing protein PsbO in the oxygen-evolving complex exhibited a similar expression trend as Aureochrome5. An increase in PsbO levels could cause the splitting of H 2 O to oxygen and protons and provide electrons for the photosynthetic electron chain (PET), contributing to the occurrence of photosynthesis.
Therefore, kelp can coordinate association aureochromes and photosynthesis in response to irradiation.
In this study, PGR5 was identi ed to compete for the common response element of miR8181-x with Aureochrome5, suggesting the ceRNA relationship between PGR5 and Aureochrome5. PGR5 could induce thermal dissipation by adjusting the production of the trans-membrane proton gradient, and the absence of the PGR5 protein in chloroplasts could inhibit NPQ occurrence (Kawashima et al. 2017;Sato et al. 2018). Upon cultivation with DCCD, NPQ was signi cantly down-regulated by a decrease in pH-regulated energy dissipation, leading to the decease in photo-protection capacity. Moreover, Aureochrome5 was also up-regulated and exhibited coexpressions with PGR5, thus con rming their ceRNA relationship. Therefore, we speculated that miR818-x may act as a bridge to contribute to the coordination between photosynthesis and the blue-light receptor aureochrome.

Conclusions
MiR8181-x and novel-m2866-5p negatively regulate Aureochrome5 in a tissue speci c manner. miR8181-x functions in the regulation of cell differentiation, cell cycle and cell development and plays an important role in kelp growth. The enhancement of Aureochrome5 under BL irradiation was coordinated with kelp photosynthesis and photoprotection. PGR5 incorporates the ceRNAs of Aureochrome5, and might act as the switch between the regulation of photo-protection and Aureochrome.

Sample collection and treatment
The S. japonica juvenile sporophytes ("Zhongke no. 1") were identi ed through appearance by us and collected from experimental aquaculture area in Rongcheng, Shandong, China. After washing the epiphytes thoroughly with sterilized seawater, the kelps were stored in cold conditions during the shipping. The kelps were pre-cultured in the dark at 10 °C for 24 h and then treated with darkness, white light (WL), or BL for 3 h. White uorescent lamps (Philips, Shanghai, China) and blue light-emitting diodes (460~475 nm wavelengths; Ichia, Japan) were applied as light sources. DCCD ( nal concentration 40 μM) was added to the culture medium before exposure to light irradiation. After treatment, the sporophytes were rapidly frozen with liquid nitrogen and then stored at -80 °C for subsequent RNA extraction. "Zhongke no. 1" was originally cultivated after 9 generations of hybridization with long and wide phenotypes S. japonica. A voucher specimen of S. japonica was deposited in the Resource-sharing Platform of Specimens Marine Biological Museum Chinese Academy of Sciences (No. MBM436715).

RNA isolation and library preparation.
Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA). The extracted RNA was qualitatively examined by 1% agarose gel electrophoresis, and the concentration was determined by a Nanodrop 2000 spectrophotometer (Thermo Scienti c, Wilmington, USA). After extraction, the RNAs, with molecules weights ranging from 18 to 30 nt, were separated by 15% polyacrylamide gel electrophoresis (PAGE). Then, the 3' adapters were added, and the 36-44 nt RNAs were enriched. Subsequently, the 5' adapters were ligated to the RNAs. The ligation products were reverse transcribed and the 140-160 bp PCR products were enriched to generate a cDNA library. Finally sequencing was conducted on an Illumina HiSeqTM 2500 at Gene Denovo Biotechnology Co. (Guangzhou, China). The RNA integrity and concentration were further measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The small RNA and mRNA were reverse transcribed to cDNA and then sequenced with a HiSeq 2500 and HiSeq 4000 (Illumina, San Diego, CA, USA), respectively.

Identi cation of miRNAs and target gene prediction
After removing reads containing adapters or low-quality bases, all of the clean tags were aligned with small RNAs in the Rfam and GenBank databases (Release 209.0), and our previous S. japonica genome data were registered in the NCBI database (accession: MEHQ00000000). The rRNA, scRNA, snoRNA, snRNA and tRNA sequences were ltered out. All of the 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 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 (MFE) 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 (Conesa et al. 2005) was employed to explore the Gene Ontology (GO) annotation terms. DAVID (Database for Annotation, Visualization and Integrated Discovery) was included to determine the pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Okuda et al. 2008). P values lower than 0.05 indicated enriched gene sets.

Veri cation of miRNAs and target genes by qRT-PCR
Candidate miRNA sequences were selected to quantitative real-time PCR (qRT-PCR) veri cation (Takara, Japan). qRT-PCR was conducted with the SYBR® PrimeScript™ miRNA RT-PCR Kit (RR716, Takara, China). Poly(A) tail addition and reverse transcription reactions were conducted in a total volume of 20 L. The miRNA PrimeScript RT enzyme mix from the SYBR® PrimeScript™ miRNA RT-PCR Kit was used in the reverse transcription reaction mixtures, and the qRT-PCR primers used are listed in Table S1. 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.

Chlorophyll uorescence measurement
The chlorophyll uorescence induction kinetics were measured under a Fluocam (Photon System Instruments, Czech Republic) with a saturating ash intensity of ca. 2,000 μmol m −2 s −1 . The uorescence intensity at 50 s was regarded as the initial uorescence, F O ; the maximum uorescence level of the uorescence transient was measured under saturating light. Before measurement of the chlorophyll uorescence induction kinetics, the kelp sporophytes were adapted to the dark for 15 min to ensure complete oxidation of the PSII reaction centre.

Declarations
Availability of data and materials All supporting data can be found within the manuscript and its additional le. The datasets used during the current study are available from the corresponding author on reasonable request. We con rmed that we have included a statement regarding material availability in the declaration section of my manuscript.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have a pending patent application based on the presented data.

Funding
This study was supported by the National Natural Science Foundation of China (31772848) Authors contributions XQY has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. DLD is involved in drafting the manuscript or revising it critically for important intellectual content.   Figure 1 Relative expression pro les of ve Aureochrome homologs transcripts in response to blue light. Values are normalized for -actin expression levels and represent means ± S.E. for n = 3.

Figure 2
The regulatory network between miRNAs and Aureochrome homologs.  qRT-PCR analyses of Aureochrome5 (a), miR8181-x, and novel-m2866-5p (b) transcripts in response to blue light (jewelry blue) and white light (light grey). Values are normalized for -actin expression levels and represent means ± S.E. for n = 3.

Figure 5
Relative expression pro les of Aureochrome5 (a), miR8181-x (b), and novel-m2866-5p (c) transcripts in different tissues. Values are normalized for -actin expression levels and represent means ± S.E. for n = 3.

Figure 6
Page 14/18 The regulatory network between miRNAs and target genes.

Figure 7
A set of diagrams showing six patterns of dynamic expression in the target genes of novel-m2866-5p in response to different light treatments.

Figure 8
A set of diagrams showing six patterns of dynamic expression in the target genes of miR-8181-x in response to different light treatments. Figure 9 9 GO annotations of cluster4 target genes of miR-8181-x with the top 10 enrichment scores of biological processes (a). Enrichment analysis of KEGG pathways of cluster4 target genes of miR8181-x with the top 10 enrichment scores of biological processes (b). The gradual color represents the P value; the size of the spot represents the gene number.
Page 16/18 Figure 10 Analysis of miR-8181-x function. Go analysis (a) and KEGG pathway analysis (b) for the target genes of miR-8181-x.

Figure 11
Analysis of novel-m2866-5p function. Go analysis (a) and KEGG pathway 38 analysis (b) for the target genes of novel-m2866-5p.

Figure 12
Analysis of the regulatory function of Aureochrome5. (a) The potential ceRNAs of Aureochrome5 that exhibited negative correlation with novel-m2866-5p and miR8181-x. (b) KEGG pathway analysis for the ceRNAs.

Figure 13
The effect of DCCD incubation on the chlorophyll a uorescence dynamics. The protocol included a dark preadaptation period of 5 min, a period of strong light (white bar; 5 min; 750 μmol·m−2·s−1) followed by a period of relaxation in the dark (black bar; 5 min). The uorescence dynamics in response to blue light was set as control (black lines).

Figure 14
The effect of DCCD incubation on the NPQ (a), Yield (b), Fv/Fm (c) and Rfd (d) of Saccharina 48 japonica in response to blue light.

Figure 15
The effect of DCCD incubation on the expression of Aureochrome5 and PGR5. qRT-PCR analyses of Aureochrome5 and PGR5 transcripts in response to blue (tulip yellow) and blue+DCCD (olive green) treatment. Values are normalized for b-actin expression levels and represent means ± S.E. for n = 3.

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