Analyses of microRNA166 gene structure, expression, and function during the early stage of somatic embryogenesis in Dimocarpus longan Lour

Plant Physiology and Biochemistry 147 (2020) 205–214

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Research article

Analyses of microRNA166 gene structure, expression, and function during the early stage of somatic embryogenesis in Dimocarpus longan Lour

T

Q.L. Zhang, L.Y. Su, S.T. Zhang, X.P. Xu, X.H. Chen, X. Li, M.Q. Jiang, S.Q. Huang, Y.K. Chen, Z.H. Zhang, Z.X. Lai, Y.L. Lin∗ Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China

ARTICLE INFO

ABSTRACT

Keywords: Dimocarpus longan Somatic embryogenesis microRNA166 Structure identification Functional analyses

MicroRNA166 (miR166) contributes to post-transcriptional regulation by binding the mRNAs of HD-ZIP III genes, which affects plant growth and development. The structural characteristics, expression, and functions of miR166 genes during the early somatic embryogenesis stage in Dimocarpus longan remain unknown. We isolated the transcripts of pri-miR166 S78 with two transcription initiation sites (TSSs) and pri-miR166 S338 with one TSS. These sequences contain potential smORFs and encode different miRNA peptides (miPEPs). Additionally, their promoters contain cis-acting elements responsive to diverse stimuli. The pre-miR166 S78 and pre-miR166 S338 expression levels were up-regulated in response to 2,4-D, abscisic acid, and ethylene. Although the expression patterns induced by hormones were similar, there were differences in the extent of the response, with pre-miR166 S338 more responsive than pre-miR166 S78. Thus, miRNA transcription and maturation are not simply linearly correlated. Moreover, pre-miR166 S78 and pre-miR166 S338 expression levels were downregulated, whereas ATHB15 (target gene) expression was up-regulated, from the longan embryonic callus to the globular embryo stages. These results are indicative of a negative regulatory relationship between miR166 and ATHB15 during the early somatic embryogenesis stage in longan. At the same stages, miR166a.2-agomir, miR166a.2-antagomir, and miPEP166 S338 increased or decreased the expression of miR166a.2 and ATHB15, but with no consistent patterns or linear synchronization, from which we've found some reasons for it.

1. Introduction MicroRNAs (miRNAs) form a conserved class of small (19–24 nucleotides), single-stranded RNA molecules that are widely distributed among eukaryotes. In terrestrial plant species, the miR165/166 (miR166) is one of the oldest and largest miRNA families (Barik et al., 2014). These miRNAs contribute to post-transcriptional regulation by specifically binding the mRNAs of HD-ZIP III genes (Zhong and Ye, 2007), which affects plant growth and development. In addition to their important roles related to the development of plant vascular tissues (Park, 2007; Zhu et al., 2011; Zhou et al., 2015), the production of lateral roots (Singh et al., 2014), and responses to stresses (e.g., drought, cold, and heavy metals) (Lv, 2015), they also help mediate the development of plant embryos and seed germination. In Arabidopsis thaliana (Wójcik et al., 2017) and Larix leptolepis (Li et al., 2017), miR166 affects the auxin biosynthesis pathway in diverse ways, thereby regulating somatic embryogenesis (SE). Promoters are important regulators of gene expression. Numerous studies have proven that miRNAs are closely related to the signal transduction of plant hormones, ∗

including auxin (Meng et al., 2009), gibberellin (Achard et al., 2004), abscisic acid (ABA) (José and Chua, 2007), and ethylene (Chen et al., 2012). However, the combined analysis of the effects of promoters and hormones has been completed in only a few model plant species (Liu et al., 2009; Zhao et al., 2012). As part of the continuing research on miRNAs, analyses of miRNA-encoded peptides (miPEPs) may be useful for further characterizing miRNAs. Lauressergues and his colleagues (Lauressergues et al., 2015) revealed the regulatory function of the A. thaliana pri-miRNA sequence. Moreover, pri-miR165a encodes miPEP165a, which increases the accumulation of miR165a, inhibits lateral root growth, and promotes primary root growth. Additionally, the development of the abaxial side of the A. thaliana leaf primordial base requires mature miR165a as well as the coordination of pri-miR165a (Lauressergues et al., 2015; Lv et al., 2016). Thus, the traditional belief that miRNAs are non-coding RNAs was proven false. Dimocarpus longan (longan), is an evergreen woody fruit tree species that grows in tropical and subtropical regions, primarily in Southeast Asia and southern China. The mature seeds are dark brown and contain many biologically active ingredients used for traditional Chinese

Corresponding author. E-mail address: [email protected] (Y.L. Lin).

https://doi.org/10.1016/j.plaphy.2019.12.014 Received 30 August 2019; Received in revised form 11 December 2019; Accepted 11 December 2019 Available online 12 December 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved.

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miR166 S53, miR166 S78, and miR166 S338 genes were revealed for the further functional characterization of the miR166 family during the early SE stage in longan.

medicine, which can improve human health and increase the immunomodulatory capacity (Yean, 2005). However, the molecular mechanism underlying longan seed development remains unclear because of the difficulties associated with sampling the early-stage embryos as well as the extreme genetic heterozygosity (Lin and Lai, 2013; Lin et al., 2015). To overcome these difficulties, longan SE, which is a model system resembling zygotic embryogenesis, has been widely used to investigate the in vivo and in vitro regulation of embryogenesis in plants (Lai et al., 2010; Lin and Lai, 2010). We have analyzed the miR166 family previously. Specifically, we identified 12 mature miR166 sequences in the miRNA data for embryogenic cultures of ‘Honghezi’ (Fujian, China) longan (Lin and Lai, 2013). In these cultures, miR166a.2 was the most highly expressed miR166 (22,387 reads), which was an order of magnitude higher than the second most highly expressed miR166 (i.e., miR166e with 1987 reads). A comparison with A. thaliana revealed that in apple, citrus, and other species, miR166a.2 (UCGGACCAGGCUUCAUUCCCC), which may be named differently depending on the species, mainly comprises 21 nucleotides. These miR166 sequences primarily target HD-ZIP III genes, among which ATHB15 is mainly targeted during SE in longan (Lin et al., 2017a). Additionally, seven miR166 precursor sequences were detected in the transcriptome database for a longan embryogenic callus (Lai and Lin, 2013), all of which could form stable secondary stem ring structures (Lin et al., 2017a). In some longan SE systems (Lai and Chen, 2002; Lai et al., 2000, 2001), pri-miR166 S53 (i.e., primary miR166) was isolated and cloned in the early SE stage, and revealed to potentially encode a smORF. We previously observed that the pre-miR166 S53 and miR166a.2 expression patterns were inconsistent in response to various hormone treatments (Zhang et al., 2018). This may be related to the other members of the miR166 family. During the early SE stage in longan, in addition to pre-miR166 S53, both pre-miR166 S78 and premiR166 S338 are also highly expressed, and the latter two sequences may also be cut and processed to produce mature miR166a.2. The objectives of this study were to clone the pri-miR166 S78 and pri-miR166 S338 sequences, analyze the transcription start sites (TSSs), and clarify whether there are smORFs encoded in the primary sequences. The longan genome database (Lin et al., 2017b) was used to detect the presence of introns and to analyze the cis-acting elements upstream of the TSS of the miR166 S78 and miR166 S338 genes. Plant hormone treatments (2,4-D, ABA, and ethylene) were used to explore the response patterns of pre-miR166 S78 and pre-miR166 S338, which were compared with that of pre-miR166 S53 (Zhang et al., 2018). Additionally, RNA oligonucleotides and a synthetic miPEP166 S338 were used to treat longan SE cells to observe their effects on morphogenesis and to explore the regulatory relationships among pre-miR166, miR166a.2, and ATHB15 (target gene) during SE. Furthermore, a series of internal regulatory links from the transcription to the maturation of

2. Material and methods 2.1. Tissue samples Synchronized D. longan ‘Honghezi’ (Fujian, China) embryogenic cultures representing different developmental stages were prepared. Specifically, the friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC), globular embryo (GE), and cotyledonary embryo (CE) were generated as described by Lai and Chen (1997). The samples were used for the subsequent extraction of total RNA and analyses. The EC was pre-cultured on agar-solidified Murashige and Skoog (MS) medium containing 2,4-D (1.0 mg/L) for 20 days. It was then subcultured in 50 mL liquid MS medium containing 2,4-D (0, 0.1, 0.5, 1, or 2 mg/L), ABA (0, 5, 50, 500, or 5000 μg/L), or ethylene (0, 25, 50, 100, or 125 mg/L). Samples were incubated on a rotary shaker (150 rpm) for 24 h in darkness. The EC pre-cultured on MS medium containing 2,4-D was also sub-cultured in 30 mL liquid MS medium lacking 2,4-D. The sample was incubated on a rotary shaker (115 rpm) for 12 days in darkness to generate the GE. This system was used for further functional studies involving RNA oligonucleotides and miPEPs. 2.2. Database searches for longan miR166 family members Mature and precursor sequences of miR166 family members were obtained from the available small RNA data (BioSample accession SAMN04120614, Bio-Project ID PRJNA297248) generated with various synchronized ‘Honghezi’ longan embryogenic cultures (Lin and Lai, 2013) (Lai and Lin, 2013). 2.3. Pri-miR166 S78 and pri-miR166 S338 3ʹ and 5ʹ RACE experiments To amplify the full-length longan pri-miR166 S78 and pri-miR166 S338 cDNA sequences, the DNAMAN6.0 program was used to design 5′ RACE and 3′ RACE primers specific for pre-miR166 S78 and premiR166 S338 sequences, which were used as templates. The appropriate 5′ RACE1 and 5′ Upml primers were used for the amplification of the 5′-end of the pri-miR166 S78 and pri-miR166 S338 cDNA sequences with the SMART™ RACE kit (TaKaRa, Japan). The amplified products were used as the template for a second amplification round involving the 5′ RACE2 and 5′ Upms primers to generate the 3′-end of the primir166 S78 and pri-mir166 S338 cDNA sequences. Details regarding the primers, which were synthesized by BGI, are listed in Table 1.

Table 1 Primers used in this study. Use

Primer

Sequence (5′→3′)

5′RACE Rapid amplification of cDNA 5′ends

Pri-miR166 S78/S338 5R1 Pri-miR166 S78 5R2 Pri-miR166 S338 5R2 Pri-miR166 S78 3R1 Pri-miR166 S78 3R2 Pri-miR166 S338 3R1 Pri-miR166 S338 3R2 Upml Upms Pre-miR166 S53 F Pre-miR166 S78 F Pre-miR166 S338 F Pre-miR166 S53/S78/S338 R miR166a.2 ATHB15 F ATHB15 R

GTTGAGGGGAATGTTGTCTGG CGGCACTAACAGATGATCGA CGAAGATGATGCCATGAACT TGTCTGGCTCGAGGACTCT GCCATGTTCTGTAGATCCAATC TCTGGTTCGAGACCTTTCG GGCGCTGACAAGTTAAGTTCAT CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT CTAATACGACTCACTATAGGGC TCTACATGAACAGTTGAGGGGA GTCTGGCTCGAGGACTCTTCT TGGGGAATGCTGTCTGGT GAATGAAGCCTGGTCCGA TCGGACCAGGCTTCATTCCCC CAAAGGCCACTGGAACTGCTGT CTAAACTCACCAGGCCGCAAG

3′RACE Rapid amplification of cDNA 3′ends Universal primer Pre-miR166 qRT-PCR

miR166 qRT-PCR Target qRT-PCR

206

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The PCR amplifications were completed in 25-μL reaction volumes comprising 1 μL template cDNA, 12.5 μL 2 × DreamTaq™ Green Master Mix, 1 μL upstream and downstream primers, and ddH2O. The PCR program was as follows: 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s; 72 °C for 10 min; preservation at 4 °C.

the miPEPs of pri-miR166 S53, pri-miR166 S78, and pri-miR166 S338 enable the functional characterization of each miR166 family member. Additionally, the miPEPs are active only in the cells that express the miRNA, which results in a more targeted and controllable analysis. MiPEP166 S338 was prepared as a powder (0.5 mg/tube) and dissolved in 1 mg/mL acetonitrile and water (2:1, v/v). The solution was then diluted and divided into aliquots for later use. The EC was treated with miPEP166 S338 (0.4 μM), with a method involving the miR166a.2-agomir/antagomir system described in section 2.5. Moreover, miPEP166 338 (MTNSISNSPLLVPSFYIYLSFFFFFFSISLLWGMLSGSRPFAYKLSSWHHLLE) was synthesized by the Nanjing Leon Biotechnology Company, but miPEP166 S53 and miPEP166 S78 could not be synthesized.

2.4. Sequence determination and promoter analysis of pri-miR166 S78 and pri-miR166 S338 in longan The amplified fragments were analyzed by agarose gel electrophoresis and then purified with a DNA recovery kit (TaKaRa, Japan). The purified fragments were ligated to the pMD18-T vector (TaKaRa). The resulting recombinant plasmid was inserted into Escherichia coli Trans1-T1 cells (Trans, China). Positive clones, which were identified based on their resistance to ampicillin (Trans), were used for a PCR amplification with the universal primers M13F and M13R. The amplified products were sequenced by the BoShang company and analyzed with the DNAMAN6.0 program (Lynnon Biosoft, USA). The pri-miR166 S78 and pri-miR166 S338 sequences were obtained with DNAMAN6.0 to splice the sequenced fragments and the precursor sequence. The resulting sequences were used to screen the longan genome data (Lin et al., 2017b) to determine whether the sequenced fragments contained introns and to predict whether they encoded smORFs or peptides (i.e., miPEPs). Furthermore, after identifying other genes/coding sequences that do not overlap in the region by Blast validation, the PlantCare bioinformatics program was used to analyze the cis-acting elements in the promoter regions approximately 3000 bp upstream of the TSS.

2.7. Quantitative real-time PCR analysis A quantitative real-time (qRT)-PCR assay was conducted to investigate the expression of pre-miR166 S53, pre-miR166 S78, premiR166 S338, miR166a.2, and ATHB15 during the early SE stage in longan in response to treatments with 2,4-D, ABA, ethylene, miR166a.2-agomir/antagomir, and miPEP166 S338. Total RNA was extracted from samples with the RNAprep Pure Plant kit (Tiangen), after which cDNA was synthesized with the SYBR ExScript (TaKaRa) and the Tip qMix (Trans) reverse transcription kits. A 1:10 dilution of the cDNA was prepared for the qRT-PCR assay. The qRT-PCR assay was completed with the LightCycler 480 system (Roche Applied Science, Switzerland) and the method described by Lin and Lai (2010). The Fe-SOD gene was used as a reference control to normalize the pre-miR166 S53, pre-miR166 S78, pre-miR166 S338, and ATHB15 expression levels. The miR166a.2 expression level was normalized against the miR408-5p1 expression data. All reactions were completed in triplicate. The data were analyzed with SPSS 19. Details regarding the qRT-PCR primers are provided in Table 1.

2.5. RNA oligonucleotide (miR166a.2-agomir and miR166a.2-antagomir) treatments during the early SE stage in longan The advantages of RNA oligonucleotide overexpression and inhibition technology over traditional Agrobacterium tumefaciens-mediated transgene technology include simplicity, increased effectiveness, and lack of a carrier structure. The overexpression and inhibition system established by Lin et al. (2016) and a liquid MS system were used to convert the longan EC to the GE in 12 days. The specific scheme is as follows: 24-well sterile plate (2 mL/well) as reaction vessel; 0.2 g suspended and filtered EC as the starting material; miR166a.2-agomir/antagomir as treatment reagent, lipofectamine 2000 (Yingweijiji Trading Company) as transfection agent assisted in the successful transfer of RNA oligonucleotide reagent into EC cells. The reaction system of 500ul is as follows: 25 μL miR166a. 2-agomir/antagomir(20 M) + 30 μL Lip + 445 μl/470 μl MS. Each sample was treated three times for 24 h and then sub-cultured in 30 mL liquid MS medium without 2,4-D. Samples were incubated on a rotary shaker (150 rpm) for 12 days in darkness. On days 1 (i.e., subculturing date), 6, and 12, samples were collected and analyzed with a microscope. Samples were also quickly frozen with liquid nitrogen and stored at −80 °C for a subsequent gene expression analysis. The UCGGACCAGGCUUCAUUCCCC miR166a.2-agomir ( ) and GGAAUGAAGCCUGGUCCGAUU miR166a.2-antagomir (GGGGAAUGAAGCCUGGUCCGA) RNA oligonucleotide sequences were synthesized by the Shanghai Gima Biopharmaceutical Technology Company.

3. Results 3.1. Analysis of the aligned longan pre-miR166 S53, pre-miR166 S78, and pre-miR166 S338 sequences To determine the similarity among family members, the pre-miR166 S53, pre-miR166 S78, and pre-miR166 S338 sequences were downloaded from a transcriptome database (Lai and Lin, 2013). A comparative analysis of the three precursor sequences revealed that the mature miR166a.2 region (arm) was highly conserved, whereas the other positions were not. Of the examined sequences, pre-miR166 S53 and pre-miR166 S338 were more similar. Additionally, pre-miR166 S78 may have undergone sequence-insertion events during its evolution (Fig. 1). Moreover, miR166 genes may have evolved to favor negative selection. 3.2. Cloning of pri-miR166 S78 and pri-miR166 S338 cDNA sequences from the early SE stage in longan and an analysis of potential smORFs We cloned pri-mir166 S53 in one of our previous studies (Zhang et al., 2018). In the current study, we attempted to clone pri-miR166 S78 and pri-miR166 S338 to analyze the TSSs of the miR166 S78 and miR166 S338 genes. Upstream and downstream RACE primers were designed based on pre-miR166 S78 and pre-miR166 S338 sequences. The cDNA derived from the longan EC, ICpEC, and GE mixed RNA samples was used as the template for the 5′ RACE and 3′ RACE cloning. By aligning the sequences from positive clones with the corresponding precursor sequences, we were able to obtain the target sequences. The pre-miR166 S78 5′ RACE yielded two fragments (329 bp and 191 bp), both of which contained a 30-bp connector primer. The 3′ RACE yielded a 300-bp fragment that included a 28-bp poly-A tail. The pre-mir166 S338 5′ RACE sequence was 206 bp, including a 52-bp complete

2.6. MiPEP166 S338 treatment during the early SE stage in longan As a new tool for studying miRNAs, miPEPs have attracted increasing attention. The disadvantage of traditional methods for transgenic research involving recombinant plasmids is that they cannot decrease the activity of only one member of the miRNA family. The expression of miRNAs across all time-points and in all tissues leads to abnormal phenotypes. The advantage of miPEPs is that the miRNA family members produce distinct miPEPs. For example, the diversity in 207

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Fig. 1. Aligned longan pre-miR166 S53, pre-miR166 78, and pre-miR166 338 sequences.

Fig. 2. Longan pri-miR166 S78 and pri-miR166 S338 sequence analysis. (A) Pri-miR166 S78. (B) Pri-miR166 S338. The gray box indicates the TSS. The translation initiation codon (ATG) and the translation termination codon (TGA) are presented in red. The longan pre-miR166 S78 and pre-miR166 338 sequences are underlined. The potential smORFs encoding miPEPs are shaded in yellow, whereas the mature miR166a.2 is shaded in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

connector primer, whereas the 3′ RACE sequence was 344 bp, including a 30-bp poly-A tail. Furthermore, the 5′ RACE and 3′ RACE sequences were aligned and spliced with the pre-miR166 S78 and pre-miR166 S338 sequences to generate complete primary sequences. The primiR166 S78 sequences produced from two TSSs were 510 bp and 372 bp long, including a 159-bp precursor sequence, a 181-bp or 43-bp 5′end, and a 170 bp 3′-end. The second TSS was 139 bp downstream of the first (Fig. 2A). The pri-miR166 S338 sequence was 436 bp long, including a 111-bp precursor sequence, 84-bp 5′-end, and a 241-bp 3′end (Fig. 2B). The pri-mir166 S53 sequence was 317 bp long, including a 128-bp precursor sequence, a 50-bp 5′-end, and a 139-bp 3′-end (Zhang et al., 2018). A comparison of the pri-miR166 S53, pri-miR166 S78, and pri-miR166 S338 cDNA sequences with the genomic DNA sequences (Lin et al., 2017b) revealed that all sequences overlapped perfectly, which indicated that the miR166 S53, miR166 S78, and miR166 S338 genes lacked introns. The DNAMAN6.0 program was used to predict whether pri-miR166 S78 and pri-miR166 S338 contain potential smORFs. The pri-miR166 S78 sequence contains an ATG codon at 12, 205, and 336 bp downstream of the first TSS, whereas the pri-mir166 S338 sequence includes the ATG codon at 1, 100, and 141 bp downstream of the TSS. However, Lauressergues and his colleagues (Lauressergues et al., 2015) reported that in A. thaliana, only the miPEP associated with the first ATG after the TSS is produced. Accordingly, the two TSSs of pri-miR166 S78 result in two miPEPs, namely miPEP166 S78-1 with only one amino acid (M) and miPEP166S78-2 with 29 amino acids (MLSGSRTLLLIIFCRSNLSIQINSGSIIC) (Fig. 2A). Additionally, pri-miR166 S338 encodes miPEP166 S338 with 53 amino acids

(MTNSISNSPLLVPSFYIYLSFFFFFFSISLLWGMLSGSRPFAYKLSSWHHLLE) (Fig. 2B). Moreover, pri-mir166 S53 encodes miPEP166 S53 with 13 amino acids (MLCFVDALFLIST) (Zhang et al., 2018). Whether these miPEPs have biological functions remains to be verified. 3.3. Analysis of the cis-acting elements in the miR166 S78 and miR166 S338 promoters in longan To analyze the cis-acting elements in the miR166 S78 and miR166 S338 promoters, a 3000-bp sequence upstream of the TSS was extracted from the longan genome database (Lin et al., 2017b) to represent the promoter region. An analysis with the PlantCARE program indicated that miR166 S78 and miR166 S338 contain a TATA-box approximately 30 bp upstream of the first TSS, with a CAAT-box located 30–60 bp upstream of the TATA-box. This important core promoter region was consistent with previously described predicted miRNA promoters (Jones-Rhoades and Bartel, 2004; Morton et al., 2014). A further analysis of the miR166 S78 and miR166 S338 promoter sequences revealed that although the promoter sequences were quite different, there were many common functional elements. In addition to a large number of CAAT and TATA sequences, the promoters also contained many lightresponsive elements and the same situation also appeared in miR166 S53. Furthermore, the three miR166 members also contained anaerobic-responsive elements (AREs), wound-responsive elements (WUNmotifs), abscisic acid-responsive elements (ABREs), and ethylene-responsive elements (EREs). The endosperm expression element (GCN4 motif), the seed-specific expression element (RY-element), and the salicylic acid-responsive element (TCA-element) were specific to the 208

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Table 2 Cis-acting elements in miR166 promoter sequences. Cis-acting elements

Function

Pri-miR166 S78

Pri-miR166 S338

TATA-box CAAT-box Light-related motifs ARE GCN4_motif MBS RY-element TC-rich repeats wun-motif ABRE AUXRR-core CGTCA-motif ERE P-box TCA-element TGA-element TGACG-motif Totals Promoter length (bp)

core promoter element around −30 of transcription start common cis-acting element in promoter and enhancer regions cis-acting regulatory element involved in light responsiveness cis-acting regulatory element essential for the anaerobic inductionnse cis-regulatory element involved in endosperm expression MYB binding site involved in drought-inducibility cis-acting regulatory element involved in seed-specific regulation cis-acting element involved in defense and stress responsiveness cis-acting element conferring wound response cis-acting element involved in the abscisic acid responsiveness auxin-responsive element cis-acting regulatory element involved in the MeJA-responsiveness ethylene-responsive element gibberellin-responsive element and part of a light responsive element cis-acting element involved in salicylic acid responsiveness auxin-responsive element cis-acting regulatory element involved in the MeJA-responsiveness

27 45 21 3 1 / 1 / 2 7 1 / 2 / 3 / / 103 3000

44 24 30 7 / 2 / 1 2 3 / 1 1 3 / 1 1 120 3000

miR166 S53 was similar to that of mature miR166a.2, with expression levels down-regulated at high ABA concentrations (Zhang et al., 2018). The expression patterns of pre-miR166 S78 and pre-miR166 S338 were inconsistent with those of other family members. The expression levels of the precursors were much higher than that of miR166a.2, suggesting there may be some functional redundancy between pre-miR166 S78 and pre-miR166 S338. The qRT-PCR analysis of the effects of various ethylene concentrations indicated that pre-miR166 S78 and pre-miR166 S338 are strongly responsive to this phytohormone. Although the expression of both sequences was considerably up-regulated by the ethylene treatment, premiR166 S338 was more responsive than pre-miR166 S78 (Fig. 3C). In contrast, under the same conditions, pre-miR166 S53 expression was down-regulated, whereas the expression of mature miR166a.2 increased slowly with increasing ethylene concentrations (Zhang et al., 2018). The similarity and diversity in the expression patterns may reflect the synergistic and complementary functions of the family members.

miR166 S78 promoter region. In contrast, the drought-responsive element (MBS), defense and stress-responsive elements (TC-rich repeats), and the gibberellin-responsive P-box were detected only in the miR166 S338 promoter region (Table 2). The miR166 S53 promoter region also included the characteristic thermal stress-responsive element (HSE) and the low-temperature-responsive element (LTR) (Zhang et al., 2018). The detected cis-acting elements may be closely related to the potential functions of the miR166 S53, miR166 S78, and miR166 S338 genes. Additionally, because of the similarity or diversity in the cis-acting elements in the promoters, the miR166a.2 generated by different family members may be associated with synergistic and specific spatio-temporal expression and function. 3.4. Analysis of the pre-miR166 S78 and pre-miR166 S338 expression patterns in the longan EC in response to different hormones (2,4-D, ABA, and ethylene) The promoter located upstream of the TSS is important for regulating gene transcription levels and controls the timing and intensity of gene expression. Hormones have crucial functions regarding plant embryo development and seed germination. Consequently, hormoneresponsive elements are distributed in promoter sequences. The binding of transcription factors to these sequences contributes to the regulation of gene expression. In this study, the EC was treated with various concentrations of 2,4D for 24 h, after which a qRT-PCR assay was completed to examine premiR166 S78 and pre-miR166 S338 expression levels. The pre-miR166 S78 and pre-miR166 S338 expression patterns were similar. Specifically, high 2,4-D concentrations up-regulated the expression of both sequences, but the extent of the increase was greater for premiR166 S338 than for pre-mir166 S78 (Fig. 3A). Under the same conditions, pre-miR166 S53 expression was essentially unaffected by 2,4-D, whereas miR166a.2 expression was slowly down-regulated (Zhang et al., 2018). The diversity in the expression patterns among the analyzed family members implied that they exhibit some functional specificity. Our qRT-PCR data indicated that treatments with different ABA concentrations similarly affected the expression patterns of pre-miR166 S78 and pre-miR166 S338. At low concentrations, the expression levels decreased with increasing ABA concentrations, but then increased with further increases in ABA concentration. We observed that pre-miR166 S338 was affected by the ABA treatments more than pre-mir166 S78 (Fig. 3B). Under the same conditions, the expression pattern of pre-

3.5. Analysis of miR166a.2-agomir/antagomir and miPEP166 S338 expression during the early SE stage in longan Although an analysis of the effects of the overexpression and inhibited expression of miRNAs may be suitable for functionally characterizing miRNAs, investigations involving RNA oligonucleotides and miPEPs represent viable alternative options. In this study, the longan EC was treated with miR166a.2-agomir, miR166a.2-antagomir, and miPEP166 S338, after which it was transferred to liquid MS medium and analyzed at specific time-points to clarify the function of miR166a.2 in SE. We observed that miR166a.2-agomir/antagomir and miPEP166 S338 did not induce any obvious morphological differences during SE. Regardless of the treatment, the EC observed on day 1 developed into the ICpEC by day 6 and the GE by day 12 (Fig. 4A–C). To further elucidate the effects of miR166a.2-agomir, miR166a.2antagomir, and miPEP166 S338, we conducted a qRT-PCR assay to examine the expression levels of miR166a.2, pre-miR166 S53, miR166 S78, miR166 S338, and ATHB15 during the early SE stage in longan (i.e., days 1, 6, and 12). The mock treatment induced similar expression patterns for the precursors and mature 166a.2, but the mature 166a.2 was obviously less responsive than the pre-miR166 sequences. In contrast, ATHB15 expression was up-regulated, indicative of a typical negative regulatory relationship (Fig. 5A–C). These results suggested that the high miR166a.2 expression level may be beneficial for the 209

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expressiont. From another way, the situation may be related to the different half-lives of agomir and antagomir or the expression of endogenous miR166a.2. A previous study confirmed that pri-miRNAs encoding miPEPs induce the accumulation of the corresponding miRNAs, which is a finding that may be relevant for miRNA research (Lauressergues et al., 2015). In the current study, the miPEP166 S338 treatment led to an increase in the pre-miR166 S338 level on day 1, but a subsequent decrease to mock control levels (Fig. 6A). As presented in Fig. 6B, miPEP166 S338 inhibited the decline in the mature miR166a.2 level before day 6. Additionally, the up-regulated expression of ATHB15 was inhibited during the early SE stage (Fig. 6B). These findings indicate that the regulation of the precursor sequence, mature miR166a.2, and ATHB15 by miPEP166 S338 may be unsynchronized because these sequences are expressed at different steps in the pathway and have different half-lives in the cell. 4. Discussion 4.1. Structural characteristics of miR166 S78 and miR166 338 genes in longan Analyses of miRNA gene family sequences, including the functional differentiation of members, is currently important for the comprehensive characterization of miRNAs. Recent increases in the available information in plant genome databases has benefited research in this field. For example, studies on the miR166 family in A. thaliana (Allen et al., 2004), rice (Barik et al., 2014), and soybean (Wang et al., 2015a) revealed that miR166 is a multi-copy gene family. However, most functional miR166s are 21 nucleotides long (including miR166a.2 in this study) and have highly conserved mature regions, whereas other regions are not conserved (Lin et al., 2017a). The data presented herein are consistent with these earlier findings. The function of miR166 in different plants may be maintained by the conserved structure of the mature region which is one of the indicators that the miR166 gene may have been under negative selection pressure during evolution. After that, we cloned pri-miR166 S78 and pri-miR166 S338. A smORF analysis revealed that pri-miR166 S78 and pri-miR166 S338 encode different miPEPs. This is in accordance with the conclusion of the study by Lauressergues and his colleagues (i.e., different miRNA family members encode different miPEPs) (Lauressergues et al., 2015). Furthermore, the two TSSs of pri-miR166 S78 may be associated with diverse regulatory modes. Some studies indicated that miRNAs encoded at different TSSs of the same gene exhibit tissue specificity and identifying TSSs may help predict miRNA functions (Georgakilas et al., 2014; Bhattacharyya et al., 2012). The post-transcriptional pri-miRNAs were traditionally believed to form mature miRNAs after two nuclease digestions (Dong et al., 2008; Jones-Rhoades et al., 2006). However, little was known regarding pri-miRNA translation. A whole-genome analysis of the ribosome footprint in A. thaliana revealed that ribosomes are associated with the miPEP165a sequence in pri-miR165a (Juntawong et al., 2014), indicating that the translation or maturation of the primary sequence involves a one-way selection. A cordycepin treatment with an RNA synthesis inhibitor can completely eliminate the miPEP-induced accumulation of pri-miRNA, suggesting that miPEP can increase the abundance of miRNA by activating the transcription of miRNA in the nucleus (Lauressergues et al., 2015). The upstream of the pri-miRNA is the TTS and promoter regions. Our study showed that miR166 S53, miR166 S78 and miR166 S338 have similar TTS structure. The results are in accordance with the findings of a previous study by Zhang et al. (2013) who predicted the TSS of a corn miRNA gene. Thus, miR166 genes appear to contain a conserved TSS region. In the promoter regions, excluding TATA and CAAT, the most common cis-acting elements detected in the current study were light-responsive elements. Similar observations were reported for a previous study on the miR166 gene of the rubber tree

Fig. 3. Analysis of pre-miR166 S78 and pre-miR166 S338 expression levels in the longan EC in response to various concentrations of 2,4-D, ABA, and ethylene at 24 h after the treatments. (A) 2,4-D control. (B) ABA control. (C) Ethylene control.

maintenance of loose longan calli, whereas the high ATHB15 expression level was conducive to the formation of a spherical embryo. The RNA oligonucleotide overexpression and inhibition system represents a simple and effective new method for investigating miRNAs (Lin et al., 2016). When miR166a.2-agomir was expressed, the miR166a.2 expression level remained high throughout the process, unlike the ATHB15 expression level, which increased on days 1 and 6, but decreased on day 12. When miR166a.2-antagomir was expressed, the ATHB15 expression level was obviously up-regulated on day 1, but decreased to the control level (i.e., mock treatment) on day 6 (Fig. 5B–C). These results were far from perfect but, to some extent, it still indicated that miR166a.2 negatively regulated ATHB15 210

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Fig. 4. Morphological changes over three sequential early SE stages in longan. (A) Friable-embryogenic callus. (B) Incomplete compact pro-embryogenic culture. (C) Globular embryo. The bars are 50 μm in (A–C).

4.3. Response of pre-mi166 (S53, S78, and S338), miR166a.2, and ATHB15 to miR166a.2-agomir, miR166a.2-antagomir, and miPEP166 S338 during the early SE stage in longan

(Kanjanawattanawong et al., 2014). Additionally, other detected promoter elements were primarily stress- or hormone-responsive elements (e.g., MBS, Wun-motif, ABRE, and ERE). Some of these are commonly distributed, whereas some are gene-specific (Table 2). Therefore, we speculate that the cis-acting elements contribute to the functional diversity of the miR166 family members as well as to their spatio-temporal expression in various tissues.

Hormone treatments can considerably increase the transcription of miR166, but the expression level of the mature miRNA may not be synchronously up-regulated. The RNA oligonucleotide overexpression and inhibition techniques involve siRNA-like approaches to deliver mature miRNA analogs or reverse-sequence RNA oligonucleotides into cells. To date, RNA oligonucleotide technology has been widely used for targeted therapy in humans, including in treatments for heart disease (Wang et al., 2015b) and cancer (Li et al., 2018). However, its application in plants has not been reported. The utility of miPEPs has provided researchers with new ways to investigate miRNAs. Lauressergues et al. (2015) determined that pri-miR165a and pri-miR171b encode miPEPs, which increase the abundance of the corresponding miRNAs to regulate the development of A. thaliana and M. truncatula roots. Additionally, Couzigou (Couzigou et al., 2015) proposed that miPEPs are universal and plant growth and development may be regulated by applying exogenous miPEPs. In A. thaliana, miR165 and miR166 influence the auxin biosynthesis pathway by regulating PHABULOSA/PHAVOLUTA (PHB/PHV)-mediated LEC2 expression (HD-ZIP III family member), thereby promoting SE (Wójcik et al., 2017). In Larix leptolepis, miR166a overexpression mediates the synthesis of indole-3-acetic acid (i.e., an auxin) and promotes the germination of late mature embryos (Li et al., 2017). In Lilium species, a negative correlation was observed between miR166 and its target genes, but the expression patterns of different family members were inconsistent during SE (Yang, 2017). In the current study, miR166a.2-agomir, miR166a.2-antagomir, and miPEP166 S338 treatments were associated with diverse overexpression and inhibition results. A qRT-PCR assay revealed that these treatments differentially regulated miR166a.2 and ATHB15 expression. Compared with the mock, miR166a.2-agomir and miR166a.2-antagomir both increased or decreased miR166a.2 expression obviously, and miPEP166 S338 increased the transcription of pre-miR166 S338 and miR166a.2 expression. However, there were no linear synchronization in the regulation of the target gene ATHB15. This may have been due to the differences the half-lives of exogenous molecules in cells. The half-life of miR166a.2-agomir is longer than that of miR166a.2-antagomir and miPEP166 S338. There may also be other cellular mechanisms responsible for our results. For example, before binding to target genes, miR166a.2 must form a silencing complex with AGO1 (Yu et al., 2017). An insufficient amount of AGO1 will lead to the excessive production of functionally redundant miR166a.2. This may partially explain the hysteresis related to the regulatory effects of miR166a.2-agomir and miPEP166 S338 on the target genes. Another theory is that excessive miR166a.2 amounts up-regulate the expression of the target genes. Additionally, AGO10 can bind miR166a.2 more specifically than AGO1 and block the function of miR166a.2 (Yu et al., 2017). In a way, the role

4.2. Responses of pre-miR166 S78 and pre-miR166 S338 to 2,4-D, ABA, and ethylene treatments during the early SE stage in longan Several studies have shown that cis-acting elements are distributed in the promoter regions and are involved in regulating gene expression (Barik et al., 2014; Li et al., 2017; Kanjanawattanawong et al., 2014; Valiollahi et al., 2014; Zhou et al., 2008). The fluctuations in miRNA expression during plant growth and development are often related to hormone responses and occur in a specific developmental period (Oh et al., 2008). Studies on the influence of phytohormones on the regulation of miRNA-mediated gene expression have been conducted for some model plant species (Chen et al., 2012). The examined hormones include the five classical plant hormones (auxin, gibberellin, cytokinin, ABA, and ethylene) as well as methyl jasmonate and brassinolide. In the current study, the response of the EC to various concentrations of 2,4-D, ABA, and ethylene during the early SE stage in longan indicated that the expression patterns of the precursors of different miR166 family members varied depending on the hormone, and pre-miR166 S338 was more responsive to 2,4-D, ABA, and ethylene than pre-miR166 S78, which is probably related to differences in the transcriptional intensity regulated by cis-acting elements in the promoters. Additionally, the ethylene treatment up-regulated the expression of pre-miR166 S78 and pre-miR166 S338, but down-regulated the expression of pre-miR166 S53 (Zhang et al., 2018). The expression pattern of miR166 S53 was highly similar to that of the mature miR166 in rubber trees under the same conditions (Kanjanawattanawong et al., 2014). Moreover, the precursor transcription level was significantly higher than that of the mature miRNA, suggesting that the transition from the transcription to the maturation of miR166 is not necessarily smooth. This is consistent with the research of Zhang et al. (2012) on L. leptolepis. They concluded that there is no linear correlation between the abundance of mature miRNA and the expression of the corresponding precursor. This may be related to the stability of the precursor and the mature miR166 in cells. Katarzyna et al. (2017) observed a significant decrease in the transcription of DCL1 and HEN1 enzymes in A. thaliana, which are involved in the processing of pri-miRNAs. Similar phenomena have been observed in translation genomics research. In A. thaliana, the translation efficiency of highly transcribed genes under hypoxic conditions reportedly does not significantly increase and may even decrease (Juntawong et al., 2014). Therefore, there may be some functional redundancy among the excessive pri-miRNA transcripts, which are directly degraded in the nucleus. 211

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Fig. 6. Analysis of the pre-miR166 S338, miR166a.2, and ATHB15 expression levels during the early SE stage in longan following the miPEP166 S338 treatment. (A) Pre-miR166 S338. (B) MiR166a.2. (C) ATHB15.

Fig. 5. Analysis of the pre-miR166, miR166a.2, and ATHB15 expression levels during the early SE stage in longan following the miR166a.2-agomir and miR166a.2-antagomir treatments. (A) Pre-miR166 S53/S78/S338. (B) MiR166a.2. (C) ATHB15.

fix nitrogen, which proves the effectiveness of exogenous miPEPs (CouzigouAndré et al., 2016). Therefore, the application of exogenous and synthetic miPEPs may be useful for regulating target miRNAs and for promoting a series of controllable changes in physiological and metabolic processes in plants to enhance plant morphogenesis and improve fruit quality (Lv et al., 2016; Couzigou et al., 2015). Here, to better understand miPEP, we propose a conjecture that the miPEP166 are synthesized by ribosomes in the cytoplasm and then bind to the promoter as a transcription factor(TF) or a unit of it to enhance transcription efficiency of miR166 genes (Fig. 7).

of miR166a.2-antagomir in the cell is similar to that of AGO10 (i.e., silencing miR166a.2) (Fig. 7). Furthermore, SE and seed germination involve the joint regulation of miR156, miR158, miR159, miR166, miR167, miR172, and other miRNAs (Huang et al., 2013). Therefore, it is reasonable that miR166a.2-agomir, miR166a.2-antagomir, and miPEP166 S338 have regulatory roles but fail to change the early morphological characteristics of longan somatoblast cells. It is not complex that the mechanism underlying miR166a.2-agomir and miR166a.2-antagomir, but how miPEPs are produced and function remains unclear. Western blotting confirmed the presence of miPEPs in cells (Lauressergues et al., 2015). In soybean, artificially synthesized miPEP172c promotes the expression of miR172c as well as ENOD40-1 and other genes. These changes enhance the ability of root nodules to

5. Conclusion The conserved region of the mature miR166 ensures the gene function remains conserved. It also helps regulate the post-transcriptional level via the specific binding of HD-ZIP III family members, 212

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Fig. 7. Basic pathways for miRNA biogenesis and metabolism in longan. The pri-miR166 transcribed by RNA polymerase II (RNA Pol II) can be processed by a dicer to generate mature miR166a.2 or translated to produce the miPEP (smORF), which in turn increases miR166 transcription. The ✘ indicates pri-miR166, pre-miR166, and miR166a.2 may be degraded, whereas the ✔ indicates miR166a.2 shears the HD-ZIP III target genes. A solid line represents the experimental treatment and related molecular mechanisms, whereas a dashed line represents an unknown molecular mechanism.

thereby affecting plant growth and development. However, miR166 must be processed and regulated from transcription to maturity which shows in a summary (Fig. 7). The cis-acting elements in the promoter regulate the transcription of pri-miR166, enabling different members of the family to be differentially expressed over time and in various tissues for the coordinated completion of a series of processes influencing growth and development. However, the transcription of pri-miR166 is only the first step, and many pri-miR166 transcripts may be produced during growth and development and in response to stresses and hormones. Transcribed pri-miR166 may have multiple fates. In addition to the normal transition to mature miR166, pri-miR166 may also be degraded because of functional redundancy. Because of an insufficiency in specific nucleases or other active factors, pri-miR166 may not be able to proceed to the next developmental stage and is degraded instead. It may also be recognized by ribosomes, ultimately resulting in the production of miPEP166, which enhances the transcription of pri-miR166 and accumulation of mature miR166. Moreover, mature miR166 must bind to AGO1 to form a silencing complex. However, AGO10 can block the binding of AGO1, which inhibits the miR166 function. Every step of the maturation of miR166 is monitored and mediated by various cellular mechanisms. Therefore, to fully functionally characterize miR166, a systematic network approach is required. Only by fully understanding the various mechanisms within cells can we truly grasp the subtle changes in the miR166 role in the life cycle.

Declaration of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Analyses of microRNA166 gene structure, expression, and function during the early stage of somatic embryogenesis in Dimocarpus longan Lour.” Acknowledgements This work was funded by Research Funds for the National Natural Science Foundation of China (31672127, 31572088), the Natural Science Funds for Distinguished Young Scholar in Fujian Province (2015J06004), and the Science and Technology Innovation Fund of the Fujian Agriculture and Forestry University (CXZX2017187). References Achard, P., Herr, A., Baulcombe, D.C., Harberd, N.P., 2004. Modulation of floral development by a gibberellin-regulatedmicrorna. Development 131, 3357–3365. Allen, E., Xie, Z., Gustafson, A.M., Sung, G.H., Spatafora, J.W., Carrington, J.C., 2004. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat. Genet. 36, 1282–1290. Barik, S., SarkarDas, S., Singh, A., Gautam, V., Kumar, P., Majee, M., Sarkar, A.K., 2014. Phylogenetic analysis reveals conservation and diversification of microRNA166 genes among diverse plant species. Genomics 103, 114–121. Bhattacharyya, M., Feuerbach, L., Bhadra, T., Lengauer, T., Bandyopadhyay, S., 2012. MicroRNA transcription start site prediction with multi-objective feature selection. Stat. Appl. Genet. Mol. Biol. 11, 1–25. Chen, L., Wang, T.Z., Zhao, M.G., Zhang, W.H., 2012. Ethylene-responsive miRNAs in roots of Medicago truncatula identified by high-throughput sequencing at whole genome level. Plant Sci. 184 0-19. Couzigou, J.M., Lauressergues, D., Bécard, Guillaume, Combier, J.P., 2015. MiRNA-encoded peptides (miPEPs): a new tool to analyse the roles of miRNAs in plant biology. RNA Biol. 12, 1178–1180. Couzigou, J.M., Olivier, André, Guillotin, B., Marlène, Alexandre, Combier, J.P., 2016. Use of microRNA-encoded peptide miPEP172c to stimulate nodulation in soybean. New Phytol. 211, 379–381.

Contributions ZQL participated in the study design, carried out the experimental work and wrote the manuscript. LYL and LZX conceived of the study, and participated in its design and coordination and helped to draft the manuscript. SLY, ZST, XXP, CXH, LX, JMQ, HSQ, CYK, and ZZH prepared the materials. All authors read and approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 213

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