- Background: Light is the main source of energy and, as such, is one of the most important environmental factors for plant growth, morphogenesis, and other physiological responses. - However, the role of miRNAs in the light response is less studied. - We used tomato seedlings that were cultured in red light then transferred to blue light for 2 min to identify miRNAs related to light response by high-throughput sequencing.. - Among them, 15 known and 5 predicted novel miRNAs were differentially expressed after blue light treatment compared with the control (red light treatment). - Zeatin biosynthesis and plant hormone signal transduction are related to plant hormones, indicating that plant hormones play important roles in the light response.. - Conclusion: Our results provide a theoretical basis for further understanding the role of miRNAs in the light response of plants.. - Tomato (Solanum lycopersicum L.) is one of the main vegetables under protected cultivation and is an import- ant part of the human diet. - MiRNAs not only regulate the development of leaf, flower, and fruit in tomato [5–9], but also are involved in the biotic and abiotic stress responses of tomato plants . - However, there are few reports on the roles of miRNAs in the light response.. - Light is the main source of energy and is one of the most important environmental factors for plant growth, morphogenesis, and other physiological re- sponses [14–16]. - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - In addition, red light inhibits the photosynthetic product from the leaves, which increases the starch accumulation of the leaves. - In this study, we cultured tomato seedlings in red light then transferred some of them to blue light for 2 min, and identified miRNAs related to the light signal response by high-throughput sequencing. - The results will provide a theoretical foundation for further understanding the role of miRNAs in the light response of plants.. - Sequence analysis, and classification and annotation of the small RNAs (sRNAs). - After removing the low-quality reads of the total) and of the total) clean reads were obtained in the red light and blue light libraries, respectively (Table 1).. - Most of the sRNAs in the red light and blue light librar- ies were unannotated, followed by those identified as. - Only a small proportion of the sRNAs were iden- tified as scRNAs, snRNAs, snoRNAs, or tRNAs (Fig. - Generally, sRNAs are 18–30 nt long, and in the control and treated libraries, most of them were 24 nt long, ac- counting for 36.57 and 38.90% of the total reads, respect- ively. - Furthermore, most of the reads (90.16 and 89.52%) in the control and treated libraries were ≤ 24 nt (Fig. - We identified 108 known miRNAs in the two libraries, and 88 of them were from 39 miRNA families. - Sly-miR159 was the most abundant with 137,039 and 129,853 counts in the control and treated libraries, respectively (Additional file 2: Table S2).. - We identified 141 candidate novel miRNAs in the two li- braries, and 79 of them were from 56 miRNA families. - Among the novel miRNAs, unconservative_1_3796 was the most abun- dant with 101,523 and 100,601 counts in the control and treated libraries, respectively (Additional file 2: Table S2).. - Overall, we identified 249 miRNAs (108 known and 141 novel) in the two libraries. - By comparing the expression levels of the miRNAs between the red light treated (con- trol) and blue light treated libraries we identified 15 known and 5 novel miRNAs that were differentially expressed. - Table 1 Quality control of the clean reads data. - Prediction and functional analysis of the target genes of the differentially expressed miRNAs. - A total of 40 target genes of the 20 differentially expressed miRNAs were identified and functionally an- notated with gene ontilogy (GO) terms and kyoto encyclopedia of genes and genomes (KEGG) pathways.. - In the blue light treated li- brary, DNA metabolic process (GO:0006259), bounding membrane of organelle (GO:0098588), and nucleic acid binding (GO:0003676) were significantly enriched (Additional file 5: Table S5).. - Verification of differentially expressed miRNAs and target genes. - The expression levels of the 20 differentially expressed miRNAs and 10 randomly selected target genes were. - The expression patterns of the miRNAs deter- mined by qRT-PCR were consistent with those from the Illumina RNA sequencing data, and the expression pat- terns of the 10 target genes were opposite to those of the corresponding miRNAs (Fig. - Therefore, in this study, tomato seedlings were cultured in red light then some were transferred to blue light for 2 min. - Then the red light treated (control) and blue light treated libraries were compared to detect miRNAs related to light signals.. - The sequencing results showed that 24 nt long sRNAs were the most abundant in the control and blue light treated libraries, accounting for 36.57 and 38.90% of the total reads respectively. - 1 Analysis of the small RNAs (sRNAs) in the blue light and red light treated libraries. - a Classification of the sRNA sequences in the two libraries. - b Length distribution of sRNA in the two libraries. - S04, S05, and S06 are blue light treated tomato leaves. - We detected 249 miRNAs in the two libraries. - Of the 108 known miRNAs, 88 belonged to 39 families. - Of the 141 novel miRNAs, 79 belonged to 56 fam- ilies, and the MIR398 family had the most members.. - Unconservative_1_3796 was the most highly expressed of the novel miRNAs, having 101,523 and 100,601 counts in the red light control and blue light treated libraries, re- spectively (Additional file 2: Table S2).. - Tsuda seedlings, blue light specifically down-regulated miR156 and miR157, and up-regulated the target genes SPL9 and SPL15. - [38] reported that blue light inhibited the expression of miR394, which promoted the expression of the target genes and the accumulation of flavonoids and epicatechins, but inhibited the synthesis of rutin. - We found that sly-miR156e-3p, sly-miR156e-5p, and sly-miR394-3p were significantly down-regulated in the blue light treated leaves, which is consistent with the above research results.. - In addition, 20 miRNAs (15 known and 5 novel) belonging to 8 families (MIR169_2, MIR169_1, MIR5302, MIR1516, MIR156, MIR394, MIR837, and MIR8005) were differentially expressed in the blue light treated leaves. - a Scatter diagram of the differential read counts of miRNA. - Each point in the figure represents a miRNA. - b Heat map of the differentially expressed miRNAs. - Previ- ous studies have shown that plant hormone signal trans- duction pathways were involved in the growth and development of sugarcane [36], maize [40], and radish [41], as well as in the responses to pathogenic micro- organism infection [42–44], salt stress [45], and drought [46, 47]. - Members of the TIFY pro- tein family are involved in many biological processes.. - ZIM) in Arabidopsis thaliana led to the extension of the petiole and hypocotyls [50], overexpression of AtTIFY4a (also known as PPD1) and AtTIFY4b (also known as PPD2) contributed to the synchronous growth of Arabi- dopsis leaves [51], and overexpression of JAZ affected the response to biotic and abiotic stresses through medi- ating jasmonic acid signal transduction [52–55]. - PP2C is a protein serine/threonine phosphatase and a key en- zyme in the regulation of reversible protein phosphoryl- ation. - 3 Gene ontology (GO) classification and kyoto encyclopedia of genes and genomes (KEGG) analysis of target genes of the differentialy expressed miRNAs. - a GO annotation of 16 candidate target genes. - the right y-axis indicates the number of target genes in a category. - Each graph represents a KEGG pathway and the pathway name is shown in the right graph. - The abscissa is the enrichment factor, indicating the proportion of the number of differentially expressed miRNA target genes annotated to a certain pathway in the total number of genes annotated to this pathway. - Thus, the larger the ordinate, the more reliable the significance of the enrichment of the differentially expressed miRNA target gene in this pathway. - c KEGG classification map of the target genes of the differentialy expressed miRNAs.. - 4 Verification of the differentially expressed miRNAs and their target genes by qRT-PCR. - b Verification of the target genes target genes in tomato. - Adenylate isoamyltransferase, a rate-limiting enzyme in cytokinin synthesis, catalyzes the transfer of the isopentenyl group of dimethylallyl pyrophosphate to the amino terminal N 6 of ATP, ADP, and AMP. - Zeatin biosynthesis and plant hormone signal transduction are both signaling pathways related to plant hormones, which indicates that plant hormones play very important roles in the light response of plants.. - Among them, 15 known and 5 novel miRNAs were differentially expressed in tomato leaves after blue light treatment. - KEGG enrichment analysis of the target genes revealed that the zeatin biosynthesis (ko00908), homologous recombination (ko03440), and plant hormone signal transduction (ko04075) pathways were obviously enriched. - Zeatin biosynthesis and plant hormone signal transduction both are related to plant hormones, which indicates plant hormones play a very important role in the light response. - When the second true leaf had fully expanded, the same growth conditions were selected and transferred to the light quality laboratory of Scientific and Technological Innovation Park in Shandong Agriculture University and grown in the same substrate under red light (657 nm) until the five-leaf stage. - Then, half of the seedlings were transferred to blue light (457 nm) for 2 min. - The sec- ond leaf from the bottom of the tomato seedlings treated with red and blue light was removed and frozen in liquid nitrogen, then stored at − 80 °C for further experiments.. - The concentra- tion and quality of the RNA samples were detected using a Nanodrop 2000 spectrophotometer (Thermo Fisher scientific, Wilmington, USA). - The concentration of the sRNAs in each library was detected using Qubit 2.0, then they were diluted to 1 ng. - The insert size was detected using an Agilent 2100 Bioanalyzer and the ef- fective concentration of each library was measured by qRT-PCR to ensure the quality of the library. - High- throughput sequencing of the libraries was carried out using an Illumina’s HiSeq X-Ten system, with single-end read length of 50 nt.. - We used the miRDeep2 (v2.0.5) package [67] to compare reads that were aligned to the tomato reference genome with known miRNA precursor sequences in the miRBase database. - Reads that did not find matches in miRBase were identified as novel miRNAs by Bayesian model grading based on the loca- tion of the reads in the precursor sequence (including mature, star, and loop) and the energy of the precursor structure determined by RNAfold randfold. - The fold change (FC) indicates the ratio of the expres- sion levels in the two libraries.. - The functions of the target genes were pre- dicted by BLAST searches against the GO [72] and KEGG [73] databases.. - Table 2 Primers used for qRT-PCR verification of the miRNAs and target genes. - Verification of miRNAs and target genes by qRT-PCR The expressions of 20 of the differentially expressed miRNAs and the corresponding 10 major target genes were detected by qRT-PCR. - Amplification curve and melting curve analyses (95 °C for 60 s, 55 °C for 30 s, and 95 °C for 30 s) were carried out to ensure the specificity of the prod- ucts. - KEGG analysis of target genes.. - We are grateful to all co-authors who participated in the studies mentioned in the text that were published by our groups. - LXW and YHD contributed in the retrieval of genes and the analysis of qRT-PCR results. - The dataset supporting the conclusions of this article is available in the NCBI ’ s BioProject database [PRJNA523681].. - The tomato fruits were cultivated and collected in the light quality laboratory of Scientific and Technological Innovation Park in Shandong Agriculture University. - 1 Vegetable and Flower Research Institute of Shandong Academy of Agricultural Sciences / Shandong Key Laboratory of Greenhouse Vegetable Biology / Shandong Branch of National Vegetable Improvement Center / Vegetable Science Observation and Experimental Station in Huang-Huai District of the Ministry of Agriculture, Jinan 250100, China. - Identification and expression profiling of microRNAs involved in the stigma exsertion under high-temperature stress in tomato. - Effects of different proportions of red and blue light on the growth and photosynthesis of tomato seedlings. - A survey of the small RNA population during far- red light-induced apical hook opening. - Exploration of the effect of blue light on microRNAs involved in the accumulation of functional metabolites of longan embryonic calli through RNA-sequencing. - Effects of blue light on flavonoid accumulation linked to the expression of miR393, miR394 and miR395 in longan embryogenic calli. - Origin and evolutionary analysis of the plant-specific TIFY transcription factor family. - Functional analyses of the ABI1-related protein phosphatase type 2C reveal. - POLTERGEIST functions to regulate meristem development downstream of the CLAVATA loci. - Differential activation of the wheat SnRK2 family by abiotic stresses. - Genome-wide identification and evolutionary analyses of the PP2C gene family with their expression profiling in response to multiple stresses in Brachypodium distachyon
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