High throughput sequencing of two celery varieties small RNAs identifies microRNAs involved in temperature stress response
© Li et al.; licensee BioMed Central Ltd. 2014
Received: 30 June 2013
Accepted: 24 March 2014
Published: 27 March 2014
MicroRNAs (miRNAs) are small, non-coding RNAs of 20 to 24 nucleotides that regulate gene expression and responses to biotic and abiotic stress. Till now, no reports have previously been published concerning miRNAs in celery.
Two small RNAs libraries were constructed from two celery varieties, ‘Jinnan Shiqin’ and ‘Ventura’, and characterized by deep sequencing. A total of 431 (418 known and 13 novel) and 346 (341 known and five novel) miRNAs were identified in celery varieties ‘Jinnan Shiqin’ and ‘Ventura’, respectively. Potential miRNA-target genes were predicted and annotated by screening diverse protein databases, including Gene Ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes. Significant differential expression between the two varieties was seen for 221 miRNAs. qRT-PCR was used to analyze the abundance of six miRNAs under cold and heat stress conditions. The results showed that miRNAs may have important functions in controlling temperature stress in celery.
A large number of miRNAs were identified in celery, and their target genes, functional annotations, and gene expression patterns have been explored.
These findings provide the first information on celery miRNAs and enhance understanding of celery miRNA regulatory mechanisms under extreme temperature stress.
MicroRNAs (miRNAs) are small, non-coding RNAs found in animals and plants, and mainly function in regulating gene expression at the post-transcriptional level . miRNAs are highly conserved in eukaryotes, and are an important component in the evolution of genetic regulation [2, 3]. Mature miRNAs are generated from primary miRNAs (pri-miRNAs) via two steps. The pri-miRNAs are cut by RNA polymerase II into precursor miRNAs (pre-miRNAs) that have a stem-loop structure and are 70 to 100 nucleotides (nt) in length. The pre-miRNAs are then cleaved into mature miRNAs of 19 to 23 nt by Dicer-Like1 in the cytoplasm .
The first two characterized miRNAs, lin-4 and let-7, were identified as regulators of the juvenile-to-adult phase transition in Caenorhabditis elegans[5, 6]. The latest miRBase database (miRBase19) contains 21,264 entries representing hairpin pre-miRNAs, expressing 25,141 mature miRNA products in 193 species. Numerous studies have revealed that miRNAs are involved in diverse biological and metabolic processes, such as regulation of cell growth and development, abiotic stress response, pathogen defense and gene translational repression [7–10]. Levels of miR156, miR167, and miR164 increase during virus intrusion [11, 12]. Furthermore, miR172 and miR159 affect flowering time regulation in plants [13, 14], whereas miR160 has important roles in the regulation of plant development and hormonal signals . In cold conditions, miR393 over-expression is induced .
Celery (Apium graveolens L.) is an annual or perennial herb belonging to the Apiaceae family. Although celery originated from the Mediterranean and the Middle East, it is now cultivated worldwide. Celery is rich in carotene, vitamins, carbohydrates, and volatile aroma compounds, and has excellent medicinal properties to regulate the digestive system and blood lipids [17–19]. However, celery is one of the most common allergenic foods in many European countries . Numerous allergenic compounds contained in celery tissue can induce allergic symptoms . miRNAs have significant biological functions in metabolic and immune responses [8, 22], and exploring the miRNAs present in celery will provide a foundation for the study of their functions. Till now, there have been no previously published reports concerning miRNA s in Apiaceae species.
Extreme temperature is one of the major limiting factors on celery growth and yield. Low temperatures can lead to early bolting, and high temperatures can induce various diseases. To investigate the roles of celery miRNAs under temperature stress, two varieties, ‘Jinnan Shiqin’ and ‘Ventura’, were selected for small RNA and transcriptome sequencing. ‘Jinnan Shiqin’ was bred by the Institute of Tianjin City, China, while ‘Ventura’ originated in the United States and was introduced to China. These two varieties have similar phenotypes, although they originated from different geographical areas. Here, the significant differential expression miRNAs were detected between two varieties, and the results provide useful information for miRNA that can response to temperature stress.
Sequence analysis of small RNAs
Distribution of small RNAs among different categories in ‘Jinnan Shiqin’ and ‘Ventura’
Identification of conserved miRNAs
Identification of novel miRNAs
Novel miRNA candidates in ‘Jinnan Shiqin’
Length of precursors(nt)
Novel miRNA candidates in ‘Ventura’
Length of precursors(nt)
Prediction and annotation of miRNA target genes
Differential expression of miRNAs between celery varieties
qRT-PCR analysis of miRNAs following cold or heat stress
High throughput sequencing technology has been extensively applied in small RNA research. Thousands of miRNAs and their functions have been identified in higher plants. To date, only a single report detailing transcriptome data in celery has been published, and thus, comprehensive miRNA information is not available for this plant . Moreover, there are no reports concerning miRNAs in other species of Apiaceae. In the present study, miRNAs were identified and characterized from two celery varieties, namely, ‘Jinnan Shiqin’ and ‘Ventura’, which come from different geographical sources but have similar morphology. Till now, this study is the first to identify and investigate small RNAs in celery, and the results provide new information for further research into the functions, biological pathways and evolution of target genes related to temperature stresses in celery.
Over six million reads of 16 to 30 nt were obtained from each library. Based on the sequence conservation of mature miRNAs, 418 and 341 conserved miRNAs were identified in ‘Jinnan Shiqin’ and ‘Ventura’ libraries, respectively. Most conserved miRNAs showed high sequence similarities to other plants, and were distributed in 37 conserved miRNA families. Axtell et al.  concluded that miRNA families were highly conserved in various plants, and that these miRNAs performed analogous regulatory functions. Some highly conserved miRNA families, including miR156, miR159, miR166 and miR396, showed relatively high numbers of reads in the two celery varieties. Several studies have reported that these miRNAs have crucial roles in biotic and abiotic stress responses, plant development and fertility, and cell proliferation [14, 34–36]. Other families, such as miR395 and miR812, had very low abundance in both celery varieties. These findings suggest that over long evolutionary timescales, the miRNAs evolved at different rates and have different roles in plant development. Putative targets gene were predicted to provide more information concerning the identified miRNAs, in which most target genes were involved in molecular functions and transcription pathways.
After searching for potential pre-miRNAs and predicting their hairpin-like structures, 13 and five novel miRNAs were identified in ‘Jinnan Shiqin’ and ‘Ventura’, respectively. Surprisingly, among all the newly identified miRNAs, only one (celery-miR-27) was present in both varieties. Therefore, the functions of these novel celery miRNAs should be analyzed in future studies.
Significantly different abundance was seen for 221 miRNAs between ‘Jinnan Shiqin’ and ‘Ventura’. Different miRNA genes accounted for a large proportion of all identified miRNAs. The two varieties have similar phenotypes, although they originate from different geographical areas: ‘Jinnan Shiqin’ from Asia and ‘Ventura’ from North America. This finding suggests that miRNA evolution in the same plant species is more conservative, and that variations in miRNA machinery in the two celery varieties may be critical for the differences in miRNA expression.
Considering the instability of the environment, plants are frequently challenged by various biotic and abiotic stresses, including temperature stress. Numerous studies have confirmed that small RNAs have important roles in biotic and abiotic stress responses in plants. Some miRNAs are temperature sensitive . For instance, in wheat, miR393 and Ta-miR2002 were up-regulated after 0.5 h of heat treatment , while ten cold-regulated miRNAs were detected in Arabidopsis by microarray . In castor bean, 41 miRNAs were down-regulated and four others were up-regulated under cold stress compared with normal growth conditions . In the present study, qRT-PCR method was used to investigate six miRNAs under cold and heat stress conditions. All miRNAs in both celery varieties showed sensitivity to temperature change, and abundance was remarkably up-regulated. This finding is consistent with results in Arabidopsis, poplar, and wheat [38, 39, 41]. The stronger response of miRNAs to cold compared to heat stress may be related to celery being a cool-season biennial plant. The abundance of most miRNAs was higher in ‘Jinnan Shiqin’ than in ‘Ventura’, suggesting that ‘Jinnan Shiqin’ is more resistant to temperature stress. This result is consistent with the phenotypes of the two plants in relation to temperature stress. Thus, these miRNAs may have important roles in temperature stress defense. However, understanding the molecular mechanisms underlying these roles requires further study.
This is the first report to investigate small RNAs in celery; a large number of small RNAs were characterized as known and novel miRNAs. Putative target genes were predicted and then annotated by GO, COG, and KEGG databases to explore gene functions. The differential expression of miRNAs under temperature stress conditions between the two varieties suggests that the miRNA machinery in the two celery varieties may be different. Taken together, this study provides useful information for understanding the functions and regulatory mechanisms of miRNAs.
Celery plants (A. graveolens L. cvs. ‘Jinnan Shiqin’ and ‘Ventura’) were grown in pots containing soil:vermiculite mixture (3:1) in a controlled-environment growth chamber programmed for 16 h/8 h at 25°C/16°C for day/night and 3000 lux of light intensity. Two month-old plants were transferred to growth chambers set at 4°C or 38°C under the same light intensity and day length as above. Leaves and stems were collected 1 h after transfer to the hot or cold chambers, immediately frozen in liquid nitrogen, and then stored at −80°C until use.
RNA isolation, small RNA library development and Solexa sequencing
Total RNA was extracted using an RNA kit (RNAsimple Total RNA Kit, Tiangen, Beijing, China) according to the manufacturer’s instructions. RNA quantity and quality were examined using gel electrophoresis and a Nanodrop ND1000 spectrophotometer. Small RNAs of 18 to 30 nt were separated from total RNAs by polyacrylamide gel electrophoresis. Small RNA molecules were then ligated to Solexa adaptors at both 5’- and 3’-ends, and then converted to cDNA by RT-PCR. The purified DNA samples were sequenced using an Illumina Cluster Station and Illumina Genome Analyzer. The data were submitted to the National Center for Biotechnology Information under accession number, SRR1125031.
Prediction of conserved and novel miRNAs
After removing the impure sequences (the low quality reads, adaptor reads, and reads with length < 16 or length > 30), unique reads were queried against ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) from GenBank (http://www.ncbi.nlm.nih.gov/). rRNA, tRNA, small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) sequences were obtained from Rfam (http://rfam.sanger.ac.uk). Unique reads were also used for a nucleotide–nucleotide Basic Local Alignment Search Tool (BLASTn) search against the miRNA database (miRBase 16.0) to identify conserved miRNAs. To identify novel miRNAs, the Mireap program was used to obtain all candidate precursors with hairpin-like structures that were perfectly mapped by sequencing tags (http://sourceforge.net/projects/mireap). The secondary structures of putative pre-miRNAs were checked using Mfold . The criteria chosen for stem-loop hairpins were described by Meyers et al. .
Prediction of potential miRNA target genes
Target genes of miRNAs were predicted using the psRNA Target program (http://plantgrn.noble.org/psRNATarget). The rules used for target prediction were based on those suggested by Allen et al.  and Schwab et al. . BLASTn hits with < 4 mismatches were chosen as candidate targets, and then nucleotide 6-frame translation-protein (blastx) was used to obtain their putative functions.
Functional annotation of the potential miRNA target genes
To investigate the putative functions of potential target genes, the target sequences were annotated using diverse protein databases, including Gene Ontology (GO), Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) [30, 31, 46]. The GO categorization results are expressed as three independent hierarchies for biological processes, cellular components, and molecular functions.
Differential expression of miRNAs between celery varieties
To select differentially expressed miRNAs between the two libraries, the frequency of miRNAs was normalized to one million of the total number of miRNA reads in each sample. The selection method used was according to Audic and Claverie . IDEG6 software  was used to analyze the differential expression.
qRT-PCR analysis of miRNA abundance under temperature stress conditions
Total RNA was extracted using an RNAsimple Total RNA Kit (Tiangen) according to the manufacturer’s instructions. Small RNAs were reverse-transcribed into cDNA using the One Step PrimeScript® miRNA cDNA Synthesis Kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qRT-PCR) was performed using the MyiQ single-color real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The reactions were carried out in a total volume of 20 μL containing 2.0 μL of diluted cDNA, 0.8 μL of each primer, and 10 μL of SYBR GreenI Mix with the following cycling profile: 95°C for 30 s; followed by 40 cycles at 95°C for 5 s, 60°C for 20 s; Melting curve analysis was performed (61 cycles at 65°C for 10 s) to verify specific amplification. Each sample was processed in triplicate, and 5.8S rRNA was used as an internal control. The qRT-PCR primers are listed in Additional file 2: Table S2.
The research was supported by National Natural Science Foundation of China (31272175); New Century Excellent Talents in University (NCET-11-0670); Graduate Educated Innovation Project of Jiangsu (CXZZ13_0297); Jiangsu Natural Science Foundation (BK20130027); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Jiangsu Shuangchuang Project.
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