- Research article
- Open Access
De novo assembly and analysis of the transcriptome of Ocimum americanum var. pilosum under cold stress
© Zhan et al. 2016
- Received: 25 September 2015
- Accepted: 19 February 2016
- Published: 9 March 2016
Ocimum americanum var. pilosum is a chilling-sensitive, widely distributed plant that is consumed as a vegetable in central and southern China. To increase our understanding of cold stress responses in this species, we performed de novo transcriptome assembly for O. americanum var. pilosum and compared the transcriptomes of plants grown under normal and low temperatures.
A total of 115,022,842 high quality, clean reads were obtained from four libraries (two replicates of control samples and two replicates of chilling-treated samples) and were used to perform de novo transcriptome assembly. After isoforms were considered, 42,816 unigenes were generated, 30,748 of which were similar to known proteins as determined by a BLASTx search (E-value < =1.0E-05) against NCBI non-redundant, Swiss-Prot, Gene Ontology, KEGG, and Cluster of COG databases. Comparative analysis of transcriptomes revealed that 5179 unigenes were differentially expressed (with at least 2-fold changes, FDR < 0.01) in chilling-treated samples, and that 2344 and 2835 unigenes were up- and down-regulated by chilling stress, respectively. Expression of the 10 most up-regulated and the five most down-regulated unigenes was validated by qRT-PCR. To increase our understanding of these differentially expressed unigenes, we performed Gene ontology and KEGG pathway enrichment analyses. The CBF-mediated transcriptional cascade, a well-known cold tolerance pathway, was reconstructed using our de novo assembled transcriptome.
Our study has generated a genome-wide transcript profile of O. americanum var. pilosum and a de novo assembled transcriptome, which can be used to characterize genes related to diverse biological processes. This is the first study to assess the cold-responsive transcriptome in an Ocimum species. Our results suggest that cold temperature significantly affects genes related to protein translation and cellular metabolism in this chilling sensitive species. Although most of the CBF pathway genes have orthologs in O. americanum var. pilosum, none of the identified cold responsive (COR) gene orthologs was induced by cold, which is consistent with the lack of cold tolerance in this plant.
- Chilling tolerance
- Gene regulation
- O. americanum
Species in the genus Ocimum are native to Africa, South America, and Asia, and are valued as aromatic and medicinal plants. Essential oils extracted from Ocimum spp., such as O. basilicum, O. sanctum, and O. americanum, have anti-inflammatory, antimicrobial, antioxidant, and larvicidal activities [1–4]. Recently, the transcriptomes of O. basilicum and O. sanctum were assembled and used to identify genes involved in the biosynthesis of essential oils and medicinal metabolites [5, 6]. In addition to being used for medicinal purposes in some parts of the world [2–4], O. americanum var. pilosum of the Lamiaceae family is one of the most popular vegetables in Anhui and Henan provinces in China, where it is frequently grown. O. americanum var. pilosum is very sensitive to chilling injury, however, which limits its growing area and can also dramatically reduce its yield, leading to economic losses for farmers.
Low temperature is a major environmental factor determining the growth and productivity of plants. Temperate plants are tolerant to chilling temperatures (0-15 °C) but are usually intolerant of freezing temperatures (<0 °C) . For many temperate plant species, a period of exposure to chilling temperatures increases plant tolerance to subsequent freezing conditions in a phenomenon known as “cold acclimation” . In contrast, plants of tropical and subtropical origins are intolerant of chilling and freezing temperatures. In response to low temperature, many biochemical and physiological processes change in plants through regulation of cold responsive (COR) gene expression as well as through posttranslational protein modifications. The ICE1-CBF-COR transcriptional cascade (inducer of CBF expression 1 and C-repeat binding factor transcriptional cascade) is the best characterized pathway for gene regulation under cold conditions in many species . In Arabidopsis, three transcription factors in the CBF family (CBF1, CBF2, and CBF3) are rapidly induced by low temperatures . These CBFs can bind to and activate downstream COR genes, such as COR15, COR47, COR78, and KIN1, to protect plant cells from freezing damage. The pathway may also be important for chilling tolerance . Under cold conditions, the expression of CBFs can be regulated by several upstream transcription factors such as ICE1, CAMTAs, MYB15 and EIN3 . The protein kinase OST1 (open stomata 1) was recently found to phosphorylate ICE1 under cold stress and to thereby stabilize and activate ICE1 activity . SIZ1 (SAP and Miz 1), a SUMO (small ubiquitin related modifier) E3 ligase, can stabilize ICE1 through sumoylation . ICE1 protein stability and activity can also be regulated by E3 ligase HOS1 (high expression of osmotically responsive gene1)-mediated protein ubiquitination and proteosomal degradation under cold stress .
RNA-seq technology is increasingly being used to characterize transcriptomic events in plants. RNA-seq can provide transcriptomic information in the absence of a reference genome, and thus it is particularly useful in non-model species, whose genomic sequences are often unavailable. For many crops and other economically important plants, a period of unexpected low temperature may cause damage to plants and result in great losses to farmers. Recently, a number of studies have characterized cold responsive transcriptomes of these plants, including important crops such as Prunus dulcis Mill. (Almond) , Beta vulgaris L. (Sugar beet)  and Poncirus trifoliata (L.) Raf. (Trifoliate orange)  and other plants with high economic value, such as Eucalyptus dunnii , Chrysanthemum nankingense , and Lilium longiflorum (Easter lily) [20, 21]. These studies suggested that the gene expression responses of plants to cold are complex and involve numerous cellular processes, such as carbohydrate metabolism, protein metabolism, calcium signaling and hormonal changes [17–20].
In this study, we performed de novo transcriptome assembly of O. americanum var. pilosum and compared its transcriptomes under normal and chilling conditions to investigate how O. americanum var. pilosum responds to low temperature stress. In the de novo assembled transcriptome of O. americanum var. pilosum, we identified 42,816 active transcribed unigenes and found that the expression of 5179 unigenes was up- or down-regulated in response to low temperature. To understand the potential involvement of the CBF pathway in the cold response of O. americanum var. pilosum, we reconstructed the CBF pathway by using our de novo assembled transcriptome and compared the expression of CBF pathway factors before and after chilling treatment. We found that none of the identified COR gene orthologs was induced by cold in O. americanum var. pilosum, which is consistent with the cold sensitive phenotype of this plant. In contrast, many of the cold regulated genes have functions related to protein translation and cellular metabolism, suggesting that cold temperature affects this chilling sensitive plant by altering protein synthesis and metabolism.
Response of O. americanum var. pilosum to chilling stress
RNA sequencing and de novo transcriptome assembly
Sequencing output statistics in four O. americanum var. pilosum leaf samples from plants that were not chilled (Control_rep1 and 2) or chilled (Chilling _rep 1 and 2)
Number of raw reads
Number of clean reads
Percentage of reads kept
Size of clean reads (bp)
Number of total clean reads
Statistics of transcriptome assembly and predicted unigenes
Total number of assembled transcripts
Number of predicted unigenes
Size of data (bp)
Minimum length (bp)
Maximum length (bp)
Mean length (bp)
N50 length (bp)
Annotation and classification of O. americanum var. pilosum unigenes
Annotation statistics of O. americanum var. pilosum unigenes
Number of DEGs
In summary, all these annotation and classification analyses can provide valuable information to further investigate gene functions, metabolic processes, and active pathways of O. americanum var. pilosum.
Differentially expressed genes in chilling-treated O. americanum var. pilosum plants
Using our de novo assembled transcriptome as a reference, we identified putative genes expressed in control and chilling-treated plants. In control_rep1 and control_rep2 samples, 40,372 and 40,407 expressed putative genes (FPKM > =1) were detected, and 39334 were expressed in both samples. In chilling_rep1 and chilling_rep2 samples, 40,004 and 40,043 expressed putative genes (FPKM > =1) were detected, and 39,028 were expressed in both samples. The high similarity between the two biological replicates for either control or chilling-treated samples indicated that the RNA-seq results were consistent. The consistency was also supported by FPKM (fragments per kilobase of gene per million mapped reads) correlation analysis between the two biological replicates (r > 0.99) (Additional file 5: Figure S2).
To begin to explore the molecular mechanisms of cold stress response of O. americanum var. pilosum, we identified genes that were differentially expressed in seedlings grown under normal vs. chilling temperatures. Compared with the control, 5,179 differentially expressed genes (DEGs) with at least 2-fold changes (false discovery rate [FDR] < 0.01) were identified in chilling-treated samples with the edgeR package. Of these DEGs, 2,344 were up-regulated and 2,835 were down-regulated in chilling-treated plants. Functional annotation with five databases was also executed on these DEGs, and about 86 % of them (4,465 DEGs) were successfully annotated (Table 3 and Additional file 1: Table S1).
About 608 up-regulated DEGs and 842 down-regulated DEGs were successfully annotated with GO (Table 3 and Additional file 1: Table S1). Their GO level 2 distributions are shown in Fig. 4. Up-regulated and down-regulated DEGs had a similar distribution pattern, which was also similar to that of all GO annotated unigenes (Fig. 4).
KEGG pathway enrichment of DEGs
Plant hormone signal transduction
Starch and sucrose metabolism
Porphyrin and chlorophyll metabolism
Stilbenoid, diarylheptanoid and gingerol biosynthesis
Photosynthesis - antenna proteins
Other glycan degradation
In general, analyses towards DEGs in response to cold will help to understand how gene expression in O. americanum var. pilosum is influenced by chilling treatment.
Validation of DEGs in chilling-treated O. americanum var. pilosum plants
The 10 most up- and down-regulated O. americanum var. pilosum genes after cold treatment
Calcium-binding protein PBP1
Zinc finger CCCH domain-containing protein 29
Brassinosteroid-regulated protein BRU1
Probable WRKY transcription factor 53
Transcription factor MYC4
Bifunctional nuclease 2
Protein REVEILLE 1
Cytochrome P450 71A8
Chlorophyll a-b binding protein 36, chloroplastic
Cyclic dof factor 3
Zinc finger protein CONSTANS-LIKE 5
Granule-bound starch synthase 1, chloroplastic/amyloplastic
Retrovirus-related Pol polyprotein from transposon TNT 1-94
Serine acetyltransferase 1, chloroplastic
O. americanum var. pilosum unigenes involved in the CBF cold response pathway
Unigenes matched to known CBF-pathway factors
O. americanum var. pilosum is an aromatic plant and a popular vegetable in central and southern parts of China. It is very sensitive to low temperatures. Here, we performed de novo transcriptome assembly of O. americanum var. pilosum using the Trinity program and obtained 42,816 assembled unigenes. By analyzing the genome-wide transcriptome under low temperature, we identified several thousand potential cold-responsive unigenes and 10 pathways containing DEGs under chilling treatment. Our analysis of the DEGs suggested that cold temperature significantly affects protein translation and cellular metabolism in this chilling sensitive species. Although most genes involved in the ICE1-CBF-COR pathway have orthologs in O. americanum var. pilosum, none of the identified orthologs of COR genes was induced by low temperature, which is consistent with the lack of cold tolerance in this plant. In summary, we have profiled the high-resolution expression pattern of O. americanum var. pilosum under normal and chilling conditions. Our results should be useful for future research concerning the molecular mechanisms of low temperature responses in O. americanum var. pilosum.
Seeds of O. americanum var. pilosum in this study were collected from Funan County of Anhui Province, China. Seeds germinated on MS agar medium. Then 5-day old seedlings were transferred to soil and grown in a growth room with a 16-h-light/8-h-dark photoperiod at 22 °C. Chilling treatment were performed in climate chamber with a 16-h-light/8-h-dark photoperiod at 4 °C. During chilling treatment, control plants were kept in the same conditions but at normal temperature (22 °C). RNA from 30-day-old plants was used in RNA-seq. For RT-PCR, young leaves of 6 individual one-month-old plants were collected at every indicated time points and immediately frozen in liquid nitrogen before RNA extraction. Plants used for RT-PCR and RNA-seq were harvested in two independent experiments. Biological replicates in RNA-seq were harvested at the same time.
RNA sequencing and de novo transcriptome assembly
Total RNA was extracted from leaves of control and chilling (4 °C for 12 h) treated 30-day-old plants with the RNApure High-purity Total RNA Rapid Extraction Kit (Bioteke). Each treatment (± chilling) was represented by two biological replicates. Residual genomic DNA was removed from the total RNA with the Turbo DNA-Free kit (Ambion) following the manufacturer's instructions. The sequencing library construction and sequencing were performed in the Genomics Core Facilities of the Shanghai Center for Plant Stress Biology, SIBS, CAS (Shanghai, China) with Illumina HiSeq2500. Clean reads were acquired from initial paired-end reads after low quality regions (Q <20), PCR duplicates, and adaptor sequences were trimmed.
Inchworm, Chrysalis, and Butterfly modules of Trinity software [15, 20] were used for de novo transcriptome assembly. We first combined the sequencing reads from the four samples and applied Inchworm to assemble the RNA-seq data into contigs (unique sequences of transcripts) with the default K-mer parameter and minimum K-mers coverage of three. The resulting Inchworm contigs were bunched by Chrysalis into clusters; Chrysalis was then used to constructed complete de Bruijn graphs for each cluster. Finally, the final assembled transcripts for alternatively spliced isoforms were reconstructed by Butterfly by reconciling individual deBruijn graphs, and only transcripts ≥ 300 bp long were retained for further analysis.
Annotation of O. americanum var. pilosum unigenes
Unigenes were first annotated by using BLASTX with an expectation value of 10−5 to search the following protein databases: NCBI nr protein database (NCBI non-redundant sequence database), SwissProt, and KEGG. Next, protein information and their functional annotations were retrieved for genes with the highest sequence similarity with O. americanum var. pilosum unigenes.
Identification and annotation of DEGs
DEGs were identified based on the negative binomial distribution with the edgeR package . We calculated the false discovery rate (FDR) values of genes firstly through edgeR, and mapped reads numbers of genes were used in this analysis. Genes with FDR ≤0.01 were considered as candidates. In addition, fragments per kilobase of gene per million mapped reads (FPKM) of these candidates was generated by using RSEM . Finally, the fold change of FPKM was computed, and genes with the over or equal to 2-fold change were characterized as DEGs. Functional enrichment analyses were then performed on identified DEGs by using GOstats . For Gene Ontology and KEGG pathway analysis, we used Hypergeometric test function (p value < 0.001) .
qRT-PCR analysis of gene expression
With the RNApure High-purity Total RNA Rapid Extraction Kit (Bioteke), total RNA was extracted from leaves of 1-month-old plants treated at 4 °C for 0, 12, 24, 36, and 48 h. Residual genomic DNA was removed from the total RNA with the Turbo DNA-Free kit (Ambion) following the manufacturer's instructions. The first-strand cDNAs were synthesized from total RNA with the SupermoIII M-MuLV RT Kit (Bioteke) and were used as templates. A CFX96 Real-Time PCR Detection System (Bio-Rad) was used for real-time quantitative RT-PCR (qRT-PCR) with TransStart Tip Green qPCR SuperMix (TransGen Biotech) to confirm the identity of up- or down-regulated genes. Each experiment had three biological replicates, and the PCR conditions were as follows: 40 cycle of 95 °C for 3 min, 95 °C for 10 s, and 58 °C for 30 s. The DNA primers for probe amplification are listed in Additional file 6: Table S4. The comparative cycle threshold (ct) method was used to calculate gene expression levels, and the TUBULIN ortholog Ob_23367 was used as reference.
Availability of supporting data
The data sets supporting the results of this article are available in the NCBI’s Gene Expression Omnibus (GSE68980).
This work was supported by the Chinese Academy of Sciences. The funders of this study played no role in the design, the collection, analysis, interpretation of data, writing of the manuscript, or the decision to submit the manuscript for publication.
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