- Original Article
- Open Access
Comparative RNA-Seq analysis on the regulation of cucumber sex differentiation under different ratios of blue and red light
© The Author(s) 2018
- Received: 2 April 2018
- Accepted: 27 August 2018
- Published: 10 September 2018
Cucumber (Cucumis sativus L.) is a typical monoecism vegetable with individual male and female flowers, which has been used as a plant model for sex determination. It is well known that light is one of the most important environmental stimuli, which control the timing of the transition from vegetative growth to reproductive development. However, whether light controls sex determination remains elusive. To unravel this problem, we performed high-throughput RNA-Seq analyses, which compared the transcriptomes of shoot apices between R2B1(Red light:Blue light = 2:1)-treated and R4B1(Red light:Blue light = 4:1)-treated cucumber seedlings. Results showed that the higher proportion of blue light in the R2B1 treatment significantly induced the formation of female flowers and accelerated female flowering time in this whole study. The genes related to flowering time, such as flowering locus T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), were up-regulated after R2B1 treatment. Furthermore, the transcriptome analysis showed that up-regulation and down-regulation of specific DEGs (the differentially expressed genes) were primarily the result of plant hormone signal transduction after treatments. The specific DEGs related with auxin formed the highest percentage of DEGs in the plant hormone signal transduction. In addition, the expression levels of transcription factors also changed after R2B1 treatment. Thus, sex differentiation affected by light quality might be induced by plant hormone signal transduction and transcription factors. These results provide a theoretical basis for further investigation of the regulatory mechanism of female flower formation under different light qualities in cucumber seedlings.
- Sex differentiation
Sex differentiation of flower buds is an important developmental process, which directly affects the product yield in plants. Cucumber (Cucumis sativus L.) is a typical monoecious plant with distinct male and female flowers. It has been served as a model system for studying physiological and molecular aspects of sex determination and differentiation in plants (Bai and Xu 2013). In the young floral buds of cucumber, both the stamen primordia and carpel primordia are initiated, with sex determination occurring just after the bisexual stage. Subsequently, male or female flowers are formed and become enlarged due to the selective arrestment of carpel or stamen development, respectively (Bai et al. 2004).
Sex differentiation of cucumber can be affected by phytohormones, such as ethylene and gibberellin. Exogenous ethylene treatment induced female flower formation in the cucumber. The ethylene content in gynoecious cucumbers is found to be higher than that of monoecious plants (Trebitsh et al. 1987; Rudich et al. 1972). Ethylene synthesis genes play an important role in the sex differentiation of the cucumber flowers, such as 1-aminocyclopropane-1-carboxylic acid oxidases (CsACO2). Organ-specific overexpression of CsACO2 driven by the organ-specific promoter P (AP3) significantly affected stamen but not carpel development, acting similarly to natural floral development of female cucumber flowers (Duan et al. 2008). A conserved-residue conversion in CsACS2 induced the formation of bisexual flowers in the cucumber (Li et al. 2009). Furthermore, ethylene signaling pathway also affects the sex differentiation. CsETR1, an ethylene receptor involved in ethylene signaling transduction, had been demonstrated to play a key role in stamen arrest in female cucumber flowers (Wang et al. 2010). Ethylene-responsive gene associated with the formation of female flowers (ERAF17), a MADS-box gene, could be induced by ethylene and might be involved in formation of female flowers in cucumbers (Ando et al. 2001). Exogenous gibberellic acid (GA3) application promoted the formation of male flowers in gynoecious plants (Pike and Peterson 1969). GA production in andromonoecious cucumbers was higher than that in gynoecious and monoecious plants (Junior et al. 1972). The GA signaling pathway is involved in stamen development in the cucumber (Zhang et al. 2014b; Fei et al. 2004). Cucumber DELLA Homolog (CsGAIP) is predominantly expressed in the male specific organs during cucumber flower development and belongs to the DELLA (the negative regulators of the GA action) family. CsGAIP inhibited stamen development through transcriptional repression of B-class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis (Zhang et al. 2014a). CsGAMYB1, a positive regulator involved in the GA signaling pathway, also mediates the sex expression of cucumbers. Knocking out the CsGAMYB1 gene in cucumbers resulted in decreased ratios of nodes with male to female flowers (Zhang et al. 2014b). The relationship between ethylene and gibberellin in mediating sex differentiation was interpreted recently, with this study finding that gibberellin mediates sex differentiation via ethylene-dependent and ethylene-independent pathways in cucumbers (Zhang et al. 2017). Apart from ethylene and gibberellin, other hormones are also involved in the flower sex differentiation of plant (Song et al. 2013). This includes the abscisic acid (ABA) induction of male flower formation (Zhu et al. 2010); Jasmonic acid (JA) signaling pathway, which might participate in the abortion of male flowers (Acosta et al. 2009); and auxin (IAA) content, which was found to increase during female flower development and decrease during male flower development (Sakata et al. 2010). Ethylene biosynthesis has probably been induced by IAA to promote female flower formation as well (Trebitsh et al. 1987). The IAA induced CsACS1 gene expression in cucumbers, which suggested that IAA promoted female flower formation through inducing ethylene synthesis (Trebitsh et al. 1997; Mibus and Tatlioglu 2004).
Environmental cues, such as temperature, photoperiod, nutrition, also affected sex determination in many species (Golenberg and West 2013; Korpelainen 1998). Light is one of the major external factors that influence plant growth and development (Chen et al. 2004). Plants respond to light through mediating light receptors. Until now, known photoreceptors were divided into three classes: the UV-B (280–320 nm light) photoreceptors; the red/far-red reversible photoreceptors, known as the phytochromes PhyA–PhyE (Mathews and Sharrock 1997) and blue UVA photoreceptors. These blue UVA photoreceptors have three classes, which are the cryptochromes (CRY1, CRY2 and CRY3) (Mathews and Sharrock 1997; Brudler et al. 2003), phototropins (PHOT1 and PHOT2) (Briggs et al. 2001) and aureochromes (AUREO1 and AUREO2) (Ishikawa et al. 2009; Takahashi et al. 2007). There is less evidence examining the effects of light on plant sex differentiation. In the gametophytes of the Her1 mutant, blue light induced male development and red light suppressed male development (Kamachi et al. 2007). Our previous studies showed that light quality affected cucumber flower formation and sex differentiation. In our previous study, we used 4R1B (Red:Blue = 4:1), R2B1 (Red:Blue = 2:1), R6G2B1 (Red:Green:Blue = 6:2:1) ratios supplemental light to treat the cucumber seedlings. Comparing to natural light, R2B1 treatment was the best light quality ratio for improving the female flower formation, location and flowering time, but none of them has effects on male flower formation (our unpublished data). However, the mechanism of how light quality regulates flower sex differentiation still remains unclear. In our study, we aimed to understand the mechanism of light regulating cucumber sexual development. We performed RNA-Seq analyses to compare the transcriptomes of shoot apices between cucumber seedlings after R2B1 treatment and R4B1 treatment. Our results built a foundation for dissecting the molecular mechanism of flower sex differentiation in cucumbers.
Effects of different LED light quality treatments on flower formation and flowering time
Effects of different light quality on the formation of female flowers and flowering time
First female flower located node
Total female flower numbers in 15 nodes
Days from transplanting to first female flowering
R2B1 (Red light:blue light = 2:1)
7.20 ± 0.23b
2.70 ± 0.13a
21.85 ± 0.17b
R4B1 (Red light:blue light = 4:1)
8.03 ± 0.19a
2.32 ± 0.10b
22.53 ± 0.12a
Analysis of differentially expressed genes (DEGs) under treatments with different ratios of blue and red light
Statistical results of expressed genes in different libraries
Numbers of known genes detected
Numbers of detected new genes
In addition, there were more DEGs in the 5 days, 10 days and 15 days library after R2B1 irradiation compared to R4B1 irradiation. We used a Venn diagram to analyze all these up-regulated or down-regulated DEGs collected from all these treatments at different time points. The results showed that 423 DEGs were found in all three stages after both R4B1 and R2B1 treatment as well as more up-regulated DEGs (1795 DEGs) at all three stages under both treatments (Fig. 1b).
Gene ontology (GO) analysis of DEGs after different ratios of blue and red light treatments
Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of DEGs after different ratios of blue and red light treatments
Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment of significantly up/down-regulated DEGs in R2B1/R4B1
DEGs with pathway annotation (543)
Plant hormone signal transduction
Up-regulated DEGs in R2B1
Protein processing in endoplasmic reticulum
Ubiquitin mediated proteolysis
Starch and sucrose metabolism
Alanine, aspartate and glutamate metabolism
Up-regulated DEGs in R4B1
Biosynthesis of amino acids
Down-regulated DEGs in R2B1
Protein processing in endoplasmic reticulum
Plant hormone signal transduction
Down-regulated DEGs in R4B1
Porphyrin and chlorophyll metabolism
Auxin plays an important role in the regulation of cucumber sex differentiation under different ratios of blue and red light treatments
Validation of RNA-Seq results by real-time quantitative PCR (qRT-PCR)
Twenty-six genes with different expression profiles were randomly selected to verify the RNA-Seq results by qRT-PCR. α-Tubulin (TUA) was used as the internal reference control to standardize the results (Wan et al. 2010). As shown in Additional file 2: Figure S1, twenty-three genes had the same expression pattern. The Pearson correlation coefficient between the RNA-Seq and qRT-PCR data were high, confirming the accuracy of the RNA-Seq data (Additional file 3: Figure S2 and Additional file 4: Figure S3).
Effect of different ratios of blue and red light treatments on light signal transduction of cucumber seedling leaves
Analysis of differentially expressed genes related to flower sex differentiation of cucumber seedlings under different ratios of blue and red light treatments
Light quality affects cucumber flowering time
Blue light is a strong signal in floral bud formation. In Chrysanthemum, flower budding was formed even after a longer photoperiod than a critical day length under blue light illumination (Jerzy et al. 2011). In petunia plants, floral bud formation and flowering occurred earlier under blue light treatment, while no floral buds were observed under low red irradiance. High red irradiance and temporal switching to blue light during long-term low red irradiance induced floral development (Fukuda et al. 2016). In our experiment, we found that the first female flower opened earlier and occurred mainly at the lower node positions under a higher proportion of blue light irradiance (Table 1). This suggested that blue light promoted flower bud formation and accelerated cucumber flowering.
Cryptochromes (CRY) are flavo-proteins that direct a diverse array of developmental processes in response to blue light in plants (Liu et al. 2016). CRY1 and CRY2 function as major blue light receptors regulating blue light-induced de-etiolation and photo-periodic flowering (Guo and Li 1998). In Arabidopsis, CRY1 and CRY2 serve both distinct and partially overlapping functions in regulating photomorphogenic responses and photoperiodic flowering. The gain-of-function mutant alleles of CRY1 exhibited an early flowering phenotype after several days (Exner et al. 2010). In our study, we detected a higher expression of CRY1 in cucumber seedling leaves after R2B1 treatment than R4B1 treatment (Fig. 7), which was consistent with the earlier flowering phenotype after R2B1 irradiation. The phytochrome (phy) family of sensory photoreceptors (phyA to phyE in Arabidopsis thaliana) also responds to inducing floral budding. Low red/far-red ratio promotes flowering in Arabidopsis through PHYA inhibiting PHYB (Chory 2003; Halliday et al. 2003). In our study, we detected 4 phytochrome orthologous genes in cucumber seedling leaves and shoot apices. Expression analysis showed that PHYA accumulated more in leaves at the R4B1-15 stage than R2B1-15 stage (Fig. 7). In comparison, the other genes displayed a higher expression in leaves at R4B1-10 stage than R2B1-10 stage (Fig. 7). Similar results were found in shoot apices, with PHYA, PHYB, PHYC, PHYE expressed more in the shoot apices of R4B1-10 sample than R2B1-10 sample (Fig. 7). The expression of CRY2 and PHYD were not detected in both cucumber leaves and shoot apices.
CONSTANS (CO) is a key transcription factor regulating flowering time. CO positively regulates two floral integrators. In Arabidopsis, CO activated SOC1 through FT to promote flowering (Rosas et al. 2014; Lee and Lee 2010). In this study, compared to R2B1-15, CO showed a higher expression in R4B1-15 (Fig. 7). Although CO transcription regulation was affected by blue light treatment, CO was not only positively regulated by CRY2 and PHYA, but also negatively regulated by PHYB (Valverde et al. 2004). The lower expression under a high proportion of blue irradiation might result from the co-regulation of the cryptochrome receptor and phytochrome receptor. In Arabidopsis, blue light could induce SOC1 and FT expression to promote flowering (Hori et al. 2011). In petunia plants, blue light induces FBP28 expression, the orthologous gene of SOC1 (Fukuda et al. 2011). SOC1 is one of the direct targets of AGL24 and is up-regulated by AGL24. In Arabidopsis, SOC1 and AGL24 are able to up-regulate each other’s expressions, both of them are MADS-domain-containing transcription factors that determine flowering time (Michaels et al. 2003). In this study, FT showed a higher expression in R2B1-10 and R2B1-15 than in R4B1-10 and R4B1-15 (Fig. 8b), which suggested that higher proportion of blue light irradiation accelerated cucumber flowering through inducing FT expression. Two SOC1 orthologous genes and seven AGLs (only AGL19 showed different expression) orthologous genes were found in cucumber shoot apices. SOC1 and AGL19 displayed the same expression pattern, with increased expression in R2B1-10 and R2B1-15 treatments (Fig. 8b). All these results suggested that a higher proportion of blue light induced cucumber flower bud formation and promoted flowering time through up-regulating expression of these flowering regulation factors.
Light quality affects flower sex differentiation of cucumber seedlings through regulating plant hormone signaling transduction
In this study, we found that under a higher proportion of blue light (R2B1) treatment, the first female flower node declined significantly and the number of female flowers within fifteen plant nodes increased markedly (Table 1). Transcriptome analysis of cucumber seedlings under different light treatment by RNA-seq technology showed that there were more DEGs under R2B1 treatment. These DEGs were mostly involved in the hormone signaling pathway (Table 3). This suggested that a higher proportion of blue light regulated female flower formation through mediating plant hormone signaling pathways.
Plant sex differentiation is closely related to plant hormones, such as ethylene, which is considered as a potent sex hormone in cucumbers that can induce formation of female flowers (Trebitsh et al. 1987; Rudich et al. 1972; Li et al. 2017). Another example is the gibberellins, which can promote the formation of male flowers via ethylene-dependent and ethylene-independent pathways in cucumber (Hao et al. 2003). ABA can promote male flower development (Zhu et al. 2010), while auxin is able to promote the female tendency of vegetables and induce female flower development through triggering ethylene synthesis (Atsmon and Tabbak 1979; Galun et al. 1962). Exogenous CTK application can turn the grape male flower into a female flower (Chang et al. 1999; Negi and Olmo 1966). JA can regulate gynoecium development (Figueroa and Browse 2015), while BR induces androecium development in maize and inhibits gynoecium development (Hartwig et al. 2011; Makarevitch et al. 2012). In this study, we found that among these DEGs related to plant hormone transduction pathways, the specific DEGs related with IAA formed the higher proportion of DEGs in plant hormone signal transduction, which was followed by the specific DEGs related with CTK, ABA, ETH, BR, JA and SA (Fig. 5).
Ethylene is considered as a potent sex hormone in cucumber that can induce formation of female flowers (Malepszy and Niemirowicz-Szczytt 1991). Ethylene content in shoot apices of gynoecious cucumbers is higher than that of monoecious plants (Rudich et al. 1972; Fujita and Fujieda 1981; Trebitsh et al. 1987). Treatment with exogenous ethylene or ethylene-releasing reagents can increase the numbers of female and bisexual flowers in monoecious and andromonoecious lines, respectively (Mcmurray and Miller 1968; Iwahori et al. 1969). Until now, the molecular mechanism of ethylene-regulated sex determination of cucumber has been well understood. Ethylene biosynthetic genes and ethylene signal transduction genes are involved in the sex expression of cucumber (Wang et al. 2010). In the ethylene signal transduction pathway, the receptors, such as ETRs, function as negative regulators, while ERFs (downstream components of receptors) act as positive transcription factors to regulate sex determination (Tao et al. 2018; Prescott et al. 2016). In our study, the expression levels of some ERFs were increased under R2B1 treatment compared to R4B1 treatment (Fig. 6a). They were probably involved in cucumber sex expression through inhibiting the differentiation into males and promoting the differentiation into females. However, this understanding was based on bioinformatics analysis. The precise roles of ERFs in cucumber flower development remained unclear and should be verified in future studies using advanced physiological and molecular techniques. CTR1 is a negative regulator of ethylene signaling, while CsCTR1 expression gradually declined during male flower development and increased during female flower development (Bie et al. 2014). In our study, CsCTR1 expression increased on the 5th day after a higher proportion of blue light treatment.
Ethylene plays a key role in plant sex determination, with several research results proving that there is communication between auxin and ethylene during plant development (Trebitsh et al. 1987; Gao et al. 2015; Wang et al. 2018). In our study, we found a considerably number of transcription factors involving in auxin signaling pathway altered their expression levels under R2B1 treatment (Fig. 6). IAA may play a key role in effect of light quality on sex differentiation in cucumber. IAA has been considered as a transcription factor activated by phytochrome, which participates in signaling transduction. During the early development of plants, there are several reactions between phytochrome and auxin, such as auxin homeostasis promoted by light (Zhang et al. 2014c). Auxin is an important regulator in the development of male flowers (Sakata et al. 2010). The auxin synthesis pathway can be regulated by aldehyde oxidases, which have been identified in cucumber (Brown and Purves 1978). Aldehyde oxidases are involved in the conversions of indole-3-acetaldehyde to IAA (Mano and Nemoto 2012). Higher activity of aldehyde oxidase has also been measured in the auxin-overproducing superroot1 (sur1) mutant of Arabidopsis (Seo et al. 1998). Aldehyde oxidase was reported to regulate auxin and ABA biosynthesis pathway (Koshiba et al. 1996; Seo et al. 1998, 2000). In our study, the expression of Aldehyde oxidase (Csa4G269130) was down-regulated in R2B1 samples (Additional file 5: Table S2), which suggested that the auxin synthesis or ABA synthesis in cucumber seedlings flowers was low under a higher proportion of blue light. This result was contradicted against the finding that more female flower were formed in the R2B1 samples which might result from higher auxin content. Meanwhile, it was also contrary to the most up-regulated auxin responsive genes SAURs (Fig. 6). While the expression of auxin transporting gene LAX1 and PIN1 were also altered indicating that the auxin transport was changed inside the cucumber flower. Even though the low biosynthesis might be found in the flowers, the higher transportation might also cause a high auxin content in the flower tissues. However, auxin content detection and gene function analysis experiments should be performed to confirm this hypothesis.
Gibberellins, a class of tetracyclic diterpenoid phytohormones, can promote the differentiation of cucumbers into male flowers. GA production in andromonoecious cucumbers is higher than that in gynoecious and monoecious plants (Junior et al. 1972). Exogenous GA3 application can increase the ratio of males to females in monoecious cucumbers and induce the formation of male flowers in gynoecious plants (Wittwer and Bukovac 1962; Pike and Peterson 1969). In our study, we identified two DEGs involved in the GA synthesis pathway. Ent-copalyl diphosphate synthase (CPS) is limiting for ent-kaurene production in the first portion of the GA synthesis pathway (Prisic and Peters 2007). CsCPS1 was down-regulated in R2B1-5 and up-regulated in R4B1-15 (Fig. 6), which suggested that a high proportion of blue light reduces levels of GA in cucumber seedling flowers through breaking down the CPS1 gene expression. GA2-oxidase (GA2ox) is also a key gene for GA synthesis pathway. It catalyzes catabolism and inactivation of bioactive GAs or their precursors (Schomburg and Amasino 2003; Lester et al. 1999; Thomas et al. 1999). In our study, CsGA2ox2 was up-regulated in R2B1-10 samples (Fig. 6), which suggested that a high proportion of blue light reduces active GA levels through inducing the expression of CsGA2ox2. In addition, the GA signaling pathway is involved in stamen and anther development in hermaphroditic plants, such as Arabidopsis and rice (Oryza sativa) (Aya et al. 2009; Plackett et al. 2011; Sun 2011). However, in this study, we did not identify any DEGs involved in GA signaling pathway.
PIF, phytochrome interaction factor, plays a central role in light signal transduction mediated by phytochrome (De and Prat 2014). PIF3 can activate the transcription factor EIN3 directly, which was involved in the ethylene signaling pathway (Zhong et al. 2012). COI1, JAZ, MYC2 and JAR1 involved in the JA signaling pathway affects the light signal response (Kazan and Manners 2011). PHYB negatively regulated ABA accumulation through mediating light signals, while PHYA positively regulated ABA signaling pathway under far-red light treatment (Gu et al. 2012; Mach 2014). Thus, it was seen that light could regulate plant sex differentiation through mediating hormones by light receptors. The phytohormones, auxin, BR, CK, ETH, GA and JA, influenced sex differentiation in flowering plants (Durand and Durand 2010; Louis and Durand 1978; Chailakhyan 1979; Irish and Nelson 1989). Our results showed that the expression of some key genes involved in ABA, auxin, CK, ETH, GA and JA biosynthesis pathways significantly changed during development and sex expression, which suggesting that these hormones might participate in these processes. Thus, it is interesting to investigate how these hormones interact with one another to regulate the abortion of male flowers in gynoecious plants.
Light quality affects flowering time of cucumber seedlings through regulating transcription factors
A total of 433 transcription factors have been identified by RNA-seq technology. Among them, ethylene transcription factor accounted for 24.02%, MYB transcription factor accounted for 20.79%, bHLH transcription factor accounted for 16.17% and WRKY transcription factor accounted for 10.62%.
Most studies have shown that MYB, bHLH and WRKY transcription factors play an important role in biotic stress and abiotic stress (Oh et al. 2012; Li et al. 2010; Jiang and Yu 2009), but some studies proved that they were also related to flower development (Wang et al. 2011; Luo et al. 2013; Zhang et al. 2014b). In Arabidopsis, JA mediates the transcription factors MYB21 and MYB24 to regulate stamen development (Song et al. 2011), while MYC5 regulated stamen development by activating JA signaling pathway through inducing MYB21 expression. PIFs belongs to bHLH family of transcription factors, which interact with activated Pr to initiate phytochrome signaling transduction. In Arabidopsis, PIF4 accelerated flowering by activating the FT gene (Kumar et al. 2016). WRKY family members are key factors for the ABA response pathway (Rushton et al. 2012). It had been shown that WRKY transcription factors regulated plant flowering. WRKY25 accelerated flowering through negatively regulating AP1 directly or indirectly (Wang et al. 2011). In pea, GsWRKY20 promoted flowering by regulating the flowering related genes FT, SOC1 and CO (Luo et al. 2013).
Plant materials and treatments
LED light produced by Kedao technology corporation (Huizhou, China) (Red light: 650–660 nm and Blue light: 450–460 nm) was chosen for this study. Cucumber (Cucumis sativus L. cv. Huaqing No. 5) seeds were cultivated on a sponge block with a Yamasaki culture solution. When cotyledon appeared, we placed these seedlings under light conditions as follows: 300 μmol/m2/s, 12/12 (light dark) at 25 °C and a relative humidity of 70–80%. There were 2 treatments with ratios of red to blue being 4:1 (R4B1) and 2:1 (R21B), respectively. Each treatment lasted 20 days. As the flower primordium appeared 10 days after light treatment, the shoot apices and leaves after 5, 10 and 15 days of light treatments were collected immediately flash frozen in liquid nitrogen and stored at − 80 °C until use. Two biological replicates of shoot apices were used for RNA-seq analysis and real-time RT-PCR. After 20 days of treatment, 30–40 seedlings from each treatment were transplanted into the greenhouse of South China Agriculture University with a relative humidity ranging from 70 to 85% and 28 °C/20 °C (14 h/10 h) day/night temperature for further flowering time analysis.
cDNA library construction and illumina sequencing
The cucumber shoot apices stored at − 80 °C were provided to Guangzhou Gene Denovo Biological Technology Co., Ltd. (Guangzhou, China). RNA isolation and RNA-Seq library preparation and sequencing were carried out by Guangzhou Gene Denovo Biological Technology Co., Ltd. (Guangzhou, China). In brief, the workflow of library construction for transcriptome analysis was as follows: After RNA was collected, poly(A)-containing mRNA was purified using oligo(dT) magnetic beads. Following this, the mRNAs were fragmented and cDNA was synthesized using a random hexamer, DNA polymerase I and RNase H. The double-stranded cDNAs were purified and ligated to adaptors for Illumina paired-end sequencing. PCR amplication was done on the purified cDNAs with ligated adaptors. PCR products were separated by 2% agarose gel. The final PCR product chosen for sequencing is 400–500 bp band, which were cut and recovered from the gel. The quality and quantity of the library were verified using an Agilent 2100 Bioanalyzer and ABI real time RT-PCR, respectively (Additional file 6: Table S3 and Additional file 2: Figure S1). The cDNA libraries were sequenced using Illumina HiSeqTM 2500 by Gene Denovo Co. (Guangzhou, China). The libraries of two biological replicates of shoot apices were prepared independently.
Sequence read mapping and assembly
The original image data produced by sequencing were transferred into sequences (raw reads). The calculating methods for raw reads was conducted according to Cock (2010). To obtain clean reads for further analysis, the raw reads were filtered by removing adaptor sequences, low-quality reads and reads with a percentage of unknown bases (N) of more than 10%. Following this, we used Bowtie to remove the ribosome reads (Langmead et al. 2009). The clean reads were mapped to the cucumber genome assembly (Chinese long) v2 (http://www.icugi.org/cgi-bin/ICuGI/index.cgi) according to Kim et al. (2013) and Trapnell et al. (2012).
Quantification and differential expression analysis of transcripts
The expression level of each gene was determined by the numbers of reads, which were uniquely mapped to the specific gene and the total number of uniquely mapped reads in the sample. Reads per kilobase of exon model per million mapped reads (RPKM) were calculated according to Mortazavi to estimate gene expression levels. The FDR was used to determine the threshold of the P-value in multiple tests. In our study, a FDR < 0.05 and an absolute value of |log2 (fold change)| > 1 were used as the threshold to determine the significant DEGs.
GO and KEGG enrichment analysis
To identify putative biological functions and pathways for the DEGs, the Gene ontology (GO) and Kyoto encyclopedia of gene and genomes (KEGG) database were searched for annotation. The methods for GO and KEGG enrichment analysis were according to Young and Mao respectively (Young et al. 2010; Mao et al. 2005).
Total RNA was isolated from 100 mg cucumber leaves and shoot apices using Huayueyang reagent kit (with DNase I step: Pipet the DNase I onto the membrane containing RNA. Incubate at 37 °C for 30 min) according to the manufacturer’s instructions. The RNA quality and purity were verified by Nanodrop 2000 and electrophoresis on 1.0% agarose gels. The first-strand cDNA was synthesized from aliquots of 1 μg of total RNA using PrimeScriptTM RT Reagent Kit with gDNA Eraser (TaKaRa, RR047A) in a reaction volume of 20 μL. The synthesized cDNA was diluted 20 times with sterile water and used as the template for real-time PCR. The reactions were carried out in a Roche LightCycler 480 system with SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio, Dalian, China). The reactions reagent mix was 5 μL SYBR Premix Ex Taq II, 1.5 μL cDNA template, 0.4 μL each primer (10 μmol/μL), and 2.7 μL nuclease-free water. The amplification program was 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melting curve analyses were performed at the end of 40 cycles (95 °C for 5 s followed by a constant increase from 60 to 95 °C). In addition, TUA was used to normalize expression levels (Wan et al. 2010), and the relative expression of genes was calculated using the 2−∆∆Ct method (Livak and Schmittgen 2001). The results were analyzed by Excel 2010. qPCR reactions in leaves were prepared in biological triplicate. qPCR reactions in shoot apices for RNA-seq validation were prepared in three biological replicates.
Genes used for Real-Time RT-PCR were designed on the NCBI primer blast website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer quality and efficiency were checked by qPCR on series of diluting cDNA. The expression of 26 genes chosen from RNA-seq were analyzed by real-time RT-PCR in shoot apex samples (R2B1 and R4B1 at 5 days, 10 days and 15 days). Meanwhile, 9 genes were used for checking light signal transduction in leaves (Additional file 7: Table S4).
JS and YZ carried out the experiment, participated in the analysis. YH drafted the manuscript. WS carried out the qRT-PCR analysis. SS and GS performed the statistical analysis. HL and YH conceived of the study, and participated in its design. RC and HL acquired of funding and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The raw RNA-seq reads have been deposited in NCBI Sequence Read Archive (SRA) under Accession Number of SRR7345617; SRR7345616; SRR7345615; SRR7345614; SRR7345621; SRR7345620; SRR7345619; SRR7345618; SRR7345623; SRR7345622; SRR7345625; SRR7345624.
Consent for publication
Ethics approval and consent to participate
This work was supported by Teamwork Projects Funded by Guangdong Natural Science Foundation (No. S2013030012842), and the Guangzhou Science & Technology Project (201605030005).
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