Transcriptome analysis of radish sprouts hypocotyls reveals the regulatory role of hydrogen-rich water in anthocyanin biosynthesis under UV-A
Hydrogen gas (H2) is the most abundant element in the universe, and has been reported to act as a novel beneficial gaseous molecule in plant adaptive responses. Radish sprouts are popular because they contain substantial amounts of antioxidants and health-promoting compounds, such as anthocyanin and glucosinolates. Although radish sprouts accumulated more anthocyanin under UV-A after treatment with hydrogen-rich water (HRW), the molecular mechanism responsible is still elusive. To explore these mechanisms, RNA-seq analysis was used.
Four cDNA libraries from radish sprout hypocotyls were constructed, and a total of 14,564 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these unigenes were found to be implicated in light signal perception and transduction, starch and sucrose metabolism, photosynthesis, nitrogen metabolism and biosynthesis of secondary metabolites. The MYB-bHLH-WD40 complex accounted for the majority of the transcription factors found to be involved in anthocyanin biosynthesis, and levels of transcripts for this complex were in accordance with the anthocyanin concentrations observed. In addition, other transcription factors (such as NAC, bZIP and TCP) might participate in HRW-promoted anthocyanin biosynthesis. Furthermore, the signaling processes of plant hormones, MAPKs and Ca2+ might be involved in HRW-promoted anthocyanin biosynthesis under UV-A. The expression patterns of 16 selected genes were confirmed using qRT-PCR analysis.
Taken together, the results of this study may expand our understanding of HRW-promoted anthocyanin accumulation under UV-A in radish sprouts.
KeywordsAnthocyanin Molecular hydrogen Radish sprouts Transcriptome UV-A
differentially expressed genes
early biosynthetic genes
Kyoto Encyclopedia of Genes and Genomes
late biosynthetic genes
phenylalanine ammonia lyase
real time quantitative PCR
Reactive oxygen species
UDP glucose:flavonoid 3-O-glucosyltransferase
Radish (Raphanus sativus L.) sprouts are commonly consumed due to their potential benefits for human health. The bioactive phytochemicals in radish sprouts include phenolics, vitamin C, chlorophylls, carotenoids and glucosinolates [1, 2]. Color is one of the most striking features of vegetables, and red-skinned radish cultivars, which are rich in anthocyanins, can attract consumers with their lovely appearance. Anthocyanins are important phenolic compounds responsible for plant organ colors such as blue, purple and red . Apart from the well-known physiological function of attracting pollinators, anthocyanins also protect plants from a variety of biotic and abiotic stresses, such as bacterial and fungal infections, herbivory, UV radiation, temperature extremes, drought and nitrogen deficiency [4, 5]. In addition, there is mounting evidence indicating that anthocyanins act as antioxidants to protect humans from cardiovascular disease, cancer, inflammation and other chronic diseases [6, 7, 8]. Thus, an increasing amount of attention has been paid to the development of anthocyanin-rich diets.
Anthocyanins are biosynthesized via the flavonoid pathway, which is controlled by two types of genes: structural genes and regulatory genes. Structural genes encode the enzymes necessary for anthocyanin biosynthesis and can be divided into the early biosynthetic genes (EBGs) and the late biosynthetic genes (LBGs) . The enzyme products of the EBGs, including PAL, C4H, 4CL, CHS, CHI, and F3H, carry out the early steps of anthocyanin biosynthesis to the formation of dihydroflavonols. The enzyme products of the LBGs, including F3’H, DFR, ANS, LDOX and UFGT, lead to the biosynthesis of anthocyanins [9, 10]. The regulatory genes, which regulate anthocyanin biosynthesis at the transcriptional level, encode transcription factors (TFs). Three types of transcription factors involved in anthocyanin biosynthesis have been reported: WD-repeat proteins and bHLH and MYB transcription factors [11, 12]. Together, these factors form a ternary complex, the MYB–bHLH–WD repeat (MBW) complex, to orchestrate anthocyanin biosynthesis. Numerous factors have been reported to regulate the biosynthesis of anthocyanins, such as light, temperature, nitrogen depletion, salt stress, sucrose and plant hormones [13, 14, 15, 16]. Previous studies have demonstrated that anthocyanin accumulation can be induced by exposure to ultraviolet (UV) light, but most of those studies focused on UV-B-induced anthocyanin biosynthesis. UV-A (320–400 nm), which is in the range of long-wave UV and has lower energy than UV-B, was also reported to induce anthocyanin biosynthesis in Arabidopsis , turnips [18, 19], tomatoes , and blueberries . It is generally recognized that UV-A plays a key role in activating the genes responsible for anthocyanin biosynthesis .
Recently, H2 has attracted attention as a selective antioxidant and signal molecule [22, 23]. Studies in animals indicated that H2 acted as a novel antioxidant that selectively reduced the levels of toxic reactive oxygen species  and exerted anti-apoptotic, anti-allergy and anti-inflammatory effects [25, 26, 27]. A growing body of evidence indicates that H2 plays an important role in plants, especially in stress defence [28, 29, 30, 31]. For example, when H2 was supplied in the form of hydrogen-rich water (HRW), it could alleviate calcium- and salt-induced oxidative stress in rice and Chinese cabbage, respectively [32, 33]. A similar protective effect was found in alfalfa, showing that HRW conferred tolerance to UV-B-induced oxidative damage partially by the manipulation of (iso)flavonoid metabolism and antioxidant defense . In another case, H2 was reported to regulate cucumber adventitious root development . Our previous study showed that UV-A-induced toxicity was alleviated by HRW via the re-establishment of ROS homeostasis and the promotion of anthocyanin synthesis in radish sprouts . However, the regulatory role of H2 in anthocyanin metabolism under UV-A radiation has not been thoroughly studied in radish sprouts.
In this study, we used RNA-seq analysis to understand the mechanisms of H2-promoted anthocyanin biosynthesis under UV-A radiation in radish sprout hypocotyls. Thus, based on the analysis of DEGs found by pairwise comparisons, a model regulatory network for H2-promoted anthocyanin biosynthesis in radish sprouts was proposed. The results of our study provide an overview of transcriptomic responses to H2 in radish and thus extend our understanding of the biological functions of H2 in plants.
Plant materials and culture conditions
Radish (Raphanus sativus L., cultivar ‘Yanghua’) sprouts were cultured in nutrient medium (1/4 Hoagland’s solution prepared with distilled water or HRW) in the dark for 1 day at 25 °C and then transferred to incubators with white light (W) or UV-A illumination (Ningbo Saifu Instrument Experimental Factory, Zhejiang, China). The light intensity of the white light was 30 ± 5 μmol m− 2 s− 1. The UV-A radiation was 5.5 W m− 2, as measured by a portable digital radiometer for UV (UV-340A, Lutron, Taiwan). The temperature and relative humidity in the growth chamber were 25 °C and 80%, respectively. The nutrient medium was replaced every 12 h. The total anthocyanin contents were measured after 12, 24 and 36 h of light treatment. Hypocotyl tissues were sampled immediately after 24 h light treatment, frozen in liquid nitrogen, and stored at − 80 °C for further analysis.
Preparation of HRW
Purified H2 gas (99.99%, v/v) was produced with a hydrogen gas generator (SHC-300, Saikesaisi Hydrogen Energy Co., Ltd., Jinan, China). In our experimental conditions, the H2 concentration in freshly prepared HRW (100% saturation) analyzed by gas chromatography was 781 μmol L− 1 . Endogenous H2 production was measured by gas chromatography (Agilent 7890A, Wilmington, DE, USA) with a thermal conductivity detector .
Determination of endogenous H2 concentrations
Endogenous H2 concentration was measured by gas chromatography (GC) (Agilent 7890A) .
Determination of anthocyanin concentration
Anthocyanin concentrations in hypocotyls of radish sprouts were measured in accordance with previously described methods . Briefly, 0.5 g of freeze-dried hypocotyl samples were extracted in 5 mL of 0.1% HCl/methanol. (A530–0.25 × A657) g− 1 dry weight (DW) was used to quantify the relative amount of anthocyanin.
RNA extraction, library construction, and transcriptome sequencing
Total RNA for RNA-seq was extracted from hypocotyls using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and concentration of RNA were checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and the Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), respectively. In addition, the integrity of RNA was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Total RNA from each sample was used for Illumina sequencing at Novogene Bioinformatics Technology Co. Ltd. (Beijing, China). Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according the manufacturer’s instructions, and index codes were added to attribute sequences to each sample.
Quantification of gene expression levels
HTSeq v0.6.1 was used to count the number of reads mapped to each gene. In addition, the FPKM (fragments per kilobase of exon model per million mapped reads) of each gene was calculated based on the length of the gene and the number of reads mapped to the gene .
Differential expression analysis
Differential expression analysis of pairs of groups was performed using the DESeq R package (1.18.0). The P-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed [39, 40].
GO and KEGG enrichment analysis of DEGs
Gene Ontology (GO) enrichment analysis of DEGs was implemented by the GOseq R package. GO terms with corrected P-values < 0.05 were considered to be significantly enriched by differentially expressed genes . KOBAS software was used to test the statistical enrichment of DEGs in KEGG pathways.
Quantitative PCR analysis
The transcription levels of 16 genes involved in anthocyanin biosynthesis pathway were analyzed using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from radish hypocotyls using Trizol reagent (Invitrogen, USA) and 1 μg aliquots were treated with RNase-free DNase I to remove genomic DNA and then reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). qRT-PCR was carried out on a Mastercycler ep realplex Real-time PCR System (Eppendorf, Hamburg, Germany) using Bestar SYBR Green qPCR Mastermix (DBI, Bioscience Inc., Germany) . Reactions were performed at 95 °C for 2 min, 40 cycles of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s. Three biological replicates were applied. The specific primers were designed using the Primer Premier 6.0 software. The primer sequences were listed in Additional file 1: Table S1 in the supporting information. Actin2 and EF1 were used as the reference genes . The relative expression levels of the target genes were calculated using the 2−△△CT approach, with normalization of data to the geometric average of two reference control genes [43, 44].
Statistical analysis was performed using SPSS statistic 17.0 software. For statistical analysis, Duncan’s multiple test (P<0.05) was chosen. Data are means ± SE from at least three independent biological replicates.
HRW promoted UV-A-induced anthocyanin accumulation
To gain insights into the underlying mechanisms of HRW-promoted anthocyanin accumulation, mRNA sequencing of the hypocotyl samples after 24 h of light exposure were conducted.
Identification of differentially expressed genes
Functional classification of the DEGs by GO and KEGG pathway analysis
Expression profiling of structural genes associated with anthocyanin biosynthesis
In this study, 5 putative OMT (O-methyltransferase) genes were identified, and the expression levels of Rsa1.0_00866.1_g00008.1, Rsa1.0_06693.1_g00002.1 and R sa1.0_14791.1_g00001.1 were increased under UV-A and further increased under UV-A + HRW relative to W. We identified 24 putative GST (glutathione S-transferase)-encoding genes here. Fifteen putative GST genes were up-regulated under UV-A + HRW compared with UV-A, including Rsa1.0_00336.1_g00025.1, Rsa1.0_02928.1_g00007.1 and Rsa1.0_00370.1_g00004.1. In addition, 33 putative MATE genes were identified, and the expression levels of Rsa1.0_02494.1_g00005.1 and Rsa1.0_00065.1_g00071.1 were significantly up-regulated under UV-A + HRW compared with UV-A.
Expression profiling of transcription factors associated with anthocyanin biosynthesis
Among the various TFs, MYB composed the largest percentage (26.60%), followed by bHLH (17.02%) and WD40 (5.32%), indicating that the MBW complex might be the major determinant of anthocyanin biosynthesis under UV-A + HRW in radish sprouts (Fig. 6 a, g, h). All 16 of the putative bHLH TFs were significantly up-regulated under UV-A treatment compared to W, and UV-A + HRW treatment further promoted this effect (Fig. 6a). In this study, 25 putative MYB genes were identified, and most of them (except Rsa1.0_00145.1_g00031.1_0) were up-regulated under UV-A + HRW treatment compared with UV-A treatment (Fig. 6h). Furthermore, 8 putative bZIPs, 10 putative NACs and 9 putative zf-HDs were identified in this study, and their expression levels increased under UV-A + HRW treatment compared to UV-A, implying that these TFs might positively regulate HRW-promoted anthocyanin biosynthesis (Fig. 6b, i, k). In addition, 5 other families (C2H2, C3H, FAR1, TCP and WRKY) showed up-regulation or down-regulation under UV-A + HRW treatment compared to UV-A, implying that these TFs might be involved in HRW-regulated anthocyanin biosynthesis (Fig. 6).
The differentially expressed genes involved in anthocyanin biosynthesis-related signaling pathways
Light signals are perceived by plants through different classes of photoreceptors. CRY and PHOT mediate blue light/UV-A signal transduction. In our study, based on the KEGG and GO pathway annotations, three putative CRYs were found, and compared to W, their expression levels increased under UV-A but were lower under UV-A + HRW. Among the eight putative PHOT genes, the expression levels of Rsa1.0_08201.1_g00003.1 and Rsa1.0_51768.1_g00001.1 increased under UV-A + HRW compared with UV-A, although there was no significant difference. The expression level of a putative COP1 (Rsa1.0_02834.1_g00003.1) increased under UV-A compared with W and remained the same under UV-A + HRW. Expression of a putative HY5 (Rsa1.0_02208.1_g00006.1) decreased under both UV-A and UV-A + HRW relative to W (Additional file 2: Table S2).
Plant hormones have a crucial role in the regulation of anthocyanin biosynthesis. It has been reported that ABA, JA, cytokinins and GA promote light-induced anthocyanin biosynthesis [45, 46, 47, 48]. In our study, eight putative cytokinin-related genes were identified, of which the expression levels of Rsa1.0_00570.1_g00019.1 and Rsa1.0_01905.1_g00006.1 increased under UV-A relative to W. The expression level of one putative auxin-related gene (Rsa1.0_02398.1_g00002.1) decreased under UV-A + HRW compared with UV-A.
We also investigated the genes of MAPK and calcium signaling pathways. We found that the expression levels of one putative MAPK signaling-related gene (Rsa1.0_00250.1_g00016.1) and two calcium signaling-related genes (Rsa1.0_00024.1_g00018.1 and Rsa1.0_07482.1_g00001.1) decreased under UV-A + HRW compared with UV-A. All of these results suggested that light and hormone signals play a vital role in UV-A-induced anthocyanin biosynthesis and might also take part in H2-promoted anthocyanin accumulation under UV-A.
qRT-PCR validation of differentially expressed genes
To validate the RNA-seq data, 16 anthocyanin biosynthesis-related genes were chosen for validation of expression by qRT-PCR analysis (Additional file 3: Figure S1). These 16 genes included those involved in UV-A photoreception and light signal perception, as well as transcription factors and structural genes involved in anthocyanin biosynthesis. In the qRT-PCR analysis, the expression of these genes showed patterns very similar to those of the FPKM values from sequencing under the corresponding treatments. These results indicated that the RNA-seq data were reliable.
Radishes are members of the Brassicaceae, and radish sprouts are important edible vegetables that receive significant attention for their high nutrient contents and health-promoting effects. Anthocyanins, which confer vivid red hypocotyls that draw consumers to radish sprouts, are among these important health-promoting compounds. Therefore, various methods and technologies have been applied to produce radish sprouts rich in anthocyanins [29, 36, 49]. Light acts as an essential stimulus and a safe regulator for anthocyanin biosynthesis. In recent decades, the understanding of the mechanisms underlying light-regulated anthocyanin biosynthesis in plants has markedly increased [50, 51]. Specifically, UV-A could induce anthocyanin accumulation with little damage to plants.
Molecular hydrogen (H2) has been identified as a novel antioxidant that can efficiently reduce oxidative stress. The progress toward therapeutic and preventive applications of hydrogen has shown that supplementation with hydrogen-rich water may have a role in preventing of insulin resistance and type 2 diabetes mellitus (T2DM) . More recently, studies on animals showed that HRW could protect against ischemic brain injury in rats, and the maintenance of parvalbumin and hippocalcin levels by HRW might contribute to neuroprotective effects against neuron damage . Therapeutic hydrogen has been administered by different delivery methods, including straightforward inhalation, injection with hydrogen-saturated saline and drinking hydrogen dissolved in water . In plants, external supplementation with H2 in the form of HRW protects plants from multiple environmental stimuli and enhances their antioxidant capacity [31, 32, 54]. Recently, it was reported that UV-B-triggered biosynthesis of (iso)flavonoids was substantially strengthened by HRW , and the accumulated (iso)flavonoids enhanced the tolerance of alfalfa to UV-B. Based on those cumulative knowledge, the beneficial biological effects of hydrogen have been preliminarily proved. In our recent study, the observation that UV-A-induced anthocyanin biosynthesis was further increased by supplementation with HRW was first reported . Furthermore, HRW significantly blocked UV-A-induced ROS accumulation and enhanced the activities of antioxidant enzymes to reestablish ROS homeostasis. However, to date, there has been no study on the identification of DEGs related to H2-promoted anthocyanin biosynthesis under UV-A, and little is known of related signaling pathways. This is the first study to investigate the transcriptional changes associated with anthocyanin accumulation under UV-A + HRW.
The present study showed that the expression of most structural genes associated with anthocyanin biosynthesis was up-regulated by UV-A and UV-A + HRW (Fig. 5). The accumulation of anthocyanin in radish sprouts hypocotyls is a complex biological process that is driven by different regulatory factors, including plant hormones, transcription factors and other signal transduction pathways. In our study, 11 UV-A photoreceptors, including three CRY and eight PHOT, were identified (Additional file 2: Table S2). The transcript levels of most of these photoreceptors increased under UV-A compared with W and did not increase when HRW was applied. Downstream of the light receptors, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as a molecular switch of light-induced plant development, mediating the degradation of various photomorphogenesis-promoting transcription factors by the Ub-proteasome system [13, 55]. ELONGATED HYPOCOTYL5 (HY5) is a bZIP transcription factor that has been linked to activation of CHS and the accumulation of flavonoids in response to light in Arabidopsis . One COP1 was identified in this study, and its transcript level increased under UV-A. Among the three HY5 found, the transcript levels of two increased under UV-A, and the other decreased. Interestingly, the transcript levels of COP1 and HY5 did not increase under UV-A + HRW compared with UV-A. These results indicated that the photoreceptors COP1 and HY5 might participate in UV-A-induced anthocyanin biosynthesis, but the expression levels of these photoreceptors were not affected by HRW.
Plant hormones may be important factors regulating the light-induced anthocyanin accumulation. For example, auxin has been found to retard anthocyanin accumulation in apple and Arabidopsis [57, 58]. In the present study, the transcript level of one auxin-associated gene (Rsa1.0_02398.1_g00002.1) decreased (Additional file 2: Table S2), implying that it may participate in anthocyanin biosynthesis under UV-A + HRW. Other plant hormones, such as abscisic acid, cytokinins, and jasmonic acid have been reported to promote anthocyanin accumulation [45, 47, 48]. However, in this study, the transcript levels of most of those hormone-related genes remained unchanged under UV-A + HRW compared with UV-A. These results are not consistent with our expectation. Recently, it was reported that H2 could be a novel signaling molecule in phytohormone signaling pathways in response to biotic and abiotic stresses . Thus, further pharmacological experiments will be necessary to verify the role of phytohormones in H2-promoted anthocyanin biosynthesis under UV-A. In addition, one transcript encoding a gene involved in MAPK signaling and two transcripts encoding genes involved in calcium signaling were down-regulated under UV-A + HRW. Anthocyanin pigmentation is modulated by changes in intracellular Ca2+ level in Arabidopsis , and application of a CaCl2 spray could enhance accumulation of anthocyanins in strawberry . These results imply that Ca2+ may take part in H2-promoted anthocyanin biosynthesis under UV-A, although additional evidence needs to be obtained.
The molecular basis of plant anthocyanin biosynthesis is well known. Transcription factors regulating the expression of flavonoid biosynthetic genes have been characterized extensively in Arabidopsis and other plants [16, 62, 63]. In Arabidopsis, three transcription factor families, including R2R3-MYB, basic helix-loop-helix (bHLH), and WD40-repeat proteins, constitute a ternary complex predominantly responsible for orchestrating anthocyanin biosynthesis . MYB proteins, which are widely distributed in all eukaryotic organisms, constitute one of the largest transcription factor families in the plant kingdom. The production of anthocyanin is predominantly regulated by R2R3-MYB . In this study, we identified 89 unique transcripts encoding members of 10 transcription factor families, which were annotated in anthocyanin biosynthesis in radish sprouts (Fig. 6). Among those TFs, 25 putative MYB-encoding genes, 16 putative bHLH-encoding genes and 2 putative WD40-encoding genes were identified. Those three TFs accounted for approximately one-half of the total number of TFs identified. This finding suggested that the MBW complex is likely to be the primary regulator of anthocyanin accumulation in radish sprouts under UV-A + HRW treatment. In peach, PpNAC1 can activate the transcription of PpMYB10.1 to promote anthocyanin pigmentation . In addition, it has been suggested that IbNAC1 has multiple biological functions in the JA response, including the inhibition of root formation and accumulation of anthocyanin in sweet potato . In our present study, 9 NACs identified in radish sprouts showed increased expression levels under UV-A + HRW treatment. In addition, the co-up-regulation of NAC and MYB implied that the activation of MYB might be associated with NAC. In our study, the expression levels of most transcription factors annotated in anthocyanin biosynthesis increased under UV-A and were further enhanced by HRW under UV-A. These findings showed that the regulatory effects of TFs might play vital roles in H2-promoted anthocyanin biosynthesis under UV-A.
In this study, the H2 production of radish sprouts was significantly increased under UV-A (Fig. 1c). Similarly, the UV-B irradiation was reported to significantly increase the H2 content in alfalfa . We observed that endogenous H2 content increased under abiotic stress in higher plants, including alfalfa , Chinese cabbage  and Arabidopsis . In bacteria and green algae, a hydrogenase is responsible for H2 production . However, no reports have clarified the mechanism of H2 production in higher plants. With current levels of technology, it is difficult to answer these questions completely. In future, these problems will gradually be solved with improved scientific methods and increased attention to the physiological role of H2 in higher plants.
Four sets of transcriptome data comprising 80,522 unigenes were identified in radish sprouts by Illumina sequencing. A total of 14,564 DEGs were identified through pairwise comparisons. GO and KEGG pathway enrichment analyses revealed the DEGs involved in HRW-promoted anthocyanin biosynthesis under UV-A. Genes encoding light signal transduction elements, anthocyanin biosynthesis-related TFs and structural genes, and anthocyanin biosynthesis-related signaling pathways were also identified. The transcriptome data provided valuable information and gene sequences to accelerate the process of revealing the regulatory mechanism of HRW-promoted anthocyanin biosynthesis under UV-A in radish sprouts. In addition, further studies are needed to confirm the precise mechanisms by which H2 regulates physiological processes.
We thank the Beijing Biomics Biotech Co. Ltd. for excellent technical help in bioinformatics analysis.
This work was supported by the National Natural Science Foundation of China (Grant No. 31572169).
Availability of data and materials
We have deposited our data in Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra/), the accession number for our submissions are: PRJNA388018.
Consent to publish
Conceived and designed the experiments: XZ and NS. Performed the experiments: XZ, LJ, JYT, HL. Analyzed the data: XZ, LJ, LH. Wrote the paper: XZ, ZS, JC. All authors have reviewed and approved the manuscript.
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The authors declare that they have no competing interests.
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