UV-B irradiation promotes anthocyanin biosynthesis in the leaves of Lycium ruthenicum Murray

Plant materials and UV-B irradiation treatment

Seeds used in this experiment of L. ruthenicum were collected from the desert area of Dulan County, Qinghai Province, China. The healthy and similar seeds were collected and germinated in a petri dish for 15 days before being transplanted into organic soil. The seedlings were then grown for 80 days in an environment with a light/dark cycle (16 h/8 h, 25/20 °C) and a humidity of 60%. Twelve plants of similar size were selected, with each group comprising four plants, resulting in a total of three groups, which were designated as three biological replicates. All data were obtained based on these three biological replicates. The samples that were not subjected to UV-B irradiation served as the control group (CK), while the samples that were irradiated with UV-B constituted the treated group.

UV-B irradiation was produced by a UV-B lamp with a main spectral line of 253.7 nm and a real-time power density of 10 µw/cm². During each light/dark cycle, the plants in the experimental group were intermittently exposed to the UV-B irradiation for 12 periods during the light cycle in each day. The irradiation duration of each period was 10 min (i.e., 8:00-8:10, 9:00-9:10,..., 19:00-19:10). After being irradiated for 0.5 days (6 periods), 1 day (12 periods), 1.5 days ((12+6) periods), 2 days ((12+12) periods), and 4 days ((12+12+12+12) periods), the leaves were collected from the same batch of plants for physiological indicators and RNA-Seq analysis.

Determination of anthocyanin content

Anthocyanin content was determined using the methods described by Neff (Neff & Chory, 1998; Ai et al., 2016). According to the laboratory optimized anthocyanin extraction method from L. ruthenicum. The Fresh leaves of L. ruthenicum (0.1 g) were ground into fine pieces and extracted with 1 mL of methanol: water (65% v/v, Methanol: >99% purity; Thermo Fisher Scientific, Waltham, MA, USA) solution (hydrochloric acid volume ratio: 1%) using ultrasonication with three replicates. The ultrasonic time was 20 min. The temperature was maintained at room temperature. The optical density of 530 nm (A₅₃₀) and 657 nm (A₆₅₇) were measured using a microplate reader, then the anthocyanin content was calculated as (A₅₃₀−0.25*A₆₅₇)/m.

Measuring of H₂O₂, malondialdehyde and antioxidant enzymes activities

The contents of malondialdehyde (MDA) were determined in the fresh leaves of L. ruthenicum by the thiobarbituric acid method, using MDA detection Kit (MDA-1-Y). H₂O₂ in leaves of L. ruthenicum were extracted by acetone and the contents were determined using the Kit (H₂O₂-1-Y). The activities of SOD, POD and catalase (CAT) were determined in the fresh leaves of L. ruthenicum under treatment. The three enzymes indexes were determined according to the manufacturer’s protocol of assay kits SOD-1-W for SOD activity; POD-1-Y for POD activity; CAT-1-Y for CAT activity. All the kits for activities were purchased from Comin Biotechnology Co., Ltd., Suzhou, China (http://www.cominbio.com) (Chen et al., 2022).

RNA-Seq analysis

Transcriptome sequencing was performed by GeneDenovo Biotechnology Co. (Guangzhou, China). Total RNA was extracted using the TIANGEN Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Cat # DP441; TIANGEN Biotech, Beijing, China). mRNA was enriched using Oligo(dT) magnetic beads and further purified. The mRNA was reverse transcribed using random primers to synthesize cDNA. A library was constructed through PCR amplification, and sequencing was conducted on the Illumina NovaSeq 6000 platform.

After sequencing, raw reads were subjected to quality control to obtain clean reads. These reads were assembled using Trinity software, resulting in high-quality unigenes. The unigene sequences were aligned against the NR, SwissProt, KEGG, and COG/KOG protein databases using BLASTx (E-value<1 × 10⁻⁵) for functional annotation. RSEM (v1.2.19) was used to quantify the assembled unigenes. DESeq2 (v1.20.0) was used to analyze the differentially expressed genes (DEGs) between two groups. DEGs were identified using thresholds of FDR<0.05 and —log2FC— ≥2. KEGG pathway enrichment analysis was performed using KOBAS software (v2.0.12) to assess the statistical significance of DEGs in KEGG pathways.

qPCR analysis

RNA was extracted using the Polysaccharide Polyphenol Plant Total RNA Extraction Kit (DP441; Tiangen, China). cDNA synthesis was performed using the Rapid Reverse Transcription Kit (Takara, RR092A). qPCR analysis was performed using a quantitative PCR kit (RR820A; Takara) with three replicates. The primer sequences are shown in Table S1. Actin from L. ruthenicum was used as the internal reference gene. The expression of genes was calculated using the 2^−ΔΔCt method.

Interaction network analysis of structural genes and transcription factors for anthocyanin synthesis

Using the STRING protein interaction database (http://string-db.org), the Omicsmart online platform of GeneDenovo Biotechnology Co. predicted the transcription factors that interact with 24 structural genes involved in anthocyanin synthesis. Out of the 12,720 genes that were differentially expressed after UV-B irradiation treatment, 424 were identified as transcription factors. An interaction network was created by using the 24 structural genes as a target gene set and the 424 transcription factors as an associated gene set. Only prediction results with a combined score >400 were retained.

UV-B induced anthocyanin accumulation and changes in antioxidant enzymes

After being exposed to UV-B irradiation for 0.5 days, 1 day, 1.5 days, 2 days, and 4 days, the color of the anthocyanin extraction solution obtained from the leaves of L. ruthenicum changed significantly, as shown in Fig. 1. The color of the anthocyanin extraction solution became darker after UV-B treatment compared to the control group (CK).

Figure 2A displays the anthocyanin content measured by spectrophotometry for varying durations of UV-B irradiation. The highest level of anthocyanin content was observed after 1 day of exposure to UV-B irradiation, with a relative content of 5.43 (A₅₃₀−0.25*A₆₅₇) g⁻¹, which is approximately 2.8 times higher than that of the control group. However, as the duration of UV-B irradiation increased, the anthocyanin content gradually decreased. Because when treated for a short time, UV-B radiation can promote the resistance of plants, but after a certain threshold, the resistance will no longer increase but begin to weaken. Furthermore, Figs. 2B–2F display the changes in the content of enzymes, i.e., H₂O₂, MDA, SOD, POD, and CAT, in the leaves of L. ruthenicum after UV-B irradiation treatment. The content of antioxidant enzymes (excluding MDA) increased after UV-B irradiation treatment, indicating that increasing antioxidant enzyme activity is a physiological response for plants to adapt to UV-B irradiation stress. Enhanced antioxidant enzyme activity and anthocyanin synthesis represent components of the plant defense system. These responses enable plants to adapt and resist the stress caused by UV-B radiation.

Differential expression genes after UV-B irradiation treatment

The anthocyanin content in the leaves of L. ruthenicum changed significantly after 1 day of UV-B irradiation treatment. Therefore, the leaves that were exposed to UV-B irradiation for 1 day were selected for transcriptome analysis. Six transcriptome libraries of L. ruthenicum leaves were constructed, which were then divided into a control group (CK) and a treatment group. The control group consisted of Lr-CK-1, Lr-CK-2, and Lr-CK-3, while the treatment group consisted of Lr-UV-B-1, Lr-UV-B-2, and Lr-UV-B-3.

After filtering the data and quality control, a total of 61∼72 million clean reads were obtained. In our libraries, the Q20 and Q30 statistics of the clean reads were greater than 97% and 93%, respectively (Table S2). The assembly produced 63,698 Unigenes with an average length of 1,125 bp and an N50 of 1945 bp (Tables S4 and S6). Over 50% of the Unigenes had lengths ranging from 200 to 2,000 bp (Fig. S1). All unigenes were subsequently annotated using BLASTx (E-value < 1 × 10⁻⁵) searches of four public databases: NCBI nr database, Swiss-Prot protein database, KEGG database, and COG database. The annotation results are presented in Table S5. A total of 63,698 unigenes were processed and low expressed genes were removed, resulting in a total of 43,989 unigenes. The statistical power of this experimental design, calculated in RnaSeqSampleSize is 0.82.

To identify differential expression genes after UV-B irradiation treatment, we used DESeq2 software to generate histograms and volcano plots of differential genes, as shown in Fig. 3. The thresholds used to identify differential expression genes between each group were DFR < 0.05 and log2 FC > log2(2). As a result, we identified a total of 12,720 differential expression genes, including 6,130 genes with increased expression and 6,617 genes with decreased expression (Table S7).

To study the functions of differential expression genes, we performed KEGG functional enrichment analysis. The unigenes were divided into 140 pathways in the KEGG functional classification, with the top 20 pathways shown in Fig. 4. The metabolic pathway were the most significantly enriched with 1,204 unigenes, followed by the biosynthesis of secondary metabolites pathway with 705 unigenes. Other enriched pathways include phenylpropanoid metabolism, flavonoid metabolism, and hormone signal transduction. These pathways play crucial roles in the leaves of L. ruthenicum response to UV-B stress by regulating the synthesis of protective compounds and mediating stress signaling.

Analysis of genes related to UV-B signal transduction

The differential expression genes in the UV-B signaling pathway were identified. The expression of seven genes was observed to change: one COP1 gene (Unigene0008780), one HY5 gene (Unigene0072293), and five UVR8 genes (Unigene0015353, Unigene0003921, Unigene0067978, Unigene0020095 and Unigene0078284). Specifically, following exposure to UV-B irradiation, the majority of UVR8 genes exhibited a decrease (Fig. 5A), while the Unigene0067978 demonstrated a significant increase. In contrast, the COP1 genes exhibited a decrease, while the expression of HY5 increased.

The results of the qPCR analysis indicated a significant increase in the expression of UVR8 (Unigene0067978) following one day of UV-B treatment, which then decreased (Fig. 5B). The expression of COP1 initially decreased and then increased significantly with the duration of treatment (Fig. 5C). The expression of HY5 increased continually with the treatment duration (Fig. 5D).

Analysis of genes related to anthocyanin synthesis

We analyzed the unigenes involved in the synthesis pathways of flavonoids (Fig. 6A) and anthocyanins under UV-B irradiation. A total of 24 enzyme-encoding genes were identified and their expression was found to be up-regulated, as shown in Fig. 6B. Among the identified genes, the two genes with the most significant increase in expression were F3’H (Unigene0009145, FPKM is 891 times that of CK) and C4H (Unigene0046607, FPKM is 18 times that of CK).

To ensure the reliability of the RNA-seq data, we selected CHS (Unigene0010556) and UFGT (Unigene0001601), which are enriched in the anthocyanin biosynthesis pathway (ko00942), for qPCR analysis. The results showed a significant increase in the expression of CHS (Unigene0010556) following one day of UV-B treatment (Fig. 6C). However, the expression decreased as the duration of UV-B irradiation treatment increased. The expression of UFGT (Unigene0001601) initially increased and then decreased with UV-B treatment time, as shown in Fig. 6D. The results of the qPCR analysis were consistent with the transcriptome analysis results.

UV-B signal transduction and anthocyanins synthesis transcription factors

A total of 424 transcription factors, belonging to 36 gene families, were identified among the differential expression genes. The largest family group was MYB transcription factors (15%), followed by ERF (14%), bHLH (10%), and WRKY (10%). The changes in expression of MYB, bHLH, and WRKY transcription factors were examined. The expression of most transcription factors was found to decrease in response to UV-B irradiation, as shown in Fig. 7. However, some transcription factors showed a significant increase in expression, such as WRKY1 (Unigene0066868, FPKM value 100 times higher than that of CK), WRKY2 (Unigene0013893, FPKM value 63 times higher than that of CK), WRKY3 (Unigene0039337, FPKM value 47 times higher than that of CK), and WRKY4 (Unigene0078996, FPKM value 14 times higher than that of CK).

The qPCR analysis was conducted with the following transcription factors: MYB (Unigene0070868), bHLH (Unigene0025729), and WRKY (Unigene0066868, Unigene0013893, Unigene0039337, Unigene0078996). The results are presented in Fig. 8, which demonstrates that the change in the expression of the six transcription factors, with the exception of MYB, is consistent with the transcriptome analysis results. Furthermore, the expression of WRKY transcription factors significantly increased following exposure to UV-B irradiation. This indicates that WRKY family genes may play a crucial role in responding to UV-B irradiation.

Network analysis of interactions between structural genes and transcription factors in anthocyanin biosynthesis

To study the regulatory role of transcription factors in anthocyanin biosynthesis, we analyzed the interactions between 24 structural genes involved in anthocyanin synthesis and transcription factors. A network graph was constructed based on protein-protein interactions with a combined score above 400. As shown in Fig. 9A, a total of 12 structural genes involved in anthocyanin synthesis formed 40 pairs of interactions with 11 transcription factors, all of which exceeded the score threshold. Notably, MYB1 (Unigene004770600) showed the highest connectivity, interacting with all 11 structural genes. Following MYB1, bHLH (Unigene0014085) and MYB308 (Unigene0053002) interacted with 9 and 5 structural genes, respectively. Among the structural genes, the CHS genes (Unigene0004160, Unigene0010556) showed the highest connectivity, interacting with 6 transcription factors, followed by ANT17 (Unigene0004160). As shown in Fig. 9B, most of the interacting transcription factors showed up-regulated expression, with the exception of JAF13, MYB4, and TAF1.