OsbZIP18 Is a Positive Regulator of Phenylpropanoid and Flavonoid Biosynthesis under UV-B Radiation in Rice

Abstract

In plants exposed to ultraviolet B radiation (UV-B; 280-315 nm), metabolic responses are activated, which reduce the damage caused by UV-B. Although several metabolites responding to UV-B stress have been identified in plants, the accumulation of these metabolites at different time points under UV-B stress remains largely unclear, and the transcription factors regulating these metabolites have not been well characterized. Here, we explored the changes in metabolites in rice after UV-B treatment for 0 h, 6 h, 12 h, and 24 h and identified six patterns of metabolic change. We show that the rice transcription factor OsbZIP18 plays an important role in regulating phenylpropanoid and flavonoid biosynthesis under UV-B stress in rice. Metabolic profiling revealed that the contents of phenylpropanoid and flavonoid were significantly reduced in osbzip18 mutants compared with the wild-type plants (WT) under UV-B stress. Further analysis showed that the expression of many genes involved in the phenylpropanoid and flavonoid biosynthesis pathways was lower in osbzip18 mutants than in WT plants, including OsPAL5, OsC4H, Os4CL, OsCHS, OsCHIL2, and OsF3H. Electrophoretic mobility shift assays (EMSA) revealed that OsbZIP18 bind to the promoters of these genes, suggesting that OsbZIP18 function is an important positive regulator of phenylpropanoid and flavonoid biosynthesis under UV-B stress. In conclusion, our findings revealed that OsbZIP18 is an essential regulator for phenylpropanoid and flavonoid biosynthesis and plays a crucial role in regulating UV-B stress responses in rice.

Keywords: UV-B radiation; metabolites; phenylpropanoid and flavonoids biosynthesis; regulator; rice.

Figure 1

Comparison of rice metabolism under different UV-B duration treatments. (A) The composition and classification of metabolites are known. (B) Principal component analysis (PCA) of known metabolites under different UV-B duration treatments. Three biological replicates were taken for the analyses from every treatment (n = 3), and each replicate was mixed with eight different plants.

Figure 2

Different patterns of metabolite levels across the four time points under UV-B treatment. (A) Cluster 1. (B) Cluster 2. (C) Cluster 3. (D) Cluster 4. (E) Cluster 5. (F) Cluster 6. k-means clustering grouped the expression profiles of the rice metabolite into six clusters. The x-axis shows different UV-B treatment times, and the y-axis depicts the Z-score standardized per metabolite. The membership factor denotes consistency with the trends in metabolite change in each cluster.

Figure 3

Comparative analysis of metabolome under different UV-B treatment duration in rice. (A) Heat map of differential metabolomes under different UV-B treatment durations. Red indicates a high abundance, and blue indicates low relative abundance metabolites. (B–D) KEGG enrichment of differential metabolites between the comparison groups (UV-B 0 h vs. UV-B 6 h/12 h/24 h). Each bubble in the plot represents a metabolic pathway whose abscissa and bubble size jointly indicate the magnitude of the impact factors of the pathway. The bubble colors represent the p-values of the enrichment analysis, with a red color showing a higher degree of enrichment.

Figure 4

Effects of UV-B treatment on transcription levels of OsbZIP18 in rice. (A) Phylogenetic tree of HY5 protein in Arabidopsis, rice, maize, wheat, soybean, and tomato, with the bootstraps values from 1000 replicates indicated. (B) Expression analysis of OsbZIP18 under control and UV-B treatments. The relative expression levels were normalized to the ubiquitin gene and were quantified by RT-qPCR. Asterisks indicate a significant difference between the control group and UV-B treatment at individual time points (n = 3, ** p < 0.01, Student’s t-test). (C) Identification of osbzip18 mutants. The osbzip18-1 is 5 bp GGACG deletion and the osbzip18-2 is 1 bp C insertion in the first exon of OsbZIP18 genome.

Figure 5

Comparative analysis of differential metabolites of osbzip18 mutants under different UV-B durations. Heat map of differential metabolites of osbzip18 mutants under different treatment duration, UV-B 0 h (A), UV-B 6 h (C), UV-B 12 h (E), and UV-B 24 h (G). Red indicates a high abundance, and blue indicates low relative abundance metabolites. KEGG pathways enriched significantly in osbzip18 mutant vs. ZH11 comparison under different treatment duration, UV-B 0 h (B), UV-B 6 h (D), UV-B 12 h (F), and UV-B 24 h (H). The bubble colors represent the p-values of the enrichment analysis, with red color showing a higher degree of enrichment.

Figure 6

Expression levels of genes involved in phenylpropanoid and flavonoid biosynthesis pathways in ZH11 and osbzip18 mutants. Abbreviations for enzymes: (A) PAL, phenylalanine ammonia lyase; (B) C4H, cinnamate 4-hydroxylase; (C) 4CL, 4-coumarate CoA ligase; (D) CHS, chalcone synthase; (E) CHIL2, chalcone isomerase; (F) F3H, flavanone 3β-hydroxylase. The relative expression levels were normalized to those of ubiquitin and were quantified by RT-qPCR. Asterisks indicate a significant difference between the control and UV-B treatments at individual time points (n = 3, * p < 0.05 and ** p < 0.01, Student’s t-test).

Figure 7

Analysis of cis-binding elements in phenylpropane and flavonoid pathway gene promoter region and electrophoretic mobility shift assays (EMSA). (A) Diagram of the OsPAL5, OsC4H, Os4CL, OsCHS, OsCHIL2, and OsF3H promoter regions showing the relative positions of the ACE and G-box cis-elements. The red rectangles represent the ACE elements and the G-box. (B) EMSA analysis of OsbZIP18 binding to the ACE and G-box motif in the OsPAL5, OsC4H, Os4CL, OsCHS, OsCHIL2, and OsF3H promoters. Twenty-five-fold molar excesses of unlabeled probes were used in the competition assay.