Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H₂O₂) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H₂O₂in rosmarinic acid (RA) production inSalvia miltiorrhizacell cultures were investigated. The results showed that SA significantly enhanced H₂O₂production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H₂O₂could also promote PAL activity and enhance RA production. If H₂O₂production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H₂O₂is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation.

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) are key signaling molecules in cells whose levels are increased in response to various stimuli and are involved in plant secondary metabolite synthesis. In this paper, the roles of H₂O₂ and NO on salvianolic acid B (Sal B) production in salicylic acid (SA)-induced Salvia miltiorrhiza cell cultures were investigated. The results showed that H₂O₂ could be significantly elicited by SA, even though IMD (an inhibitor of NADPH oxidase) or DMTU (a quencher of H₂O₂) were employed to inhibit or quench intracellular H₂O₂. These elicited H₂O₂ levels significantly increased NO production by 1.6- and 1.46 fold in IMD+SA and DMTU+SA treatments, respectively, and induced 4.58- and 4.85-fold Sal B accumulation, respectively. NO was also markedly elicited by SA, in which L-NNA (an inhibitor of NO synthase) and cPTIO (a quencher of NO) were used to inhibit or quench NO within cells, and the induced NO could significantly enhance H₂O₂ production by 1.92- and 1.37-fold in L-NNA+SA and cPTIO+SA treatments, respectively, and 3.27- and 1.50-fold for Sal B accumulation, respectively. These results indicate that elicitation of SA for either H₂O₂ or NO was independent, and the elicited H₂O₂ or NO could act independently or synergistically to induce Sal B accumulation in SA-elicited cells.

Background: Our previous study finds that male sterility in Salvia miltiorrhiza could result in stunted growth and reduced biomass, but their molecular mechanisms have not yet been revealed. In this article, we investigate the underlying mechanism of male sterility and its impact on plant growth and metabolic yield by using physiological analysis and mRNA sequencing (RNA-Seq). Results: In this study, transcriptomic and physiological analysis were performed to identify the mechanism of male sterility in mutants and its impact on plant growth and metabolic yield. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, it is found that the pathways are mainly enriched in processes including organ development, primary metabolic process and secondary metabolic process. Physiological analysis show that the chloroplast structure of male sterile mutants of S. miltiorrhiza is abnormally developed, which could result in decrease in leaf gas exchange (A, E and g_s), chlorophyll fluorescence (F_v, F_m and F_v/F_m), and the chlorophyll content. Expression level of 7 differentially expressed genes involved in photosynthesis-related pathways is downregulated in male sterile lines of S. miltiorrhiza, which could explain the corresponding phenotypic changes in chlorophyll fluorescence, chlorophyll content and leaf gas exchange. Transcriptomic analysis establishes the role of disproportionating enzyme 1 (DPE1) as catalyzing the degradation of starch, and the role of sucrose synthase 3 (SUS3) and cytosolic invertase 2 (CINV2) as catalyzing the degradation of sucrose in the S. miltiorrhiza mutants. The results also confirm that phenylalanine ammonialyase (PAL) is involved in the biosynthesis of rosmarinic acid and salvianolic acid B, and flavone synthase (FLS) is an important enzyme catalyzing steps of flavonoid biosynthesis. Conclusions: Our results from the physiological and transcriptome analysis reveal underlying mechanism of plant growth and metabolic yield in male sterile mutants, and provide insight into the crop yield of S. miltiorrhiza.

Male sterility is a common phenomenon in flowering plants, and it has been widely used in hybrid seed production in a number of economically important crops. In 2002, our team discovered a natural male sterile mutant of Salvia miltiorrhiza. It provided us with the possibility of obtaining stable and controllable quality. To study the molecular mechanism of male sterility in S. miltiorrhiza, we generated proteomic profiles comparing the male sterile mutant type (MT) and wild type (WT) using iTRAQ sequencing. We found a total of 639 differential abundant proteins (DAPs) between MT and WT buds. The DAPs associated with male sterility were mainly involved in (1) carbohydrate and energy metabolism, and (2) protein synthesis and degradation. Based on a comparison between the protein expression profiles of MT and WT, we elucidated a potential protein interaction network involved in male sterility. These results provide new potential biomarkers and insights into the molecular mechanism of male sterility in S. miltiorrhiza.

DNA methylation plays important roles through the methyl-CpG-binding domain (MBD) to realize epigenetic modifications. Thirteen AtMBD proteins have been identified from the Arabidopsis thaliana genome, but the functions of some members are unclear. AtMBD3 was found to be highly expressed in pollen and seeds and it preferably binds methylated CG, CHG, and unmethylated DNA sequences. Then, two mutant alleles at the AtMBD3 locus were obtained in order to further explore its function using CRISPR/Cas9. When compared with 92.17% mature pollen production in the wild type, significantly lower percentages of 84.31% and 78.91% were observed in the mbd3-1 and mbd3-2 mutants, respectively. About 16–21% of pollen from the mbd3 mutants suffered a collapse in reproductive transmission, whereas the other pollen was found to be normal. After pollination, about 16% and 24% of mbd3-1 and mbd3-2 mutant seeds underwent early or late abortion, respectively. Among all the late abortion seeds in mbd3-2 plants, 25% of the abnormal seeds were at the globular stage, 31.25% were at the transition stage, and 43.75% were at the heart stage. A transcriptome analysis of the seeds found 950 upregulated genes and 1128 downregulated genes between wild type and mbd3-2 mutants. Some transcriptional factors involved in embryo development were selected to be expressed, and we found significant differences between wild type and mbd3 mutants, such as WOXs, CUC1, AIB4, and RGL3. Furthermore, we found a gene that is specifically expressed in pollen, named PBL6. PBL6 was found to directly interact with AtMBD3. Our results provide insights into the function of AtMBD3 in plants, especially in sperm fertility.