Genome-wide identification and expression analysis reveals spinach brassinosteroid-signaling kinase (BSK) gene family functions in temperature stress response.

BACKGROUND: Brassinosteroid (BR)- signaling kinase (BSK) is a critical family of receptor-like cytoplasmic kinase for BR signal transduction, which plays important roles in plant development, immunity, and abiotic stress responses. Spinach (Spinacia oleracea) is cold- tolerant but heat- sensitive green leafy vegetable. A study on BSK family members and BSKs- mediated metabolic processes in spinach has not been performed. RESULTS: We identified and cloned seven SoBSKs in spinach. Phylogenetic and collinearity analyses suggested that SoBSKs had close relationship with dicotyledonous sugar beet (Beta vulgaris) rather than monocotyledons. The analyses of gene structure and conserved protein domain/ motif indicated that most SoBSKs were relative conserved, while SoBSK6 could be a truncated member. The prediction of post-translation modification (PTM) sites in SoBSKs implied their possible roles in signal transduction, redox regulation, and protein turnover of SoBSKs, especially the N-terminal myristoylation site was critical for BSK localization to cell periphery. Cis-acting elements for their responses to light, drought, temperature (heat and cold), and hormone distributed widely in the promoters of SoBSKs, implying the pivotal roles of SoBSKs in response to diverse abiotic stresses and phytohormone stimuli. Most SoBSKs were highly expressed in leaves, except for SoBSK7 in roots. Many SoBSKs were differentially regulated in spinach heat- sensitive variety Sp73 and heat- tolerant variety Sp75 under the treatments of heat, cold, as well as exogenous brassinolide (BL) and abscisic acid (ABA). The bsk134678 mutant Arabidopsis seedlings exhibited more heat tolerance than wild- type and SoBSK1- overexpressed seedlings. CONCLUSIONS: A comprehensive genome- wide analysis of the BSK gene family in spinach presented a global identification and functional prediction of SoBSKs. Seven SoBSKs had relatively- conserved gene structure and protein function domains. Except for SoBSK6, all the other SoBSKs had similar motifs and conserved PTM sites. Most SoBSKs participated in the responses to heat, cold, BR, and ABA. These findings paved the way for further functional analysis on BSK- mediated regulatory mechanisms in spinach development and stress response.

Nano-priming as emerging seed priming technology for sustainable agriculture-recent developments and future perspectives.

Nano-priming is an innovative seed priming technology that helps to improve seed germination, seed growth, and yield by providing resistance to various stresses in plants. Nano-priming is a considerably more effective method compared to all other seed priming methods. The salient features of nanoparticles (NPs) in seed priming are to develop electron exchange and enhanced surface reaction capabilities associated with various components of plant cells and tissues. Nano-priming induces the formation of nanopores in shoot and helps in the uptake of water absorption, activates reactive oxygen species (ROS)/antioxidant mechanisms in seeds, and forms hydroxyl radicals to loosen the walls of the cells and acts as an inducer for rapid hydrolysis of starch. It also induces the expression of aquaporin genes that are involved in the intake of water and also mediates H2O2, or ROS, dispersed over biological membranes. Nano-priming induces starch degradation via the stimulation of amylase, which results in the stimulation of seed germination. Nano-priming induces a mild ROS that acts as a primary signaling cue for various signaling cascade events that participate in secondary metabolite production and stress tolerance. This review provides details on the possible mechanisms by which nano-priming induces breaking seed dormancy, promotion of seed germination, and their impact on primary and secondary metabolite production. In addition, the use of nano-based fertilizer and pesticides as effective materials in nano-priming and plant growth development were also discussed, considering their recent status and future perspectives.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in halophyte Puccinellia tenuiflora.

Alkaligrass (Puccinellia tenuiflora) is a monocotyledonous halophyte pasture, which has strong tolerance to saline-alkali, drought, and chilling stresses. We have reported a high-quality chromosome-level genome and stress-responsive proteomic results in P. tenuiflora. However, the gene/protein function investigations are still lacking, due to the absent of genetic transformation system in P. tenuiflora. In this study, we established a higher efficient Agrobacterium-mediated transformation for P. tenuiflora using calluses induced from seeds. Agrobacterium strain EHA105 harbors pANIC 6B vectors that contain GUS reporter gene and Hyg gene for screening. Ten mg·L-1 hygromycin was used for selecting transgenic calluses. The optimized condition of vacuum for 10 min, ultrasonication for 10 min, and then vacuum for 10 min was used for improvement of conversion efficiency. Besides, 300 mg·L-1 timentin was the optimum antibiotics in transformation. PCR amplification exhibited that GUS gene has been successfully integrated into the chromosome of P. tenuiflora. Histochemical GUS staining and qRT-PCR analysis indicated that GUS gene has stably expressed with ß-glucuronidase activity in transgene seedlings. All these demonstrated that we have successfully established an Agrobacterium-mediated transformation system of P. tenuiflora, which provides a good platform for further gene function analysis and lays a solid foundation for molecular breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01247-8.

Phosphorylation of a malate transporter promotes malate excretion and reduces cadmium uptake in apple.

Heavy metal contamination is a major environmental and human health hazard in many areas of the world. Organic acids sequester heavy metals and protect plant roots from the effects of toxicity; however, it is largely unknown how these acids are regulated in response to heavy metal stress. Here, protein kinase SOS2L1 from apple was functionally characterized. MdSOS2L1 was found to be involved in the regulation of malate excretion, and to inhibit cadmium uptake into roots. Using the DUAL membrane system in a screen of an apple cDNA library with MdSOS2L1 as bait, a malate transporter, MdALMT14, was identified as an interactor. Bimolecular fluorescence complementation, pull-down, and co-immunoprecipitation assays further indicated the interaction of the two proteins. Transgenic analyses showed that MdSOS2L1 is required for cadmium-induced phosphorylation at the Ser358 site of MdALMT14, a modification that enhanced the stability of the MdALMT14 protein. MdSOS2L1 was also shown to enhance cadmium tolerance in an MdALMT14-dependent manner. This study sheds light on the roles of the MdSOS2L1-MdALMT14 complex in physiological responses to cadmium toxicity.

An apple sucrose transporter MdSUT2.2 is a phosphorylation target for protein kinase MdCIPK22 in response to drought.

Sugars increase with drought stress in plants and accumulate in the vacuole. However, the exact molecular mechanism underlying this process is not clear yet. In this study, protein interaction and phosphorylation experiments were conducted for sucrose transporter and CIPK kinase in apple. The specific phosphorylation site of sucrose transporter was identified with mass spectrometry. Transgenic analyses were performed to characterize their biological function. It was found that overexpression of sucrose transporter gene MdSUT2.2 in apple plants promoted sugar accumulation and drought tolerance. MdSUT2.2 protein was phosphorylated at Ser381 site in response to drought. A DUALmembrane system using MdSUT2.2 as bait through an apple cDNA library got a protein kinase MdCIPK22. Bimolecular fluorescence complementary (BiFC), pull-down and co-immunoprecipitation (Co-IP) assays further demonstrated that MdCIPK22 interacted with MdSUT2.2. A series of transgenic analysis showed that MdCIPK22 was required for the drought-induced phosphylation at Ser381 site of MdSUT2.2 protein, and that it enhanced the stability and transport activity of MdSUT2.2 protein. Finally, it was found that MdCIPK22 overexpression promoted sugar accumulation and improved drought tolerance in an MdSUT2.2-dependent manner in transgenic apple plants. MdCIPK22-MdSUT2.2 regulatory module shed light on the molecular mechanism by which plant accumulates sugars and enhances tolerance in response to drought stress.

The Glucose Sensor MdHXK1 Phosphorylates a Tonoplast Na+/H+ Exchanger to Improve Salt Tolerance.

Glc regulates many vital processes, including plant growth, development, metabolism, and responses to biotic and abiotic stress. However, the molecular mechanism by which Glc acts as a signal to regulate salinity tolerance remains unclear. In this study, we found that the apple (Malus domestica Borkh.) Glc sensor hexokinase1 (MdHXK1) contributes to Glc-mediated salinity tolerance. A combination of split ubiquitin system, pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays demonstrated that MdHXK1 interacts with and phosphorylates the Na+/H+ exchanger MdNHX1 at its Ser-275 residue. Phosphorylation improved the stability of MdNHX1 and enhanced its Na+/H+ transport activity in MdNHX1 overexpression transgenic apple and yeast complementation cells. Furthermore, Ser-275 of MdNHX1 was found to be crucial for MdHXK1-mediated phosphorylation. Finally, a series of transgenic analyses demonstrated that salt tolerance mediated by MdHXK1 partially depended on MdNHX1. Overall, our findings provide insights into how sugar recruits and regulates MdNHX1 in response to high salinity in plants.

A CIPK protein kinase targets sucrose transporter MdSUT2.2 at Ser254 for phosphorylation to enhance salt tolerance.

Soil salinity is one of the major abiotic stressors that negatively affect crop growth and yield. Salt stress can regulate antioxidants and the accumulation of osmoprotectants. In the study, a sucrose transporter MdSUT2.2 was identified in apple. Overexpression of MdSUT2.2 gene increased salt tolerance in the transgenic apple, compared with the WT control "Gala." In addition, it was found that protein MdSUT2.2 was phosphorylated at Ser254 site in response to salt. A DUAL membrane yeast hybridization system through an apple cDNA library demonstrated that a protein kinase MdCIPK13 interacted with MdSUT2.2. A series of transgenic analysis in apple calli showed that MdCIPK13 was required for the salt-induced phosphorylation of MdSUT2.2 protein and enhanced its stability and transport activity. Finally, it was found that MdCIPK13 improved salt resistance in an MdSUT2.2-dependent manner. These findings had enriched our understanding of the molecular mechanisms underlying abiotic stress.

The biosynthesis of phenolic acids is positively regulated by the JA-responsive transcription factor ERF115 in Salvia miltiorrhiza.

Phenolic acids are important secondary metabolites produced in the Chinese medicinal plant Salvia miltiorrhiza, but little is known about the transcription factors involved in the regulation of tanshinone and phenolic acid biosynthesis. Here, a novel AP2/ERF transcription factor SmERF115 was isolated and functionally characterized. SmERF115 was most responsive to methyl jasmonate (MeJA) treatment and was localized in the nucleus. The phenolic acid production was increased in SmERF115-overexpressing hairy roots, but with a decrease in tanshinone content. In contrast, silencing of SmERF115 reduced the phenolic acid level, but increased tanshinone content. The expression of the key biosynthetic gene SmRAS1 was up-regulated in SmERF115 overexpression lines but was down-regulated in SmERF115-RNAi lines. Yeast one-hybrid (Y1H) assay and EMSA showed that SmERF115 directly binds to the promoter of SmRAS1, while dual-luciferase assays showed that SmERF115 could activate expression of SmRAS1 in vivo. Furthermore, global transcriptomic analysis by RNA sequencing revealed that expression of other genes such as PAL3, 4CL5, TAT3, and RAS4 was also increased in the overexpression line, implying that they were potentially involved in the SmERF115-mediated pathway. Our data show that SmERF115 is a positive regulator of phenolic acid biosynthesis, and may be a potential target for further metabolic engineering of phenolic acid biosynthesis in S. miltiorrhiza.

Protein S-acyl transferase 15 is involved in seed triacylglycerol catabolism during early seedling growth in Arabidopsis.

Seeds of Arabidopsis contain ~40% oil, which is primarily in the form of triacylglycerol and it is converted to sugar to support post-germination growth. We identified an Arabidopsis T-DNA knockout mutant that is sugar-dependent during early seedling establishment and determined that the ß-oxidation process involved in catabolising the free fatty acids released from the seed triacylglycerol is impaired. The mutant was confirmed to be transcriptional null for Protein Acyl Transferase 15, AtPAT15 (At5g04270), one of the 24 protein acyl transferases in Arabidopsis. Although it is the shortest, AtPAT15 contains the signature 'Asp-His-His-Cys cysteine-rich domain' that is essential for the enzyme activity of this family of proteins. The function of AtPAT15 was validated by the fact that it rescued the growth defect of the yeast protein acyl transferase mutant akr1 and it was also auto-acylated in vitro. Transient expression in Arabidopsis and tobacco localised AtPAT15 in the Golgi apparatus. Taken together, our data demonstrate that AtPAT15 is involved in ß-oxidation of triacylglycerol, revealing the importance of protein S-acylation in the breakdown of seed-storage lipids during early seedling growth of Arabidopsis.

Transcription Factor AREB2 Is Involved in Soluble Sugar Accumulation by Activating Sugar Transporter and Amylase Genes.

Sugars play important roles in plant growth and development, crop yield and quality, as well as responses to abiotic stresses. Abscisic acid (ABA) is a multifunctional hormone. However, the exact mechanism by which ABA regulates sugar accumulation is largely unknown in plants. Here, we tested the expression profile of several sugar transporter and amylase genes in response to ABA treatment. MdSUT2 and MdAREB2 were isolated and genetically transformed into apple (Malus domestica) to investigate their roles in ABA-induced sugar accumulation. The MdAREB2 transcription factor was found to bind to the promoters of the sugar transporter and amylase genes and activate their expression. Both MdAREB2 and MdSUT2 transgenic plants produced more soluble sugars than controls. Furthermore, MdAREB2 promoted the accumulation of sucrose and soluble sugars in an MdSUT2-dependent manner. Our results demonstrate that the ABA-responsive transcription factor MdAREB2 directly activates the expression of amylase and sugar transporter genes to promote soluble sugar accumulation, suggesting a mechanism by which ABA regulates sugar accumulation in plants.

An apple CIPK protein kinase targets a novel residue of AREB transcription factor for ABA-dependent phosphorylation.

Phytohormone abscisic acid (ABA) regulates many important processes in plants. It is a major molecule facilitating signal transduction during the abiotic stress response. In this study, an ABA-inducible transcription factor gene, MdAREB2, was identified in apple. Transgenic analysis was performed to characterize its function in ABA sensitivity. Overexpression of the MdAREB2 gene increased ABA sensitivity in the transgenic apple compared with the wild-type (WT) control. In addition, it was found that the protein MdAREB2 was phosphorylated at a novel site Thr411 in response to ABA. A yeast two-hybridization screen of an apple cDNA library demonstrated that a protein kinase, MdCIPK22, interacted with MdAREB2. Their interaction was further verified with Pull Down and Co-IP assays. A series of transgenic analyses in apple calli and plantlets showed that MdCIPK22 was required for ABA-induced phosphorylation at Thr411 of the MdAREB2 protein and enhanced its stability and transcriptional activity. Finally, it was found that MdCIPK22 increased ABA sensitivity in an MdAREB2-dependent manner. Our findings indicate a novel phosphorylation site in CIPK-AREB regulatory module for the ABA signalling pathway, which would be helpful for researchers to identify the functions of uncharacterized homologs in the future.

Overexpression of MdSOS2L1, a CIPK protein kinase, increases the antioxidant metabolites to enhance salt tolerance in apple and tomato.

Soil salinity hinders the growth of most higher plants and becomes a gradually increasing threat to the agricultural production of such crops as the woody plant apple. In this study, a calcineurin B-like protein (CBL)-interacting protein kinase, MdCIPK24-LIKE1 (named as MdSOS2L1), was identified. Quantitative real-time polymerase chain reaction (qRT-PCR) assay revealed that the expression of MdSOS2L1 was upregulated by CaCl2 . Yeast two-hybrid (Y2H) assay and transiently transgenic analysis demonstrated that the MdSOS2L1 protein kinase physically interacted with MdCBL1, MdCBL4 and MdCBL10 proteins to increase salt tolerance in apple. Furthermore, iTRAQ proteome combined with liquid chromatography-tandem mass spectrometry (LC/MS) analysis found that several proteins, which are involved in reactive oxygen species (ROS) scavenging, procyanidin biosynthesis and malate metabolism, were induced in MdSOS2L1-overexpressing apple plants. Subsequent studies have shown that MdSOS2L1 increased antioxidant metabolites such as procyanidin and malate to improve salt tolerance in apple and tomato. In summary, our studies provide a mechanism in which SOS2L1 enhances the salt stress tolerance in apple and tomato.

MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

KEY MESSAGE: Salt-induced phosphorylation of MdVHA-B1 protein was mediated by MdSOS2L1 protein kinase, and thereby increasing malate content in apple. Salinity is an important environmental factor that influences malate accumulation in apple. However, the molecular mechanism by which salinity regulates this process is poorly understood. In this work, we found that MdSOS2L1, a novel AtSOS2-LIKE protein kinase, interacts with V-ATPase subunit MdVHA-B1. Furthermore, MdSOS2L1 directly phosphorylates MdVHA-B1 at Ser(396) site to modulate malate accumulation in response to salt stress. Meanwhile, a series of transgenic analyses in apple calli showed that the MdSOS2L1-MdVHAB1 pathway was involved in the regulation of malate accumulation. Finally, a viral vector-based transformation approach demonstrated that the MdSOS2L1-MdVHAB1 pathway also modulated malate accumulation in apple fruits with or without salt stress. Collectively, our findings provide a new insight into the mechanism by which MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

New features of triacylglycerol biosynthetic pathways of peanut seeds in early developmental stages.

The peanut (Arachis hypogaea L.) is one of the three most important oil crops in the world due to its high average oil content (50 %). To reveal the biosynthetic pathways of seed oil in the early developmental stages of peanut pods with the goal of improving the oil quality, we presented a method combining deep sequencing analysis of the peanut pod transcriptome and quantitative real-time PCR (RT-PCR) verification of seed oil-related genes. From the sequencing data, approximately 1500 lipid metabolism-associated Unigenes were identified. The RT-PCR results quantified the different expression patterns of these triacylglycerol (TAG) synthesis-related genes in the early developmental stages of peanut pods. Based on these results and analysis, we proposed a novel construct of the metabolic pathways involved in the biosynthesis of TAG, including the Kennedy pathway, acyl-CoA-independent pathway and proposed monoacylglycerol pathway. It showed that the biosynthetic pathways of TAG in the early developmental stages of peanut pods were much more complicated than a simple, unidirectional, linear pathway.

A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves.

BACKGROUND: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens­mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported. RESULTS: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB. CONCLUSION: This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.

Na2CO3-responsive mechanism insight from quantitative proteomics and SlRUB gene function in Salix linearistipularis seedlings.

Mongolian willow (Salix linearistipularis) is a naturally occurring woody dioecious plant in the saline soils of north-eastern China, which has a high tolerance to alkaline salts. Although transcriptomics studies have identified a large number of salinity-responsive genes, the mechanism of salt tolerance in Mongolian willow is not clear. Here, we found that in response to Na2CO3 stress, Mongolian willow regulates osmotic homeostasis by accumulating proline and soluble sugars and scavenges reactive oxygen species (ROS) by antioxidant enzymes and non-enzymatic antioxidants. Our quantitative proteomics study identified 154 salt-sensitive proteins mainly involved in maintaining the stability of the photosynthetic system and ROS homeostasis to cope with Na2CO3 stress. Among them, Na2CO3-induced rubredoxin (RUB) was predicted to be associated with 122 proteins for the modulation of these processes. The chloroplast-localized S. linearistipularis rubredoxin (SlRUB) was highly expressed in leaves and was significantly induced under Na2CO3 stress. Phenotypic analysis of overexpression, mutation and complementation materials of RUB in Arabidopsis suggests that SlRUB is critical for the regulation of photosynthesis, ROS scavenging and other metabolisms in the seedlings of Mongolian willow to cope with Na2CO3 stress. This provides more clues to better understand the alkali-responsive mechanism and RUB functions in the woody Mongolian willow.

Jasmonic acid regulates the biosynthesis of medicinal metabolites via the JAZ9-MYB76 complex in Salvia miltiorrhiza.

Jasmonic acid (JA) signaling pathway plays an important role in tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza. However, the specific regulatory mechanism remains largely unclear. Previous work showed that a JASMONATE ZIM-domain (JAZ) protein, SmJAZ9, acted as a repressor of tanshinone production in S. miltiorrhiza. In this study, we revealed that SmJAZ9 reduced both phenolic acid accumulation and related biosynthetic gene expression, confirming that SmJAZ9 also negatively affected phenolic acid biosynthesis. Then, we identified a novel MYB transcription factor, SmMYB76, which interacted with SmJAZ9. SmMYB76 repressed phenolic acid biosynthesis by directly downregulating SmPAL1, Sm4CL2, and SmRAS1. Further investigation demonstrated that JA mediated phenolic acids biosynthesis via SmJAZ9-SmMYB76 complex. Taken together, these findings state the molecular mechanism that SmJAZ9-SmMYB76 regulated phenolic acid biosynthesis at the transcriptional and protein levels, which provided new insights into JA signaling pathway regulating plant metabolism.

Cysteine-rich receptor-like protein kinases: emerging regulators of plant stress responses.

Cysteine-rich receptor-like kinases (CRKs) belong to a large DUF26-containing receptor-like kinase (RLK) family. They play key roles in immunity, abiotic stress response, and growth and development. How CRKs regulate diverse processes is a long-standing question. Recent studies have advanced our understanding of the molecular mechanisms underlying CRK functions in Ca2+ influx, reactive oxygen species (ROS) production, mitogen-activated protein kinase (MAPK) cascade activation, callose deposition, stomatal immunity, and programmed cell death (PCD). We review the CRK structure-function relationship with a focus on the roles of CRKs in immunity, the abiotic stress response, and the growth-stress tolerance tradeoff. We provide a critical analysis and synthesis of how CRKs control sophisticated regulatory networks that determine diverse plant phenotypic outputs.

Pto Interaction Proteins: Critical Regulators in Plant Development and Stress Response.

Pto interaction (Pti) proteins are a group of proteins that can be phosphorylated by serine/threonine protein kinase Pto, which have diverse functions in plant development and stress response. In this study, we analyzed the phylogenetic relationship, gene structure, and conserved motifs of Pti1s and predicted the potential cis-elements in the promoters of Pti1 genes using bioinformatics methods. Importantly, we systematically summarized the diverse functions of Pti1s in tomato, rice, Arabidopsis, potato, apple, and cucumber. The potential cis-elements in promoters of Pti1s decide their functional diversity in response to various biotic and abiotic stresses. The protein kinase Pti1 was phosphorylated by Pto and then modulated the downstream signaling pathways for PTI and ETI in the disease insistence process. In addition, some transcription factors have been defined as Ptis (e.g., Pti4, Pti5, and Pti6) originally, which actually were ethylene-response factors (ERFs). Pti4, Pti5, and Pti6 were modulated by salicylic acid (SA), jasmonate (JA), and ethylene signaling pathways and regulated diverse defense-related gene expression to cope with Pst infection and insect wounding.

SmbHLH60 and SmMYC2 antagonistically regulate phenolic acids and anthocyanins biosynthesis in Salvia miltiorrhiza.

INTRODUCTION: Salvia miltiorrhiza is a renowned traditional Chinese medicinal plant with extremely high medicinal value, especially for cardiovascular and cerebrovascular diseases. The jasmonic acid (JA) signaling pathway plays an important role in the improved biosynthesis of secondary metabolites, which is mediated by a major transcriptional regulator, MYC2. However, the JA regulatory mechanism of secondary metabolites biosynthesis in S. miltiorrhiza is still largely unknown. OBJECTIVES: Our work focuses on the dissection of the molecular mechanism of transcriptional regulation in MeJA-mediated biosynthesis of medicinal components of S. miltiorrhiza. We examined the role of MeJA-responsive bHLH transcription factors (TFs) in improving bioactive secondary metabolites accumulation in S. miltiorrhiza. METHODS: Hairy root transformation based on CRISPR/Cas9 technique was used to decipher gene function(s). Changes in the content of phenolic acids were evaluated by HPLC. Y1H, EMSA and dual-LUC assays were employed to analyze the molecular mechanism of SmbHLH60 in the regulation on the biosynthesis of phenolic acids and anthocyanins. Y2H, BiFC and pull-down affinity assays were used to corroborate the interaction between SmbHLH60 and SmMYC2. RESULTS: Being one of the most significantly negatively regulated bHLH genes by MeJA, a new transcription factor SmbHLH60 was discovered and characterized. Over-expression of SmbHLH60 resulted in significant inhibition of phenolic acid and anthocyanin biosynthesis in S. miltiorrhiza by transcriptionally repressing of target genes such as SmTAT1 and SmDFR, whereas CRISPR/Cas9-generated knockout of SmbHLH60 resulted in the opposite effect. In addition, SmbHLH60 and SmMYC2 formed a heterodimer to antagonistically regulate phenolic acid and anthocyanin biosynthesis. CONCLUSION: Our results clarified that SmbHLH60 is a negativeregulator on the biosynthesis of phenolic acids and anthocyanins. SmbHLH60 competed with SmMYC2 in an antagonistic manner, providing new insights for the molecular mechanism of MeJA-mediated regulation on the biosynthesis of secondary metabolites in S. miltiorrhiza.