Insight into saffron associated microbiota from different origins and explore the endophytes for enhancement of bioactive compounds.

Crocus sativus L. is a perennial crop for its valuable active compounds. Plant-associated microbes impact on the quality and efficacy of medicinal herbs by promoting bioactive components accumulation. However, how microbes influence the accumulation of bioactive components in saffron have not been well studied. Here, the microbiome in C. sativus derived from 3 core production areas were deciphered by 16S rDNA sequencing and the relationship between endophytes and bioactive ingredients were further investigated. The main results are as follows: (1) Both Comamonadaceae and Burkholderiaceae were positively correlated with the content of bioactive components in the stigmas. (2) The synthesis of crocin was positively correlated with Xanthomonadaceae, negatively correlated with Lachnospiraceae and Prevotellaceae. Therefore, further investigation is required to determine whether Xanthomonadaceae plays an unknown function in the synthesis of crocin. These findings provide guidelines for disentangling the function of endophytes in the production of bioactive ingredients and thus for microbe-mediated breeding.

Identification and Functional Validation of Two Novel Antioxidant Peptides in Saffron.

Saffron (Crocus sativus L.) is one of the most expensive spices in the world, boasting rich medicinal and edible value. However, the effective development of active natural substances in saffron is still limited. Currently, there is a lack of comprehensive studies on the saffron stigma protein, and the main effect peptides have not been identified. In this study, the total protein composition of saffron stigmas was analyzed, and two main antioxidant peptides (DGGSDYLGK and VDPYFNK) were identified, which showed high antioxidant activity. Then, the stability of two peptides was further evaluated. Furthermore, our results suggested that these two peptides may protect HepG2 cells from H2O2-induced oxidative damage by significantly improving the activity of endogenous antioxidant enzymes and reducing the malondialdehyde (MDA) content. Collectively, we identified two peptides screened from the saffron protein possessing good antioxidant activity and stability, making them promising candidates for use as functional foods, etc., for health promotion. Our findings indicated that proteomic analysis together with peptide identification is a good method for exploitation and utilization of spice plants.

Evaluation of whitening and antimicrobial activity of two strains of Bletilla striata WT and HL20.

ETHNOPHARMACOLOGICAL RELEVANCE: Bletilla striata (Thunb.) Reixchb.f. is a perennial herb of the Orchid-aceae Bletilla and have various ethnopharmacological uses. As a traditional astringent hemostatic Chinese herbal medicine, B. striata has been widely used in the treatment of 127 different kinds of hemorrhagic diseases. Moreover, B. striata has been a beauty medicine since ancient times, with the first ancient records dating back to 2000 years ago, traditionally used to removing freckle and smooth the skin. Because of the high content of polysaccharides, which is considered the primary active substance of B. striata and having anti-aging, whitening, and anti-oxidation functions, this is also widely used in the cosmetics industry. AIM: We screened the germplasm resources of B. striata in the early stage and the superior HL20 strain was obtained. Our research aims to analyze and compare the whitening and antimicrobial activities of different extracts (aqueous extract, ethanol extract, and aqueous extract from ethanol extract filter residue) of the selected superior varieties (HL20) and the control (WT). MATERIALS AND METHODS: L-tyrosine and L-dopa were used as substrates to establish a tyrosinase inhibition system with arbutin as the positive control and the whitening activity was measured by the inhibition rate of TYR-M and TYR-D. Besides, an in vitro antimicrobial susceptibility test was performed to assess the antimicrobial activity of the B. striata extracts. In a nutshell, the method of punching diffusion was used to thoroughly examine the effects of three extracts from two strains on the antimicrobial activity of five types of microorganism in cosmetics microbiological testing products. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different extracts were also assessed. RESULTS: Results showed that the whitening and antimicrobial properties of the HL20 strain were found to be more potent than those of the WT strain. Compared with the other two extraction methods, the aqueous extract from ethanol extract filter residue of B. striata exhibited better inhibition of tyrosinase activity. The antimicrobial assay manifested that only the ethanol extract of B. striata had an inhibitory effect and had a potent antimicrobial impact on E. faecalis. CONCLUSIONS: In summary, we evaluated the pharmacological activity of the pre-selected excellent variety (HL20) in terms of whitening and antimicrobial activity. Our results reveal that the selected strain (HL20) has certain advantages over the control (WT). These characteristics make it a candidate additive for whitening cosmetics. Our study also provides a further contribution to the product application of B. striata in cosmetics and antimicrobial agents and the selected HL20 also lays a foundation for the breeding of superior B. striata varieties.

Two polyamines -responsive WRKY transcription factors from Anoectochilus roxburghii play opposite functions on flower development.

Anoectochilus roxburghii is a rare and precious plant with medicinal and healthcare functions. Embryo abortion caused the lack of resources. Polyamine promoted its flowering and stress resistance in our previous study. But the mechanism remains unclear. The WRKY transcription factor family has been linked to a variety of biological processes in plants. In this study, two WRKY TFs (ArWRKY5 and ArWRKY20) of A. roxburghii, which showed significant response to Spd treatment, were identified and functionally analyzed. Tissue specific expression analyzation showed both of them mostly present in the flower. And ArWRKY5 expressed highest in the flower bud stage (-1 Flowering), while ArWRKY20 showed the highest expression in earlier flower bud stage (-2 Flowering) and the expression gradually decreased with flowering. The transcriptional activation activity assay and subcellular localization revealed that ArWRKY5 and ArWRKY20 were located in the nucleus and ArWRKY20 showed transcriptional activity. The heterologous expression of ArWRKY5 in Arabidopsis thaliana showed earlier flowering, while overexpression of ArWRKY20 delayed flowering. But the OE-ArWRKY20 lines had a robust body shape and a very significant increase in the number of rosette leaves. Furthermore, stamens and seed development were positively regulated by these two ArWRKYs. These results indicated that ArWRKY5 and ArWRKY20 not only play opposite roles in the floral development, but also regulate the plant growth and seed development in A. thaliana. But their specific biological functions and mechanism in A. roxburghii need to be investigated further.

Rapid Measurement of Residual Kanamycin Using Highly Specific Biomimetic Recognition Paper-Based Chip.

The development of highly specific biomimetic recognition material is a challenge for rapid detection of harmful residues in foodstuff. In this study, a paper-based boronate affinity metal-organic framework/molecularly imprinted polymer microfluidic chip (FZS-BA@MIP) was constructed based on the in situ construction strategy, which was also designed as a highly specific biomimetic recognition module. Here, the homogeneous zeolitic imidazole framework-8 (ZIF-8) membrane served as a great scaffold and enrichment layer. Besides, the recognition layer of MIP was prepared based on a highly oriented boronate affinity surface imprinting strategy. With the aid of the liquid flow channel, the highly specific enrichment and visual detection for antibiotic residues like kanamycin in actual products were achieved on the paper chip module of an integrated lateral flow platform. The whole analysis process could be accomplished within 30 min. In brief, this study offered a new integrated biomimetic recognition platform for visually detecting harmful veterinary residues containing cis-diols, which demonstrated promising commercial value in point-of-care testing of foodborne hazardous compounds.

Biocontrol: Endophytic bacteria could be crucial to fight soft rot disease in the rare medicinal herb, Anoectochilus roxburghii.

Microbial destabilization induced by pathogen infection has severely affected plant quality and output, such as Anoectochilus roxburghii, an economically important herb. Soft rot is the main disease that occurs during A. roxburghii culturing. However, the key members of pathogens and their interplay with non-detrimental microorganisms in diseased plants remain largely unsolved. Here, by utilizing a molecular ecological network approach, the interactions within bacterial communities in endophytic compartments and the surrounding soils during soft rot infection were investigated. Significant differences in bacterial diversity and community composition between healthy and diseased plants were observed, indicating that the endophytic communities were strongly influenced by pathogen invasion. Endophytic stem communities of the diseased plants were primarily derived from roots and the root endophytes were largely derived from rhizosphere soils, which depicts a possible pathogen migration image from soils to roots and finally the stems. Furthermore, interactions among microbial members indicated that pathogen invasion might be aided by positively correlated native microbial members, such as Enterobacter and Microbacterium, who may assist in colonization and multiplication through a mutualistic relationship in roots during the pathogen infection process. Our findings will help open new avenues for developing more accurate strategies for biological control of A. roxburghii bacterial soft rot disease.

Proteomics and metabolomics reveal the mechanism underlying differential antioxidant activity among the organs of two base plants of Shiliang tea (Chimonanthus salicifolius and Chimonanthus zhejiangensis).

The leaves and branches of Chimonanthus salicifolius and Chimonanthus zhejiangensis are the base ingredients of Shiliang tea. In this study, proteomics and metabolomics were performed to understand the molecular mechanisms underlying antioxidant activity (AA) in the leaves and branches of the two species. Stress and redox related proteins are differentially expressed among organs. The abundance of isoprenoid pathway-related proteins is higher in leaves while the abundance of phenylpropanoid and flavonoid pathway-related proteins is higher in branches in both species. Metabolomics revealed the flavonoid composition and demonstrated that procyanidins are more abundant in branches. Superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and AA are stronger in branches than leaves. Overall, branches might contribute to redox homeostasis through SOD/GSH-PX and flavonoids. Furthermore, the high level of AA of branches might be largely due to their increased accumulation of procyanidins.

Transcriptional regulation of plant seed development.

Plant seeds, which are unique reproductive organs of gymnosperms and angiosperms, are used for edible, medicinal, and industrial purposes. Transcription factors (TFs) are master regulators of plant growth, development, and stress responses. This review describes, in detail, the functions of TFs in regulating seed development. Different TFs, or even different TF families, may have similar functions in seed development. For example, WUSCHEL-related homeobox, LEC2/FUS3/ABI3, and HEME ACTIVATOR PROTEIN3 families can control plant seed embryonic initiation and development. In contrast, some members of the same TF family may have completely opposite roles. For instance, AtMYB76 and AtMYB89 inhibit the accumulation of seed oil, whereas AtMYB96 promotes seed fatty acid accumulation in Arabidopsis thaliana. Compared with the number of studies that have addressed regulation by single TFs, only a few have focused on multiple-TF regulatory networks. This review should be useful as a reference for future studies on regulatory networks of TF complexes.

Phytochemistry, pharmacology, and potential clinical applications of saffron: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, the dried red stigma of the perennial herb Crocus sativus L. (Iridaceae), is one of the most important and expensive spices in the world. It is used as a traditional Chinese medicine with demonstrated effects in promoting blood circulation and suppressing blood stasis, cooling blood detoxification, and relieving depression. It is mainly used for the treatment of depression, irregular menstruation, postpartum thrombosis, and bruises. AIM OF THE STUDY: This review aims to provide a systematic and up-to-date overview of the phytochemistry, pharmacology, and clinical applications of saffron. We hope it could provide useful references and guidance for the future directions of research on saffron. MATERIALS AND METHODS: The online database, such as Web of Science, Google Scholar, Science Direct, PubMed, SpringerLink, Wiley Online Library, SciFinder and Chemical book, and CNKI were used to collect relevant literature. And the classic books about Chinese herbal medicine were also being referenced. RESULTS: More than 150 chemical compounds, including carotenoids, flavonoids and flavonoid glycosides, monoterpenes and monoterpenoid derivatives, monocyclic aromatic hydrocarbons, amino acids, alkaloids and others, were revealed. The pharmacological activities study of saffron were focused on the antioxidant, anti-inflammatory, antitumor, antidepressant, hypoglycemic, hypolipidemic, memory-enhancing, and so on. Currently, saffron is mainly used for the treatment of diabetes, Alzheimer's disease, depression, anxiety disorders, cardiovascular diseases, learning and memory disorders, cancer, and other conditions. CONCLUSIONS: Phytochemical and pharmacological analyses of saffron have been revealed in recent studies. However, clinical studies have focused mainly on AD, depression and anxiety disorders. Therefore, a large number of clinical trials are needed to study the efficacy of saffron and its major chemical components against other diseases including hypertension, hyperlipidemia, and cancer. Further studies of the mechanism of action and toxicological properties of saffron are also required, especially research to establish an effective dose of saffron and its long-term toxicity in vivo.

SmKFB5 protein regulates phenolic acid biosynthesis by controlling the degradation of phenylalanine ammonia-lyase in Salvia miltiorrhiza.

Phenolic acids are the major secondary metabolites and significant bioactive constituents of the medicinal plant Salvia miltiorrhiza. Many enzyme-encoding genes and transcription factors involved in the biosynthesis of phenolic acids have been identified, but the underlying post-translational regulatory mechanisms are poorly understood. Here, we demonstrate that the S. miltiorrhiza Kelch repeat F-box protein SmKFB5 physically interacts with three phenylalanine ammonia-lyase (PAL) isozymes and mediates their proteolytic turnover via the ubiquitin-26S proteasome pathway. Disturbing the expression of SmKFB5 reciprocally affected the abundance of SmPAL protein and the accumulation of phenolic acids, suggesting that SmKFB5 is a post-translational regulator responsible for the turnover of PAL and negatively controlling phenolic acids. Furthermore, we discovered that treatment of the hairy root of S. miltiorrhiza with methyl jasmonate suppressed the expression of SmKFB5 while inducing the transcription of SmPAL1 and SmPAL3. These data suggested that methyl jasmonate consolidated both transcriptional and post-translational regulation mechanisms to enhance phenolic acid biosynthesis. Taken together, our results provide insights into the molecular mechanisms by which SmKFB5 mediates the regulation of phenolic acid biosynthesis by jasmonic acid, and suggest valuable targets for plant breeders in tailoring new cultivars.

Systematic Analysis of Kelch Repeat F-box (KFB) Protein Gene Family and Identification of Phenolic Acid Regulation Members in Salvia miltiorrhiza Bunge.

S. miltiorrhiza is a well-known Chinese herb for the clinical treatment of cardiovascular and cerebrovascular diseases. Tanshinones and phenolic acids are the major secondary metabolites and significant pharmacological constituents of this plant. Kelch repeat F-box (KFB) proteins play important roles in plant secondary metabolism, but their regulation mechanism in S. miltiorrhiza has not been characterized. In this study, we systematically characterized the S. miltiorrhiza KFB gene family. In total, 31 SmKFB genes were isolated from S. miltiorrhiza. Phylogenetic analysis of those SmKFBs indicated that 31 SmKFBs can be divided into four groups. Thereinto, five SmKFBs (SmKFB1, 2, 3, 5, and 28) shared high homology with other plant KFBs which have been described to be regulators of secondary metabolism. The expression profile of SmKFBs under methyl jasmonate (MeJA) treatment deciphered that six SmKFBs (SmKFB1, 2, 5, 6, 11, and 15) were significantly downregulated, and two SmKFBs (SmKFB22 and 31) were significantly upregulated. Tissue-specific expression analysis found that four SmKFBs (SmKFB4, 11, 16, and 17) were expressed preferentially in aerial tissues, while two SmKFBs (SmKFB5, 25) were predominantly expressed in roots. Through a systematic analysis, we speculated that SmKFB1, 2, and 5 are potentially involved in phenolic acids biosynthesis.

SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.

Phenolic acids and tanshinones are the two groups of pharmaceutically active metabolites in Salvia miltiorrhiza Bunge. Their contents are the key quality indicator to evaluate S. miltiorrhiza. bHLH transcription factors have important roles in regulation of plant specialised metabolism. In this study, an endogenous bHLH transcription factor, SmbHLH3, was identified and functionally analyzed. SmbHLH3 was presented in all the six tissues and mostly expressed in fibrous roots and flowers. It was localized to the nucleus. Overexpression of SmbHLH3 decreased both phenolic acids and tanshinones contents. Contents of caffeic acid and rosmarinic acid were both decreased to 50% of the control. And accumulation of salvianolic acid B was decreased as much as 62%. Content of cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA in SmbHLH3-overexpression lines were reduced 97%, 62%, 86% and 91%, respectively. In the transgenic lines, expression of C4H1, TAT and HPPR in phenolic acids pathways were reduced to about 43%, 66% and 77% of the control, respectively. For tanshinone biosynthetic pathways, transcripts of DXS3, DXR, HMGR1, KSL1, CPS1 and CYP76AH1 were reduced to 46%, 65%, 78%, 57%, 27% and 62% of the control, respectively. There was an E/G-box specific binding site in SmbHLH3, which may bind the E/G-box present in promoter region of these biosynthetic pathway genes. Y1H results indicated that SmbHLH3 could bind the promoter of TAT, HPPR, KSL1 and CYP76AH1. These findings indicated that SmbHLH3 downregulate both phenolic acids and tanshinone accumulation through directly suppressing the transcription of key enzyme genes.

Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots.

The bHLH transcription factors have important role in regulation of plant growth, development, and secondary metabolism. Tanshinones are the major pharmaceutical components present in Salvia miltiorrhiza Bunge. It has been reported that bHLHs have functions in terpenoids biosynthesis. Here, we got a bHLH family member named SmbHLH10 which could positively regulate tanshinones biosynthesis in S. miltiorrhiza hairy roots. In the SmbHLH10-overexpressing line 6, four major tanshinones contents were reaching 2.51-fold (dihydrotanshinone I), 2.84-fold (cryptotanshinone), 2.89- fold (tanshinone I), 2.68-fold (tanshinone II A) of WT, respectively. The variation in tanshinones biosynthetic pathway gene transcription was generally consistent with tanshinones content. DXS2, DXS3 and DXR of MEP pathway were induced substantially, reaching 10-fold, 3-fold, 5.74-fold higher of the WT, respectively. The downstream pathway genes CPS1, CPS5 and CYP76AH1 were highest in line OE-SmbHLH10-6, reached 4.93, 16.29 and 3.27-fold of the WT, respectively, while KSL1's expression was highest in line OE-SmbHLH10-4, 4.64-fold of WT. Yeast one-hybrid assays results showed that SmbHLH10 could binds the predicted G-box motifs within the promoters of DXS2, CPS1 and CPS5. These findings indicated that SmbHLH10 could directly binds to G-box in the pathway genes' promotor, activate their expression and then upregulate tanshinones biosynthesis.

Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: SmbHLH148 activated the whole biosynthetic pathways of phenolic acids and tanshinones, thus upregulated the production of both the two groups of pharmaceutical ingredients in Salvia miltiorrhiza. Phenolic acids and tanshinones are the two important groups of pharmaceutical ingredients presented in Salvia miltiorrhiza Bunge. The bHLH transcription factors could regulate secondary metabolism efficiently in plants. However, there are only some MYCs have been studied on regulation of either phenolic acids or tanshinones biosynthesis. In this study, a bHLH TF named SmbHLH148, which is homologous to AtbHLH148, AtbHLH147 and CubHLH1, was isolated and functionally characterized from S. miltiorrhiza. Transcription of SmbHLH148 could be intensely induced by ABA and also be moderately induced by MeJA and GA. SmbHLH148 is present in all the six tissues and mostly expressed in fibrous root and flowers. Subcellular localization analysis found that SmbHLH148 was localized in the nucleus. Overexpression of SmbHLH148 significantly increased not only three phenolic acids components accumulation but also three tanshinones content. Content of caffeic acid, rosmarinic acid and salvianolic acid B were reached to 2.87-, 4.00- and 5.99-fold of the control in the ObHLH148-3, respectively. Content of dihydrotanshinone I, cryptotanshinone, and tanshinone I were also present highest in ObHLH148-3, reached 2.5-, 5.04- and 3.97-fold of the control, respectively. Expression analysis of pathway genes of phenolic acids and tanshinones in transgenic lines showed that most of them were obviously upregulated. Moreover, transcription of AREB and JAZs were also induced in SmbHLH148 overexpression lines. These results suggested that SmbHLH148 might be taken part in ABA and MeJA signaling and activated almost the whole biosynthetic pathways of phenolic acids and tanshinones, thus the production of phenolic acids and tanshinones were upregulated.

Study on Hybrid Characteristics of Medicinally Used Cultivated Codonopsis Species Using Ribosomal Internal Transcribed Spacer (ITS) Sequencing.

Codonopsis taxa, as a traditional Chinese medicinal and edible plant, has found expanding domestic and foreign applications in recent decades. However, the poor management in germplasm resources market has inevitably caused an unnecessary hybrid of the provenances. In order to clarify the hybrid characteristics of germplasm resources in the main production area, the Codonopsis cultivars collected from the provinces Gansu, Shannxi, Shanxi, and Hubei of China were researched, using internal transcribed spacer (ITS) sequence technology. The confirmation of additive nucleotides based on the ITS sequencing of polymerase chain reaction (PCR) mixture was optimized and used to study the hybrid of Codonopsis cultivars. The results showed that when the ratio of PCR mixture increased up to 15 percent, the presence of a double peak in the sequencing electrophoresis map could be confirmed, suggesting the existence of additive nucleotides. According to the method above, 46 samples of Codonopsis cultivars collected during 2016 and 2017 were studied and compared with the samples collected from the year 2009 to 2010. All of the samples collected during 2016 and 2017 were hybridized and no genetic pure lines were found. In addition, the sites of variable base reduced greatly, concentrating at positions 122 and/or 226. These phenomena suggested that the genetic diversity of Codonopsis cultivars declined and the germplasm resources gradually converged. More attention should be paid to the reasonable exploitation and genetic breeding of Codonopsis taxa.

Salvia castanea Hairy Roots are More Tolerant to Phosphate Deficiency than Salvia miltiorrhiza Hairy Roots Based on the Secondary Metabolism and Antioxidant Defenses.

Salvia miltiorrhiza is a well-known traditional Chinese herb which is used to treat heart disease. Salvia castanea is a substitute product for S. miltiorrhiza in the medicinal field. Previous study has shown that phosphate (Pi) deficiency could promote the accumulation of secondary metabolism in herbs, and it has also developed a strategy for saving Pi resources and increasing the yield of active substances in herbs. In the present study, the hairy roots of S. miltiorrhiza and S. castanea were used to identify the Pi deficiency response mechanisms of these two Salvia species. The results showed that Pi deficiency increased the accumulation of specifically secondary metabolites, such as phenolic acids and tanshinones, which were caused by promoting the expression levels of key enzyme genes. In addition, Pi deficiency promoted the antioxidant activity in these two Salvia species. The data demonstrated that Pi deficiency increased the quality of the medicinal material in the plant. The hairy roots of S. castanea were more adaptive to Pi deficiency than those of S. miltiorrhiza in terms of biomass, secondary metabolism, and antioxidant activity. The results of this study provide insights into breeding herbs that are better adapted to Pi deficiency, which could increase the yield of active ingredients in herbs and save Pi resources.

Ag+ as a more effective elicitor for production of tanshinones than phenolic acids in Salvia miltiorrhiza hairy roots.

Phenolic acids and tanshinones are two groups of bioactive ingredients in Salvia miltiorrhiza Bunge. As a heavy metal elicitor, it has been reported that Ag+ can induce accumulations of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. In this study, the effects of Ag+ treatment on accumulations of six phenolic acids and four tanshinones in S. miltiorrhiza hairy roots were investigated. To further elucidate the molecular mechanism, expressions of key genes involved in the biosynthesis of these ingredients were also detected. The results showed that although the total phenolic acids content was almost not affected by Ag+, accumulations of rosmarinic acid (RA), caffeic acid and ferulic acid were significantly increased, while accumulations of salvianolic acid B (LAB), danshensu (DSU) and cinnamic acid were decreased. We speculate that LAB probably derived from the branch pathway of DSU biosynthesis. Contents of four tanshinones were enhanced by Ag+ and their accumulations were more sensitive to Ag+ than phenolic acids. Genes in the upstream biosynthetic pathways of these ingredients responded to Ag+ earlier than those in the downstream biosynthetic pathways. Ag+ probably induced the whole pathways, upregulated gene expressions from the upstream pathways to the downstream pathways, and finally resulted in the enhancement of ingredient production. Compared with phenolic acids, tanshinone production was more sensitive to Ag+ treatments. This study will help us understand how secondary metabolism in S. miltiorrhiza responds to elicitors and provide a reference for the improvement of the production of targeted compounds in the near future.