A New Quinazolinone Alkaloid along with Known Compounds with Seed-Germination-Promoting Activity from Rhodiola tibetica Endophytic Fungus Penicillium sp. HJT-A-6.

A new quinazolinone alkaloid named peniquinazolinone A (1), as well as eleven known compounds, 2-(2-hydroxy-3-phenylpropionamido)-N-methylbenzamide (2), viridicatin (3), viridicatol (4), (±)-cyclopeptin (5a/5b), dehydrocyclopeptin (6), cyclopenin (7), cyclopenol (8), methyl-indole-3-carboxylate (9), 2,5-dihydroxyphenyl acetate (10), methyl m-hydroxyphenylacetate (11), and conidiogenone B (12), were isolated from the endophytic Penicillium sp. HJT-A-6. The chemical structures of all the compounds were elucidated by comprehensive spectroscopic analysis, including 1D and 2D NMR and HRESIMS. The absolute configuration at C-13 of peniquinazolinone A (1) was established by applying the modified Mosher's method. Compounds 2, 3, and 7 exhibited an optimal promoting effect on the seed germination of Rhodiola tibetica at a concentration of 0.01 mg/mL, while the optimal concentration for compounds 4 and 9 to promote Rhodiola tibetica seed germination was 0.001 mg/mL. Compound 12 showed optimal seed-germination-promoting activity at a concentration of 0.1 mg/mL. Compared with the positive drug 6-benzyladenine (6-BA), compounds 2, 3, 4, 7, 9, and 12 could extend the seed germination period of Rhodiola tibetica up to the 11th day.

Regulation by fungal endophyte of Rhodiola crenulata from enzyme genes to metabolites based on combination of transcriptome and metabolome.

BACKGROUND: The contents of some its crucial metabolites tend to decrease when Rhodiola crenulata is cultured at low altitude. Interestingly, it was found that an endophyte, Phialocephala fortinii, could alleviate this problem. RESULTS: There were 16 151 differential genes including 14 706 up-regulated and 1445 down-regulated unigenes with significant differences (P < 0.05), and a total of 1432 metabolites exhibited statistically significant (P < 0.05) metabolic differences comprising 27 different marker metabolites which showed highly significant values of VIP > 5 and P < 0.01. Results highlight differential regulation of 20 enzymatic genes that are involved in the biosynthesis of five different marker metabolites including acetaldehyde, homocysteine, cyclopropylamine, 1-pyrrolinium and halistanol sulfate. CONCLUSIONS: The positive physiological effect of P. fortinii on R. crenulata encompasses differential regulation in carbohydrate metabolism, lipid metabolism and secondary metabolite synthesis. © 2020 Society of Chemical Industry.

Fungal endophyte-induced salidroside and tyrosol biosynthesis combined with signal cross-talk and the mechanism of enzyme gene expression in Rhodiola crenulata.

Endophyte is a factor that affects the physiology and metabolism of plant. However, limited information is available on the mechanism of interaction between endophyte and plant. To investigate the effects of endophytic fungus ZPRs-R11, that is, Trimmatostroma sp., on salidroside and tyrosol accumulations in Rhodiola crenulata, signal transduction, enzyme gene expression, and metabolic pathway were investigated. Results showed that hydrogen peroxide (H2O2), nitric oxide (NO), and salicylic acid (SA) involved in fungus-induced salidroside and tyrosol accumulations. NO acted as an upstream signal of H2O2 and SA. No up- or down-stream relationship was observed, but mutual coordination existed between H2O2 and SA. Rate-limiting enzyme genes with the maximum expression activities were UDP-glucosyltransferase, tyrosine decarboxylase (TYDC), monoamine oxidase, phenylalanine ammonialyase (PAL), and cinnamic-4-hydroxylase sequentially. Nevertheless, the genes of tyrosine transaminase and pyruvate decarboxylase only indicated slightly higher activities than those in control. Thus, TYDC and PAL branches were the preferential pathways in ZPRs-R11-induced salidroside and tyrosol accumulation. Trimmatostroma sp. was a potential fungus for promoting salidroside and tyrosol accumulations. The present data also provided scientific basis for understanding complex interaction between endophytic fungus and R. crenulata.

Potential of the Endophytic Fungus Phialocephala fortinii Rac56 Found in Rhodiola Plants to Produce Salidroside and p-Tyrosol.

2-(4-Hydroxyphenyl)ehyl-ß-D-glucopyranoside (salidroside) and 4-(2-hydroxyethyl)phenol (p-tyrosol) are famous food and medicine additives originally derived from alpine Rhodiola plants. Salidroside or p-tyrosol production by the endophytic fungus Rac56 (Phialocephala fortinii) was confirmed by UPLC/Q-TOF-MS and ¹H-NMR. The fermentation conditions were optimized by orthogonal design using data processing system software. The broth fermentation results showed that salidroside and p-tyrosol extraction yields from Rac56 were stable and reached 1.729 ± 0.06 mg and 1.990 ± 0.05 mg per mL of culture medium, respectively. The optimal conditions for salidroside and p-tyrosol production in fermentation culture of Rac56 were determined to be 25 °C, pH values of 7 and 5, Czapek-Dox culture medium volumes of 150 mL and 50 mL in 250 mL flasks, rotation speeds of 100× g and 200× g, and fermentation durations of 7 and 15 days, respectively. Under these optimal conditions, stable yields of 2.339 ± 0.1093 mg and 2.002 ± 0.0009 mg per mL of culture medium of salidroside and p-tyrosol, respectively, were obtained, indicating that the P. fortinii Rac56 strain is a promising source of these compounds.

[Effect of endophytic fungus on expression of key enzyme genes in pathway of salidroside biosynthesis in Rhodiola crenulata].

One strain of endophytic fungus ZPRa-R-1 was obtained for the capacity of promoting production of salidroside in Rhodiola crenulata. To explain the mechanism of salidroside biosynthesis in host plant, eight housekeeping genes were evaluated, and the evaluation method was created for the expression activities of four key enzyme genes PAL (phenylalanine ammonia-lyase), TyDC (tyrosine decarboxylase), TAT (tyrosine transaminase), UDPGT (UDP-glucosyltransferase) referenced double reference genes in biosynthesis pathway of salidroside in R. crenulata. Stabilities of housekeeping genes were confirmed by real-time fluorescent quantitative PCR technology and three softwares including geNorm, NormFinder and BestKeeper, then relative expressions of key enzyme genes were analysized by the 2-ΔΔCt method. The results showed that the most stable gene was GAPDH, followed by PCS, and the most appropriate reference of internal genes were combination with two genes in R. crenulata inoculated with endophytic fungus ZPRa-R-1. Under symbiosis conditions, regularity changes of key enzyme genes affected by endophytic fungus ZPRa-R-1 were as follows: the relative expression activity of PAL attached to peak value, which was 4.9 times as that of control group when inoculated ten days. The relative expression of TyDC reached the maximum value, which was 2.8 times of that control after inoculating 12 days. The relative expression of UDPGT actually reach 17.1 times than that of control after inoculating 8 days. However, the relative expression of TAT was not affected by this fungus. The changes of four key enzyme genes are positively correlated with the changes of salidroside content in R. crenulata.

Diversity and antioxidant activity of culturable endophytic fungi from alpine plants of Rhodiola crenulata, R. angusta, and R. sachalinensis.

Rhodiola spp. are rare and endangered alpine plants widely used as medicines and food additives by many civilizations since ancient times. Their main effective ingredients (such as salidroside and p-tyrosol) are praised to exhibit pharmacologic effects on high-altitude sickness and possess anti-aging and other adaptogenic capacities based on their antioxidant properties. In this study, 347 endophytic fungi were isolated from R. crenulata, R. angusta, and R. sachalinensis, and the molecular diversity and antioxidant activities of these fungi were investigated for the first time. These fungi were categorized into 180 morphotypes based on cultural characteristics, and their rRNA gene ITS sequences were analyzed by BLAST search in the GenBank database. Except for 12 unidentified fungi (6.67%), all others were affiliated to at least 57 genera in 20 orders of four phyla, namely, Ascomycota (88.89%), Basidiomycota (2.78%), Zygomycota (1.11%), and Glomeromycota (0.56%), which exhibited high abundance and diversity. Antioxidant assay showed that the DPPH radical-scavenging rates of 114 isolates (63.33%) were >50%, and those of five isolates (Rct45, Rct63, Rct64, Rac76, and Rsc57) were >90%. The EC50 values of five antioxidant assays suggested significant potential of these fungi on scavenging DPPH•, O2-•, and OH• radicals, as well as scavenging nitrite and chelating Fe2+, which showed preference and selection between endophytic fungi and their hosts. Further research also provided the first evidence that Rac12 could produce salidrosides and p-tyrosol. Results suggested that versatile endophytic fungi associated with Rhodiola known as antioxidants could be exploited as potential sources of novel antioxidant products.

Tyrosinase inhibitory effect and antioxidative activities of fermented and ethanol extracts of Rhodiola rosea and Lonicera japonica.

This is the first study to investigate the biological activities of fermented extracts of Rhodiola rosea L. (Crassulaceae) and Lonicera japonica Thunb. (Caprifoliaceae). Alcaligenes piechaudii CC-ESB2 fermented and ethanol extracts of Rhodiola rosea and Lonicera japonica were prepared and the antioxidative activities of different concentrations of samples were evaluated using in vitro antioxidative assays. Tyrosinase inhibition was determined by using the dopachrome method with L-DOPA as substrate. The results demonstrated that inhibitory effects (ED50 values) on mushroom tyrosinase of fermented Rhodiola rosea, fermented Lonicera japonica, ethanol extract of Lonicera japonica, and ethanol extract of Rhodiola rosea were 0.78, 4.07, 6.93, and >10 mg/ml, respectively. The DPPH scavenging effects of fermented Rhodiola rosea (ED50 = 0.073 mg/ml) and fermented Lonicera japonica (ED50 = 0.207 mg/ml) were stronger than effects of their respective ethanol extracts. Furthermore, the scavenging effect increases with the presence of high content of total phenol. However, the superoxide scavenging effects of fermented Rhodiola rosea was less than effects of fermented Lonicera japonica. The results indicated that fermentation of Rhodiola rosea and Lonicera japonica can be considered as an effective biochemical process for application in food, drug, and cosmetics.

[Construction of expression vector driven by tobacco root-specific promoter and transformation into Rhodiola hairy root].

OBJECTIVE: To construct root-specific promoter of plant expression vector and transformate into Rhodiola hairy root. METHODS: The expression vector pCA-Tob7: UGTR, driven by tobacco root-specific promoter TobRB7, was constructed from pCAM-BIA1301 by substituting the CaMV 35S promoter and GUS gene with TobRB7 and UGTR(a glycosyltransferase gene), respectively. The pCA-Tob7: UGTR vector was introduced into Agrobacterium tumefaciens strain, and the hairy root of Rhodiola were then transformed. RESULTS: Some Kanamycin resistant plants were positive,which indicated that the expression vector had integrated into Rhodiola hairy root genome. CONCLUSION: Root-specific promoter of plant expression vector is constructed and the hairy root of Rhodiola are transformed for future research.