A Transcriptomic and Metabolomic Study on the Biosynthesis of Iridoids in Phlomoides rotata from the Qinghai-Tibet Plateau.

Phlomoides rotata is a traditional Chinese herbal medicine that grows in the Qinghai-Tibet Plateau region at a 3100-5000 m altitude. Iridoid compounds are the main active compounds of the P. rotata used as medical ingredients and display anti-inflammatory, analgesic, and hepatoprotective properties. To better understand the biological mechanisms of iridoid compounds in this species, we performed a comprehensive analysis of the transcriptome and metabolome of P. rotata leaves from four different regions (3540-4270 m). Global metabolome profiling detected 575 metabolites, and 455 differentially accumulated metabolites (DAMs) were detected in P. rotata leaves from the four regions. Eight major DAMs related to iridoid metabolism in P. rotata leaves were investigated: shanzhiside methyl ester, 8-epideoxyloganic acid, barlerin, shanzhiside, geniposide, agnuside, feretoside, and catalpin. In addition, five soil physical and chemical indicators in P. rotata rhizosphere soils were analyzed. Four significant positive correlations were observed between alkaline nitrogen and geniposide, exchangeable calcium and geniposide, available potassium and shanzhiside, and available phosphorus and shanzhiside methyl ester. The transcriptome data showed 12 P. rotata cDNA libraries with 74.46 Gb of clean data, which formed 29,833 unigenes. Moreover, 78.91% of the unigenes were annotated using the eight public databases. Forty-one candidate genes representing 23 enzymes involved in the biosynthesis of iridoid compounds were identified in P. rotata leaves. Moreover, the DXS1, IDI1, 8-HGO1, and G10H2 genes associated with iridoid biosynthesis were specifically expressed in P. rotata. The integration of transcriptome and metabolome analyses highlights the crucial role of soil physical and chemical indicators and major gene expression related to iridoid metabolism pathways in P. rotata from different areas. Our findings provide a theoretical foundation for exploring the molecular mechanisms underlying iridoid compound accumulation in P. rotata.

Metabolomic and transcriptomic reveal flavonoid biosynthesis and regulation mechanism in Phlomoides rotata from different habitats.

Phlomoides rotata is a traditional medical plant at 3100-5200 m altitude in the Tibet Plateau. In this study, flavonoid metabolites were investigated in P. rotata from Henan County (HN), Guoluo County (GL), Yushu County (YS), and Chengduo County (CD) habitats in Qinghai. The level of kaempferol 3-neohesperidoside, sakuranetin, and biochanin A was high in HN. The content of limocitrin and isoquercetin was high in YS. The levels of ikarisoside A and chrysosplenol D in GL were high. Schaftoside, miquelianin, malvidin chloride, and glabrene in CD exhibited high levels. The results showed a significant correlation between 59 flavonoids and 29 DEGs. Eleven flavonoids increased with altitude. PAL2, UFGT6, COMT1, HCT2, 4CL4, and HCT3 genes were crucial in regulating flavonoid biosynthesis. Three enzymes CHS, 4CL, and UFGT, were crucial in regulating flavonoid biosynthesis. This study provided biological and chemical evidence for the different uses of various regional plants of P. rotata.

Flavonoid synthesis in Lamiophlomis rotata from Qinghai-Tibet Plateau is influenced by soil properties, microbial community, and gene expression.

Lamiophlomis rotata is a medicinal plant in Qinghai-Tibet Plateau, in which flavonoid compounds are the major medicinal components. However, it remains unclear how flavonoid metabolism of L. rotata is influenced by soil properties and microbial community. In this study, we collected L. rotata seedlings and rhizosphere soils from five habitats ranging from 3750 to 4270 m of altitude and analyzed the effects of habitat conditions on flavonoid metabolism. The activities of peroxidase, cellulase, and urease were increased with altitude, while those of alkaline phosphatase, alkaline protease, and sucrase were decreased with altitude. Analysis of OTUs showed that the total number of bacterial genera was higher than that of fungal genera. The highest number of fungal genera was 132, and that of bacterial genera was 33 in Batang (BT) town in Yushu County at an altitude of 3880 m, suggesting that the fungal communities may play a critical role in L. rotata rhizosphere soils. Flavonoids in leaves and roots of L. rotata shared a similar pattern, with a trend of increasing levels with altitude. The highest flavonoid content measured, 12.94 mg/g in leaves and 11.43 mg/g in roots, was from Zaduo (ZD) County at an altitude of 4208 m. Soil peroxidases affected quercetin content in leaves of L. rotata, while the fungus Sebacina affected flavonoid content in leaves and roots of L. rotata. The expression of PAL, F3'H, FLS, and FNS genes showed a declining trend in leaves with altitude, while F3H showed an increasing trend in both leaves and roots. Overall, soil physicochemical properties and microbial community affect flavonoid metabolism in L. rotata in Qinghai-Tibet Plateau. The variations in flavonoid content and gene expression as well as their associations with soil factors revealed the complexity of the growth conditions and genetic makeup in L. rotata habitats of Qinghai-Tibet Plateau.

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata.

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai-Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

Analysis of flavonoid metabolism during fruit development of Lycium chinense.

Lycium chinense is an important medicinal plant in the northwest of China. Flavonoids are the major pharmacological components of L. chinense fruits. However, flavonoid metabolism during fruit development of L. chinense remains to be studied. Here, we analyzed the change of flavonoid contents, enzyme activity, and gene expression during fruit development of L. chinense. We found that flavonoids, anthocyanins, and catechins are the most important components of L. chinense fruits. Flavonoid content was increased with fruit development and was high at the late developmental stage. PAL, CHS, and F3H enzymes played a significant role in flavonoid accumulation in fruits. Transcriptomic analysis showed that anthocyanin pathway, flavonol pathway, flavonoid biosynthesis, and phenylpropanoid synthesis pathway were the major pathways involved in flavonoid metabolism in L. chinense. Gene expression analysis indicated that PAL1 and CHS2 genes were critical for flavonoid metabolism in L. chinense fruits. These discoveries help us understand the dynamic changes in flavonoids during fruit development and enhance the use of L. chinense fruits.