Purple Rehmannia: Investigation of the activation of R2R3-MYB transcription factors involved in anthocyanin biosynthesis.

Engineering anthocyanin biosynthesis in herbs could provide health-promoting foods for improving human health. Rehmannia glutinosa is a popular medicinal herb in Asia, and was a health food for the emperors of the Han Dynasty (59 B.C.). In this study, we revealed the differences in anthocyanin composition and content between three Rehmannia species. On the 250, 235 and 206 identified MYBs in the respective species, six could regulate anthocyanin biosynthesis by activating the ANTHOCYANIDIN SYNTHASE (ANS) gene expression. Permanent overexpression of the Rehmannia MYB genes in tobacco strongly promoted anthocyanin content and expression levels of NtANS and other genes. A red appearance of leaves and tuberous/roots was observed, and the total anthocyanin content and the cyanidin-3-O-glucoside content were significantly higher in the lines overexpressing RgMYB41, RgMYB42, and RgMYB43 from R. glutinosa, as well as RcMYB1 and RcMYB3 in R. chingii and RhMYB1 from R. henryi plants. Knocking out of RcMYB3 by CRISPR/Cas9 gene editing resulted in the discoloration of the R. chingii corolla lobes, and decreased the content of anthocyanin. R. glutinosa overexpressing RcMYB3 displayed a distinct purple color in the whole plants, and the antioxidant activity of the transgenic plants was significantly enhanced compared to WT. These results indicate that Rehmannia MYBs can be used to engineer anthocyanin biosynthesis in herbs to improve their additional value, such as increased antioxidant contents.

[Identification and biological characteristics of a pathogen causing leaf blight of Rehmannia glutinosa].

Leaf blight outbroke in Rehmannia glutinosa plantation in Wenxian county, Henan province in 2019. R. glutinosa plants with diseased leaves were collected from the plantation, and three strains were isolated from the diseased leaf samples. Pathogenicity test, morphological observation, and phylogenetic analysis of ITS, EF1-α, and Tub suggested that they were respectively Fusarium proliferatum, F. oxysporum, and F.acuminatum. Among them, F. acuminatum, as a pathogen of R. glutinosa leaf disease, had never been reported. To clarify the biological characteristics of F. acuminatum, this study tested the influence of light, pH, temperature, medium, carbon source, and nitrogen source on the mycelial growth rate of the pathogen during a 5-day culture period, and explored the lethal temperature. The results showed that the mycelia grew well under the photoperiod of 12 h light/12 h darkness, at 5-40 ℃(optimal temperature: 25 ℃), at pH 4-11(optimal pH: 7.0), on a variety of media(optimal medium: oatmeal agar), and in the presence of diverse carbon and nitrogen sources(optimal carbon source: soluble starch; optimal nitrogen source: sodium nitrate). The lethal temperature was verified to be 51 ℃(10 min). The conclusion is expected to lay a scientific basis for diagnosis and control of R. glutinosa leaf diseases caused by F. acuminatum.

[Effects of density on growth and gene transcription characteristics of Rehmannia glutinosa].

The present study analyzed the effects of planting density on the development, quality, and gene transcription characte-ristics of Rehmannia glutinosa using 85-5 and J9 as materials with three planting densities of 5 000, 25 000, and 50 000 plants/Mu(1 Mu≈667 m~2). The agronomic characteristics of leaves and tuberous roots, the content of catalpol and acteoside, and the changes of gene expression were determined. The results showed that the leaf size, the diameter of tuberous root, leaf biomass, tuberous root number, and tuberous root biomass per plant at low density were significantly higher than those of medium and high densities. The content of catalpol and acteoside in leaves was higher at high density. The content of catalpol in tuberous roots was higher at low density, and the change trend was similar to that in leaves, while the content of acteoside in tuberous roots was higher at high density. Transcriptome analysis found that about 1/2 of the expansin genes could change regularly in response to density treatment, which was rela-ted to the development of tuberous roots. The change trend of the gene expression of multiple catalytic enzymes involved in the biosynthesis of catalpol and acteoside was consistent with that of their content, which was presumedly involved in the accumulation and regulation of density-responsive medicinal components. Based on the analysis of the development, medicinal components, and gene expression characteristics of R. glutinosa at different densities, this study is expected to provide an important basis for regulating the quality and yield of medicinal materials of R. glutinosa by managing the planting density.

[Cloning and expression analysis of a tyrosine decarboxylase gene from Rehmannia glutinosa].

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.

[Numerical analysis of morphological variation of germplasm resources of dioscorea].

OBJECTIVE: Botanical characters of germplasm resources of Dioscorea were observed and compared, which could to offer reference for its genetic improvement, germplasm resource identification and classification. METHOD: Based on field cultivation, twenty-four morphological traits of ninety-four Dioscorea germplasm resources were observed or determined. And the morphological differences among germplasm resources were compared by principal component analysis and cluster analysis. RESULT: There were ample morphological diversity in the twenty-four traits, in especially in leaf size and tuber characters of the ninety-four Dioscorea germplasm resources. The first seven principal components which accounted for 80. 957% of total variance were extracted from the principal component analysis. The ninety-four germplasm resources could be divided into four clusters, which belonging to Dioscorea opposite, D. persimili, D. fordii and D. alata respectively. CONCLUSION: There were large morphological variation among germplasm resources on Dioscorea. Identification of germplasm resources of Dioscorea should focus on leaf size and tuber characters.

[Effects of leaf removal on growth and physiological characteristics of Rehmannia glutinosa].

Effects of the leaf-clipped treatment on growth and physiological properties of Rehmannia glutinosa were studied. Result showed that with the increase of the cutting leaves degree, growth rates of shoots and roots were decreased, sugar contents declined, chlorophyll contents decreased and root activities also inhibited. Compared with the normal plant (CK), the root inhibit rates in T1, T2, T3 were 17.53%, 33.41%, 59.47%, respectively. Physiological indexes including chlorophyll contents, root activities and sugar contents also were impacted by the leaf-clipped treatment. The results indicate that to balance source-sink relationship is a very essential method for improving production of R. glutinosa.

[Dynamic change of phenolic acids in soils around rhizosphere of replanted Rehmannia glutinosa].

OBJECTIVE: To determine 5 phenolic acids in the soils around rhizosphere of Rehmannia glutinosa in the field under normal rotation and successive cropping. METHOD: Phenolic acids related to allelopathy effect in the soils around rhizosphere of R. glutinosa were determined by HPLC. RESULT: The growth of R. glutinosa under normal rotation was strong. During harvest, the dry weight of the root tube and the volume of the R. glutinosa under normal rotation were 6.02 and 7.71 times of the ones under successive cropping. CONCLUSION: The seeding stage and elongating stage are the crucial periods for the autotoxic effect of the R. glutinosa under continuous cropping. During these periods, the content of coumaric acid, 4-hydroxybenzoic acid, syringic acid, and ferulic acid of R. glutinosa under successive cropping are notably negative correlation with the growth of the leaf and root tuber of R. glutinosa under successive cropping. Among them, ferulic acid plays a major role in the restriction effect on R. glutinosa under successive cropping.

[Effects of continuous cropping obstacle on growth of Rehmannia glutinosa].

OBJECTIVE: To study the effects of continuous cropping obstacles on growth of Rehmannia glutinosa. METHOD: The growth indexes, activity of root ATPase, root activity and mineral nutritional absorption were determined. RESULT: Continuous copping decreased growth rate and declined the size of leaves. Activity of root ATPase and root activity were also inhibited. CONCLUSION: The deficiency of source capacity is an important factor to restrain the root development of R. glutinosa with continuous cropping, the decrease of root activity and ATPase activity as well as nutritional stress of potassium and nitrogen are the reasons for the effects of continuous cropping on the growth and development of R. glutinosa.

Transcriptome Analysis of Salicylic Acid Treatment in Rehmannia glutinosa Hairy Roots Using RNA-seq Technique for Identification of Genes Involved in Acteoside Biosynthesis.

Rehmannia glutinosa is a common bulk medicinal material that has been widely used in China due to its active ingredients. Acteoside, one of the ingredients, has antioxidant, antinephritic, anti-inflammatory, hepatoprotective, immunomodulatory, and neuroprotective effects, is usually selected as a quality-control component for R. glutinosa herb in the Chinese Pharmacopeia. The acteoside biosynthesis pathway in R. glutinosa has not yet been clearly established. Herein, we describe the establishment of a genetic transformation system for R. glutinosa mediated by Agrobacterium rhizogenes. We screened the optimal elicitors that markedly increased acteoside accumulation in R. glutinosa hairy roots. We found that acteoside accumulation dramatically increased with the addition of salicylic acid (SA); the optimal SA dose was 25 µmol/L for hairy roots. RNA-seq was applied to analyze the transcriptomic changes in hairy roots treated with SA for 24 h in comparison with an untreated control. A total of 3,716, 4,018, and 2,715 differentially expressed transcripts (DETs) were identified in 0 h-vs.-12 h, 0 h-vs.-24 h, and 12 h-vs.-24 h libraries, respectively. KEGG pathway-based analysis revealed that 127 DETs were enriched in "phenylpropanoid biosynthesis." Of 219 putative unigenes involved in acteoside biosynthesis, 54 were found to be up-regulated at at least one of the time points after SA treatment. Selected candidate genes were analyzed by quantitative real-time PCR (qRT-PCR) in hairy roots with SA, methyl jasmonate (MeJA), AgNO3 (Ag+), and putrescine (Put) treatment. All genes investigated were up-regulated by SA treatment, and most candidate genes were weakly increased by MeJA to some degree. Furthermore, transcription abundance of eight candidate genes in tuberous roots of the high-acteoside-content (HA) cultivar QH were higher than those of the low-acteoside-content (LA) cultivar Wen 85-5. These results will pave the way for understanding the molecular basis of acteoside biosynthesis in R. glutinosa, and can serve as a basis for future validation studies.