Fengqing Wang

Identification and Characterization of 40 Isolated Rehmannia glutinosa MYB Family Genes and Their Expression Profiles in Response to Shading and Continuous Cropping

The v-myb avian myeloblastosis viral oncogene homolog (MYB) superfamily constitutes one of the most abundant groups of transcription factors (TFs) described in plants. To date, little is known about the MYB genes in Rehmannia glutinosa. Forty unique MYB genes with full-length cDNA sequences were isolated. These 40 genes were grouped into five categories, one R1R2R3-MYB, four TRFL MYBs, four SMH MYBs, 25 R2R3-MYBs, and six MYB-related members. The MYB DNA-binding domain (DBD) sequence composition was conserved among proteins of the same subgroup. As expected, most of the closely related members in the phylogenetic tree exhibited common motifs. Additionally, the gene structure and motifs of the R. glutinosa MYB genes were analyzed. MYB gene expression was analyzed in the leaf and the tuberous root under two abiotic stress conditions. Expression profiles showed that most R. glutinosa MYB genes were expressed in the leaf and the tuberous root, suggesting that MYB genes are involved in various physiological and developmental processes in R. glutinosa. Seven MYB genes were up-regulated in response to shading in at least one tissue. Two MYB genes showed increased expression and 13 MYB genes showed decreased expression in the tuberous root under continuous cropping. This investigation is the first comprehensive study of the MYB gene family in R. glutinosa.

Differential proteomic analysis of replanted Rehmannia glutinosa roots by iTRAQ reveals molecular mechanisms for formation of replant disease

BackgroundThe normal growth of Rehmannia glutinosa, a widely used medicinal plant in China, is severely disturbed by replant disease. The formation of replant disease commonly involves interactions among plants, allelochemicals and microbes; however, these relationships remain largely unclear. As a result, no effective measures are currently available to treat replant disease.ResultsIn this study, an integrated R. glutinosa transcriptome was constructed, from which an R. glutinosa protein library was obtained. iTRAQ technology was then used to investigate changes in the proteins in replanted R. glutinosa roots, and the proteins that were expressed in response to replant disease were identified. An integrated R. glutinosa transcriptome from different developmental stages of replanted and normal-growth R. glutinosa produced 65,659 transcripts, which were accurately translated into 47,818 proteins. Using this resource, a set of 189 proteins was found to be significantly differentially expressed between normal-growth and replanted R. glutinosa. Of the proteins that were significantly upregulated in replanted R. glutinosa, most were related to metabolism, immune responses, ROS generation, programmed cell death, ER stress, and lignin synthesis.ConclusionsBy integrating these key events and the results of previous studies on replant disease formation, a new picture of the damaging mechanisms that cause replant disease stress emerged. Replant disease altered the metabolic balance of R. glutinosa, activated immune defence systems, increased levels of ROS and antioxidant enzymes, and initiated the processes of cell death and senescence in replanted R. glutinosa. Additionally, lignin deposition in R. glutinosa roots that was caused by replanting significantly inhibited tuberous root formation. These key processes provide important insights into the underlying mechanisms leading to the formation of replant disease and also for the subsequent development of new control measures to improve production and quality of replanted plants.

Transcriptome analysis reveals metabolic alteration due to consecutive monoculture and abiotic stress stimuli in Rehamannia glutinosa Libosch

Key messageWe deeply investigated the mechanism underlying metabolic regulation in response to consecutive monoculture (replanting disease) and different abiotic stresses that unfolded the response mechanism to consecutive monoculture problem through RNA-seq analysis.AbstractThe consecutive monoculture problem (CMP) resulted of complex environmental stresses mediated by multiple factors. Previous studies have noted that multiple stress factors in consecutive monoculture soils or plants severely limited the interpretation of the critical molecular mechanism, and made a predict that the specifically responding factor was autotoxic allelochemicals. To identify the specifically responding genes, we compared transcriptome changes in roots of Rehamannia glutinosa Libosch using consecutive monoculture, salt, drought, and ferulic acid as stress factors. Comparing with normal growth, 2502, 2672, 2485, and 1956 genes were differentially expressed in R. glutinosa under consecutive monoculture practice, salt, drought, and ferulic acid stress, respectively. In addition, 510 genes were specifically expressed under consecutive monoculture, which were not present under the other stress conditions. Integrating the biological and enrichment analyses of the differentially expressed genes, the result demonstrated that the plants could alter enzyme genes expression to reconstruct the complicated metabolic pathways, which used to tolerate the CMP and abiotic stresses. Furthermore, most of the affected pathway genes were closely related to secondary metabolic processes, and the influence of consecutive monoculture practice on the transcriptome genes expression profile was very similar to the profile under salt stress and then to the profile under drought stress. The outlined schematic diagram unfolded the putative signal regulation mechanism in response to the CMP. Genes that differentially up- or down-regulated under consecutive monoculture practice may play important roles in the CMP or replanting disease in R. glutinosa.

Identification and expression analysis of Rehmannia glutinosa mediator complex genes in response to continuous cropping

The mediator complex, a conserved assembly of multi-functional proteins, is required for the transcription of almost all protein-encoding genes in eukaryotes. In this report, 36 mediator complex subunit transcripts in Rehmannia glutinosa were identified from a leaf and tuberous root EST database. Nine of these transcripts had integrated open-reading frames (ORFs). A comparison of predicted amino acid sequences with putative mediator complex subunit homologues from another six flowering plants revealed high identities and several conserved domains. Phylogenetic analysis of the nine mediator complex genes in seven flowering plants revealed a closer evolutionary relationship between R. glutinosa and Solanum lycopersicum. The relative abundances of the 36 transcripts were analysed in the leaves and tuberous roots of R. glutinosa. qRT-PCR analysis revealed that the transcripts of approximately one-third of the mediator complex genes investigated in this study were expressed at similar levels. This result suggested that these transcripts might contribute to the formation, maturation or functions of the analysed tissues. The effects of consecutive monoculture on the transcription of R. glutinosa mediator complex genes were also analysed by qRT-PCR. This analysis revealed that one-third of the mediator complex genes presented differential expression patterns in the leaves of second-year plants, whereas over half of the mediator genes were differentially expressed in the tuberous roots of second-year plants. These patterns suggest that one of the subunits of the mediator complex regulates the consecutive monoculture problem in 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.