A Rehmannia glutinosa cinnamate 4-hydroxylase promotes phenolic accumulation and enhances tolerance to oxidative stress.

KEY MESSAGE: RgC4H promotes phenolic accumulation in R. glutinosa, activating the molecular networks of its antioxidant systems, and enhancing the tolerance to oxidative stresses exposed to drought, salinity and H2O2 conditions. Rehmannia glutinosa is of great economic importance in China and increasing R. glutinosa productivity relies, in part, on understanding its tolerance to oxidative stress. Oxidative stress is a key influencing factor for crop productivity in plants exposed to harsh conditions. In the defense mechanisms of plants against stress, phenolics serve an important antioxidant function. Cinnamate 4-hydroxylase (C4H) is the first hydroxylase in the plant phenolics biosynthesis pathway, and elucidating the molecular characteristics of this gene in R. glutinosa is essential for understanding the effect of tolerance to oxidative stress tolerance on improving yield. Using in vitro and in silico methods, a C4H gene, RgC4H, from R. glutinosa was isolated and characterized. RgC4H has 86.34-93.89% amino acid sequence identity with the equivalent protein in other plants and localized to the endoplasmic reticulum. An association between the RgC4H expression and total phenolics content observed in non-transgenic and transgenic R. glutinosa plants suggests that this gene is involved in the process of phenolics biosynthesis. Furthermore, the tolerance of R. glutinosa to drought, salinity and H2O2 stresses was positively or negatively altered in plants with the overexpression or knockdown of RgC4H, respectively, as indicated by the analysis in some antioxidant physiological and molecular indices. Our study highlights the important role of RgC4H in the phenolics/phenylpropanoid pathway and reveals the involvement of phenolic-mediated regulation in oxidative stress tolerance in R. glutinosa.

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.

Transcriptome-wide identification of the genes responding to replanting disease in Rehmannia glutinosa L. roots.

The development of the medicinal plant Rehmannia glutinosa L. are severely declined when are replanted on the soil of the preceding crops being themselves. The biological basis of this so called "replanting disease" is unknown. Here, we have exploited the parallel sequencing capacity of both RNA-seq and DGE technology to ascertain what genes are responsive to the replanting disease in roots of R. glutinosa. RNA-seq analysis generated 99,708 non-redundant consensus sequences from the roots of the first year (R1) and the second year (R2) replanted R. glutinosa plants. From this set, a total of 48,616 transcripts containing a complete or partial encoding region was identified. Based on this resource, two DGE tag libraries were established to capture the transcriptome differences between the R1 and R2 libraries. Finally, a set of 2,817 (1,676 up- and 1,141 down-regulated) differentially transcribed genes was screened, and 114 most strongly differentially transcribed genes were identified by DGE analysis between first year and replanted plants. Furthermore, a more detailed examination of 16 selected candidates was carried out by qRT-PCR. The indication was that replanting could promote Ca(2+) signal transduction and ethylene synthesis, resulting in forming of the replanting disease. We analyzed the biomass indexes of replanted R. glutinosa roots by irrigating Ca(2+) signal blockers. The results suggested that the alleviation of the disease impairment could be the decrease of Ca(2+) signal transduction. This study provided a global survey of the root transcriptome in replanted R. glutinosa roots at the tuberous root expansion stage. As a result, a number of candidate genes underlying the replanting disease have been identified.

[Effect of continuous cropping of Rehmannia on its morphological and physiological characteristics].

OBJECTIVE: In contrast to the newly-planted plants, through measuring and analyzing the chlorophyll content, photosynthetic characteristics, root activity and enzyme activity of Rehmannia glutinosa in growth stages, the differentiation manifestation of R. glutinosa physiological activity mediated by continuous cropping was studied. METHODS: SPAD-502 chlorophyll meter was used to measure chlorophyll content and LI-6400 portable photosynthetic apparatus to determine plant photosynthetic characteristics. Plant root vigor and enzyme system were measured following reference literature. RESULTS: The problems of Rehmannia caused by continuous cropping had happened since the early stage of its growth period, and lasted throughout the whole growth period. Under the condition of continuous cropping, the chlorophyll content, photosynthetic characteristics and root activity remained at a lower level compared with the newly-planted plants, among which, the chlorophyll content and the root activity (100 days after planting) had significant differences. CONCLUSION: The insufficient photosynthesis source and the reducing of the storage capacity (root tuber) under the condition of continuous cropping might be the main reasons for these problems of R. glutinosa.

[Molecular cloning and expression analysis of an Aux/IAA gene (RgIAA1) from Rehmannia glutinosa].

To clone and analyze a member of the Auxin/indole-3-acetic acid (Aux/IAA) gene family, RgIAA1, from Rehmannia glutinosa. The transcriptional EST database of R. glutinosa was used to clone the new Aux/IAA gene by cDNA probe of AtIAA14. Bioinformatics was applied to analyze the sequence characteristics of RgIAA1 protein and construct phylogenetiC trees. Quantitative RT-PCR has been applied to detect the transcription level of RgIAA1 in seven tissues as well as in leaves under three stresses. The results showed that, the cDNA sequence of RgIAA1 contains 903 bp was obtained. The open reading frame (ORF) of RgIAA1 was 681 bp encoding 226 amino acids, which has typical structural domains and characteristic sequence of Aux/IAA family proteins. RgIAA1 showed the highest expression level in unfolded leaf, followed by the stem. And the expression of RglAA1 was quickly decreased with leaf growing up. The transcription level increased under continuous cropping conditions while it reduced both in salinity and waterlogging stresses. RgIAA1, an Aux/IAA gene from R. glutinosa has been obtained for the first time, which can lay the foundation for further studies about its molecular function in development and responses to stress.

[Responses of physiological ecology and quality evaluation of Rehmannia gltinosa in continuous cropping].

OBJECTIVE: To study responses of physiological ecology and quality evaluation of Rehmannia glutinosa in continuous cropping. METHOD: The potted plant R. glutinosa which consists of first cropping, 1 year continuous cropping and 2 year continuous cropping were used as experimental materials. The photosynthetic activity, descending axis vitality, the protective enzymes system and MDA content were measured, the quality was evaluated by FTIR and HPLC. RESULT: Continuous cropping reduced the content of chlorophyll in the non-first cropping R. glutinos, the photosynthetic activity and descending axis vitality were weakened. Because of the increase of the free radical in the R. glutinos due to the continuous cropping, the activity of protective enzymes including POD, SOD and CAT were enhanced and MDA content were increased, more importantly the medical potency declined . And along with the increasing years of the continuous cropping, this effect becomes even stronger. CONCLUSION: Continuous cropping affects the descending axis ability of absorbing water and nutrition and photosynthesis are inhibited R. glutinosa, at the same time, it also causes the disorders of antioxidation systems in R. glutinos, resulting in continuous cropping obstacle and decline of the medicinal materials quality.

[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.

Rehmannia glutinosa: review of botany, chemistry and pharmacology.

Rehmannia glutinosa, a widely used traditional Chinese herb, belongs to the family of Scrophulariaceae, and is taken to nourish Yin and invigorate the kidney in traditional Chinese medicine (TCM) and has a very high medicinal value. In recent decades, a great number of chemical and pharmacological studies have been done on Rehmannia glutinosa. More than 70 compounds including iridoids, saccharides, amino acid, inorganic ions, as well as other trace elements have been found in the herb. Studies show that Rehmannia glutinosa and its active principles possess wide pharmacological actions on the blood system, immune system, endocrine system, cardiovascular system and the nervous system. Currently, the effective monomeric compounds or active parts have been screened for the pharmacological activity of Rehmannia glutinosa and the highest quality scientific data is delivered to support the further application and exploitation for new drug development.

[Hairy root induction and plant regeneration of Rehmannia glutinosa Libosch. f. hueichingensis (Chao et Schih) Hsiao transformed by Agrobacterium rhizogenes].

An efficient system of genetic transformation and plant regeneration was established in Rehmannia glutinosa Libosch. f. hueichingensis (Chao et Schih) Hsiao by infecting the segments of leaves, stems and petioles of young regenerated plantlets with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or on liquid 1/2 MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency of 46.7% was achieved by pre-treating the A. rhizogenes with 100 micromol/L acetosyringone at logarithmic phase (OD600 = 1.8). The calluses with 100% induction frequency were induced from hairy roots on 1/2 MS medium containing 0.2 mg/L KT and 3.0 mg/L 6-BA, from which the shoots with 51.49% differentiation frequency was produced. These shoots could take root at a percentage of 100% and develope into four transformed plantlets when transferred on 1/2 MS medium, which had differences in morphological characters such as dwarfing, shortened nodals and abundant literal branching roots, and which survived vigorously after transplantation. The content of catalpol in an transformed hairy root clone was 0.557 mg/g. FW by means of HPLC, 48.5% and 18% of that in fresh and dried Rehmannia root, respectively. PCR and Southern blot analyses confirmed that rolB gene (564 bp) of TL-DNA was inserted in the genome of transformed hairy roots and their regenerated plantlets. RT-PCR analysis and opine paper electrophoresis detection revealed that TR-DNA containing opine synthetase gene was integrated and expressed in the genome of transformed hairy roots and their regenerated plantlets.

[Studies on organ phytomorphology of leaves from Rehmannia glutinosa].

OBJECTIVE: To set up the fittest medium and optimum condition for leaves from Rehmannia glutinosa to form regenerated plantlets directly and to induce microtubers. METHOD: Leaves of different phases obtained from the virus-free plantlets were incubated in some kinds of media on the back side. RESULT AND CONCLUSION: The fittest medium for the formation of regenerated plantlet was MS + 6-BA 2.0 mg.L-1 and the fittest medium for the induction of microtuber was MS + 6-BA 1.0 mg.L-1 + NAA 0.5 mg.L-1. The optimum incubation temperaue was 25 degrees C and the light length was (2,000-3,000 lx) 12 h.d-1.