Yanqing Zhou

Naphthylacetic acid (NAA) was used to increase the tuberous root yield of Rehmannia glutinosa, but the differences between its NAA-treated and control tuberous roots (NT and CG) and the regulatory mechanism of NAA effect remain unclear. In order to investigate them, NTs and CGs were used as materials, and both yield-related indices were measured; the metabolomics and transcriptomics were used to capture differentially accumulated metabolites (DAM) and to validate them via mining differentially expressed genes (DEGs), respectively. The effects of NAA treatment: increased NT mass per plant by 21.14%, through increasing the number of roots and increasing the mean root diameter; increased catalpol content by 1.2234% (p < 0.05); up-regulated 11DAMs and 596DEGs; and down-regulated 18 DAMs and 517DEGs. In particular, we discovered that NAA regulated its DAMs and biomass via 10 common metabolic pathways, and that the number of NAA-down-regulated DAMs was more than that of NAA-up-regulated DAMs in its tuberous root. Furthermore, HPLC validated the changes of several DAMs and 15 DEGs (4CL, ARF, CCoAOMT, ARGOS, etc.) associated with the yield increase and DAMs were verified by RT-qPCR. This study provided some valuable resources, such as tuberous root indices, key genes, and DAMs of Rehmannia glutinosa in response to NAA for distinguishing the CGs from NTs, and novel insights into the regulatory mechanism of NAA effects on both at the transcriptomic and metabolomic levels, so it will lay a theoretical foundation for NAA-regulated plant yield and quality, and provide references for prohibiting the uses of NAA as a swelling agent in medicinal tuber plants in China.

Rehmannia glutinosa has important medicinal value; terpenoid is one of the main active components in R. glutinosa. In this study, iTRAQ technique was used to analyze the relative abundance of proteins in roots of R. glutinosa, and 6,752 reliable proteins were quantified. GO enrichment results indicated that most proteins were involved in metabolic process or cellular process, 57.63% proteins had catalytic activity, and 65.80% proteins were enriched in membrane-bounded organelle. In roots of R. glutinosa, there were 38 KEGG enrichments with significance, more DEPs were found in some pathways, especially the proteasome pathway and TCA cycle with 15.0% DEPs between elongation stage and expansion stage of roots. Furthermore, five KEGG pathways of terpenoid synthesis were found. Most prenyltransferases belong to FPP/GGPP synthase family, involved in terpenoid backbone biosynthesis, and all interacted with biotin carboxylase CAC2. Compared with that at the elongation stage, many prenyltransferases exhibited higher expression at the expansion stage or maturation stage of roots. In addition, eight FPP/GGPP synthase encoding genes were cloned from R. glutinosa, namely FPPS, FPPS1, GGPS, GGPS3, GGPS4, GGPS5, GPPS and GPPS2, introns were also found in FPPS, FPPS1, GGPS5 and GGPS2, and FPP/GPP synthases were more conservative in organisms, especially in viridiplantae, in which the co-occurrence of GPPS or GPPS2 was significantly higher in plants. Further analysis found that FPP/GGPP synthases of R. glutinosa were divided into three kinds, GGPS, GPPS and FPPS, and their gene expression was significantly diverse in different varieties, growth periods, or tissues of R. glutinosa. Compared with that of GGPS, the expression of GPPS and FPPS was much higher in R. glutinosa, especially at the expansion stage and maturation stage. Thus, the synthesis of terpenoids in roots of R. glutinosa is intricately regulated and needs to be further studied.

Rehmannia glutinosa is an important medicinal plant producing many bioactive compounds such as catalpol, acteoside and so on. Tyrosine aminotransferase (TAT) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived branch pathway of acteoside biosynthesis. To confirm its role for acteoside accumulation, we isolated a full-length cDNA from Rehmannia glutinosa Libosch. Sequence analysis indicated that it contained a 1266 bp open reading frame, encoding a TAT of 421 amino acid residues. Multiple sequence alignment revealed that the homology of RgTAT amino acid sequence to that of Sesamum indicum(XP_011100354.1) was the highest (89.94%). Evolutionary tree showed that Sesamum indicum TAT and RgTAT were grouped together. Quantitative real-time PCR analysis indicated that the expression of RgTAT in leaves was much higher than in roots and stems,and that the expression levels of RgTAT in the tuberous roots, stems and leaves of high-acteoside cultivar BJ-3 were higher than in that of low-acteoside cultivar Wen85-5. A plant expression vector was constructed containing the RgTAT and hygromycin resistance gene (Hyg). Transgenic Rehmannia glutinosa Libosch overexpressing RgTAT was obtained via an Agrobacterium tumefaciens-mediated transformation system, in which Hyg expression was confirmed by PCR. RgTAT expression in transgenic plantlets measured by real-time quantitative PCR was 7.72 ± 0.17 times greater than its expression in the untransformed plantlets. Moreover, HPLC analysis indicated that enhanced RgTAT expression corresponded to significantly increased acteoside for transgenic plantlets. Our results elucidate the role of RgTAT in the acteoside biosynthesis in Rehmannia glutinosa.

Differential metabolites between tuberous roots from cultivated variety (ZP) and wild variety (YS) of Rehmannia glutinosa were analyzed by widely targeted metabolomics, and annotated to KEGG pathways. 228 secondary metabolites (SM) in ZP and YS were detected, of which 58 were differential metabolites (DM), including 41 flavonoids, 10 phenolic acids, 3 terpenoids, 2 alkaloids and 2 others, and 170 were unchanged; Among 58 DMs, 44 (75.9%) were up-regulated in YS, of which 30 were unique to YS, while 14 (24.1%) were down-regulated in YS, of which 10 were unique to ZP; Among flavonoids, 33 (80.5%) were more highly expressed in YS than in ZP; Among phenolic acids, 7 (70%) were more highly expressed in YS than in ZP; 12 of 58 DMs were annotated into 17 types of KEGG pathways. Among them, benzoic acid and p-Coumaryl alcohol were up-regulated in YS, and annotated into 10 pathways (58.8%) and 4 pathways (23.5%), respectively. In addition, much of DMs possess various pharmacological effects. These results indicated better quality of YS than ZP and the necessity of YS domestication. Taken together, this study will provide a reference for the scientific introduction, comprehensive development and utilization of wild Rehmannia glutinosa.

DNA methylation plays an important role in the growth and development of plant, and would change under different environments. In this study, 5-methyl cytosine (5mC) content and methylation level exhibited tissue specificity in genomic DNA of wheat seedling, and increased significantly in leaf along with the increase of water deficit, which was especially significant in leaf of wheat AK58. Full-methylation might dominate in genomic DNA of wheat seedling, the increase of full-methylation level under water deficit was significantly higher than that of hemi-methylation level. Under water deficit, DNA methylation of wheat seedling showed significant polymorphism, this polymorphism was always higher in root, especially was higher in root of wheat AK58. Further analysis appeared that changes of DNA methylation in wheat seedling took methylation as principle and demethylation as supplement under water deficit. Therefore, under water deficit, the degree, level and polymorphism of DNA methylation in wheat seedling showed tissue specificity and species specificity, and were higher in wheat AK58 compared with those of wheat XM13, perhaps wheat AK58 could more rapidly respond to water deficit by changes of DNA methylation, which would contribute to reveal molecular mechanism of wheat adapting to water deficit.

The quality and yield of wheat (Triticum aestivum L.) are dramatically affected by drought. We used morphological and physiological characteristics and degree of DNA methylation to compare the responses of two wheat cultivars under osmotic stress, and found that the two cultivars behaved differently. Root development, leaf growth, and the accumulation of proline and soluble carbohydrate in wheat cv. AK58 all showed drought tolerance. Drought tolerance of wheat cv. XM13 was mainly improved by accumulation of proline and soluble carbohydrate. The degree of DNA methylation in wheat showed tissue specificity and increased significantly in leaf tissue with increasing osmotic stress, but decreased significantly in root tissue under mild osmotic stress. In addition, changes of DNA methylation differed between two wheat cultivars under osmotic stress, and this change was especially significant in AK58. Therefore, wheat AK58 may have stronger self-adjustment ability under osmotic stress compared with XM13, and might respond more rapidly to osmotic stress through the change of DNA methylation. This finding could be significant for revealing drought-tolerance mechanisms of plants.

In this study, ITS, ITS2, matK, rbcL and psbA-trnH in Rehmannia were successfully amplified and sequenced, but some ITS sequences need to be proofread according to ITS2 sequences. Compared with rbcL, matK and psbA-trnH, ITS and ITS2 had higher mutation rate and more information sites, and ITS2 had higher interspecific diversity and lower intraspecific variation in Rehmannia, but the interspecific genetic variation of rbcL and matK was lower. Furthermore, the obvious barcoding gap was found in psbA-trnH or ITS2 + psbA-trnH, and the overlap between interspecific and intraspecific variation of ITS, ITS2 or matK was less. In addition, the phylogenetic tree based on ITS or ITS2 indicated that R. glutinosa, R. chingii or R. henryi with obvious monophyly could be successfully identified, but R. piasezkii and R. elata were clustered into one branch, R. solanifolia could not be distinguished from R. glutinosa, and R. chingii was closer to R. henryi. In phylogenetic tree based on psbA-trnH or ITS2 + psbA-trnH, cultivars and wild varieties of R. glutinosa could be distinguished, were clearly separated from other Rehmannia species, and cultivars or wild varieties of R. glutinosa could be also distinguished by matK. Taken together, ITS2 has great potential in systematic study and species identification of Rehmannia, the combination of ITS2 and psbA-trnH might be the most suitable DNA barcode for Rehmannia species.

Rehmannia glutinosa root contains many compounds with important medicinal properties and nutritional benefits, but only more than 140 compounds have been reported so far. Many other compounds and their accumulation and metabolic networks during its development remain unclear. In order to clarify them, its metabolic profiles at three different developmental stages were analyzed using untargeted LC-MS analysis. Multivariate analysis revealed that 434 metabolites differently accumulated in its different stages, suggesting different change trends. The metabolites having the same trend share common metabolic pathways, the metabolites showing increasing contents during its development have medical and nutritional values, and some mature root-specific metabolites may be better candidates for its quality control; 434 metabolites were mapped onto 111 KEGG pathways including 62 enzymes, whose increasing and decreasing patterns were shown during its development. Some metabolites complicatedly interacted with some enzymes and the top-10 pathways enriched from 111 KEGG pathways in network analysis. These findings extended the dataset of its identified compounds, and revealed that its development and quality were associated with the accumulation of different metabolites. Our work will lay the foundation for the better understanding of its chemical constituents, quality and developmental mechanism.

RghBNG, a gene of unknown function, was cloned from Rehmannia glutinosa by reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA of RghBNG was 548 bp with a282-bp open reading frame. It encoded a polypeptide of 93 amino acids with a predicted molecular weight of 10.5 kDa and a theoretical isoelectric point of 9.25. Bioinformatics analysis indicated that RghBNG had no homology to any known plant genes, whereas the RghBNG polypeptide was highly similar to other plant proteins and possessed one conserved B12D protein family functional domain. Phylogenetic analysis revealed that RghBNG encoded for a dicot protein. RghBNG spatial and temporal expression patterns and responses to abiotic stresses and plant growth regulators were investigated by qRT-PCR. RghBNG transcripts were detected in roots, stems, leaves, petals, receptacles, stamens and pistils with the highest and lowest levels respectively observed in petals and leaves of mature plants. Additionally, RghBNG transcripts were detected at three developmental stages of roots, stems and leaves; the highest levels were observed in roots at seedling stage; Transcript levels changed to varying degrees in different tissues and stages; We also studied the effects of abiotic stress and plant growth regulators in roots and leaves. RghBNG expression was significantly increased (p < 0.01) by chromium, gibberellic acid and NaCl, with the highest levels induced by chromium stress; In contrast, 6-benzyladenine reduced expression. These results strongly suggest that RghBNG is involved in R. glutinosa growth, development and response to plant growth regulators and abiotic stresses.

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time.