Dynamic Changes in Metabolite Accumulation and the Transcriptome during Leaf Growth and Development in Eucommia ulmoides.

Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome.

Identification of the Sex-Biased Gene Expression and Putative Sex-Associated Genes in Eucommia ulmoides Oliver Using Comparative Transcriptome Analyses.

Eucommia ulmoides is a model representative of the dioecious plants with sex differentiation at initiation. Nevertheless, the genetic mechanisms of sexual dimorphism and sex determination in E. ulmoides remain poorly understood. In this study de novo transcriptome sequencing on Illumina platform generated >45 billion high-quality bases from fresh leaves of six male and female individuals of E. ulmoides. A total of 148,595 unigenes with an average length of 801 base-pairs (bp) were assembled. Through comparative transcriptome analyses, 116 differentially expressed genes (DEGs) between the males and the females were detected, including 73 male-biased genes and 43 female-biased genes. Of these DEGs, three female-biased genes were annotated to be related with the sexually dimorphic gutta content in E. ulmoides. One male-biased DEG was identified as putative MADS box gene APETALA3, a B class floral organ identity gene in the flowering plants. SNPs calling analyses further confirmed that the APETALA3-like gene was probably involved in the sex determination in E. ulmoides. Four other male-biased DEGs were potential sex-associated genes as well with segregated SNPs in accord with sex type. In addition, the SNPs density was 1.02 per kilobase (kb) in the expressed genes of E. ulmoides, implying a relatively high genetic diversity.

Genome-Wide Identification of Mitogen-activated Protein Kinase Cascade Genes and Transcriptional Profiling Analysis during Organ Development in Eucommia ulmoides.

The mitogen-activated protein kinase (MAPK) cascades, which play crucial roles in plant development processes, are universal modules of signal transduction in eukaryotes and consist of a core module of three sequentially phosphorylated kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). This is the first report on the identification and analysis of MAPK cascades in Eucommia ulmoides. We conducted a genome-wide screening and identified 13 EuMAPKs, five EuMAPKKs, and 57 EuMAPKKKs. The construction of phylogenetic trees revealed that EuMAPKs and EuMAPKKs were divided into four groups (A, B, C, and D), and EuMAPKKKs were divided into three subfamilies (MEKK, RAF, and ZIK). These subfamilies were further confirmed by conserved domain/motif analysis and gene structure analysis. Based on the expression profiles of all identified EuMAPK cascades in various organs at different developmental stages, three genes (EuRAF22-2, EuRAF34-1, and EuRAF33-2) with stable expression patterns at all stages of fruit or leaf development, three genes (EuRAF2-3, EuMPK11, and EuMEKK21) with differential expression patterns, and two highly expressed genes (EuZIK1 and EuMKK2) were screened and validated by qRT-PCR. Overall, our results could be used for further research on the precise role of MAPK cascades during organ development in E. ulmoides.

Quantitative trait locus analysis of leaf morphological characters, yield-related traits, and secondary metabolite contents in Eucommia ulmoides.

Eucommia ulmoides is cultivated for the production of eucommia rubber and Chinese herbal drugs. Molecular breeding methods, such as marker-assisted selection (MAS), have the potential to improve the efficiency of E. ulmoides breeding. Quantitative trait locus (QTL) analysis was applied to identify marker-trait associations for E. ulmoides using an F1 mapping population of 152 individuals derived from a cross between the wild genotype Xiaoye and the cultivar Qinzhong No. 1. A total of 78 QTLs were identified for 12 leaf traits involving morphology, yield, and secondary metabolites. Phenotypic variance explained by individual QTLs ranged from 10.4 to 53.3%. Fifteen QTL clusters, each harboring loci controlling at least two leaf traits, were detected across nine linkage groups. Co-location of these QTLs may be due to pleiotropy or linkage. Three main QTL regions for secondary metabolites were consistently identified each year. QTL information from this study furthers our understanding of the genetic architecture of these economically important traits and of MAS in E. ulmoides breeding.

[Study on ecological suitability regionalization of Eucommia ulmoides in Guizhou].

OBJECTIVE: To study the ecological suitability regionalization of Eucommia ulmoides, for selecting artificial planting base and high-quality industrial raw material purchase area of the herb in Guizhou. METHODS: Based on the investigation of 14 Eucommia ulmoides producing areas, pinoresinol diglucoside content and ecological factors were obtained. Using spatial analysis method to carry on ecological suitability regionalization. Meanwhile, combining pinoresinol diglucoside content, the correlation of major active components and environmental factors were analyzed by statistical analysis. RESULTS: The most suitability planting area of Eucommia ulmoides was the northwest of Guizhou. The distribution of Eucommia ulmoides was mainly affected by the type and pH value of soil, and monthly precipitation. The spatial structure of major active components in Eucommia ulmoides were randomly distributed in global space, but had only one aggregation point which had a high positive correlation in local space. The major active components of Eucommia ulmoides had no correlation with altitude, longitude or latitude. CONCLUSION: Using the spatial analysis method and statistical analysis method, based on environmental factor and pinoresinol diglucoside content, the ecological suitability regionalization of Eucommia ulmoides can provide reference for the selection of suitable planting area, artificial planting base and directing production layout.

A molecular genetic linkage map of Eucommia ulmoides and quantitative trait loci (QTL) analysis for growth traits.

Eucommia ulmoides is an economically important tree species for both herbal medicine and organic chemical industry. Effort to breed varieties with improved yield and quality is limited by the lack of knowledge on the genetic basis of the traits. A genetic linkage map of E. ulmoides was constructed from a full-sib family using sequence-related amplified polymorphism, amplified fragment length polymorphism, inter-simple sequence repeat and simple sequence repeat markers. In total, 706 markers were mapped in 25 linkage groups covering 2133 cM. The genetic linkage map covered approximately 89% of the estimated E. ulmoides genome with an average of 3.1 cM between adjacent markers. The present genetic linkage map was used to identify quantitative trait loci (QTL) affecting growth-related traits. Eighteen QTLs were found to explain 12.4%-33.3% of the phenotypic variance. This genetic linkage map provides a tool for marker-assisted selection and for studies of genome in E. ulmoides.

The Ca2+ -dependent DNases are involved in secondary xylem development in Eucommia ulmoides.

Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca(2+) -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca(2+) -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca(2+) -dependent DNases are involved in secondary xylem development.

[Study on the quality standards of decoction pieces of salt Eucommia].

OBJECTIVE: To establish the quality criteria of decoction pieces of salt Eucommia. METHODS: Decoction pieces of salt Eucommia were measured with moisture, total ash and alcohol-extract, and were analyzed by TLC and HPLC for research. RESULTS: We obtained 13 batches of decoction pieces of salt Eucommia moisture, total ash and alcohol-extract measurements; Meanwhile we set the TLC and HPLC method. CONCLUSION: By studying the development of the decoction pieces of salt Eucommia, we established the quality control standards.

[Hairy root culture optimization and aucubin medical composition production of Eucommia ulmoides].

OBJECTIVE: To introduce the hairy root of Eucommia ulmoides, and screen put the excellent roots. METHOD: Hairy roots were obtained by infecting the different explants of E. ulmoides. Aucubin content was detected by HPLC. RESULT: The best transformation condition was embryonic axis infected by twenty minutes bacillus water dip-dye and three-days co-culture. Liquid culture was better than solid culture, and advantageous to accumulating of biomass and medical contents of hairy root. The E1 was the excellent roots. CONCLUSION: Hairy root of E. ulmoides will be useful for the production of active components in aucubin.

[Dynamics of soil water under Eucommia ulmodes plantation in hilly red soil region of southern China].

By using time domain reflectometry (TDR), a fixed-position monitoring research on the dynamics of soil water under Eucommia ulmoides plantation was conducted in a hilly red soil region of southern China. The results showed that there was a significant difference in the soil water storage among different months, and the dynamics of soil water could be divided into the phases of reduction, increase, and more reduction. Soil water varied significantly in its vertical distribution, and the variation pattern also differed with seasons. The vertical distribution of soil water could be divided into two phases, i.e., accumulation and depletion based on the seasonal variation of soil water, or rainy season and dry season based on the monthly variation of rainfall. Soil water was correlated significantly (P < 0.05) with relative humidity (RH), air temperature (t), vapor pressure deficit (VPD) and rainfall (R), and regulated by these meteorological factors synthetically, among which, rainfall was the most important factor, followed by air temperature. The soil water loss rate after rain had a significant hyperbolic relationship with durative droughty days, while soil water storage had a significant linear negative relationship with this duration (P < 0.05). With the extension of drought duration after rain, soil water loss tended to vary gently with increasing soil depth.

[Effects of growth regulator, full exposure and mist spray on the cutting of Eucommia ulmoides].

OBJECTIVE: To study the effects of growth regulator on the cutting method of Eucommia ulmoides seedlings. METHODS: Treated with H2O (CK), NAA, 6-BA and NAA + 6-BA for 4 hours, respectively, cuttings of 4-year and 20-year trees were cultured for 45-60 days under the shed or full exposure and mist spray. The growth of the cuttings was observed periodically. The number of the roots, the root length, the callus and the death rate were investigated after harvest. RESULTS: Compared with the cutting from 4-year-old tree, the cutting from 20-year-old tree developed better. The rate of stroking root in treated group was 14 times as much as CK group. Other agronomic traits such as the number of the roots, the root length, the callus, the number of the leaves and the death rate of the cuttings in treated group were all better than that of the cuttings in CK group. The cultural time in treated group reduced by 15 days than that of CK group. CONCLUSIONS: The present study shows that both full exposure mist spray and growth regulators have remarkable effects on cuttings of tree with different ages for rooting or forming callus.