Eucommia ulmoides, also called hardy rubber tree, is an economically important tree; however, the lack of its genome sequence restricts the fundamental biological research and applied studies of this plant species. Here, we present a high-quality assembly of its ∼1.2-Gb genome (scaffold N50 = 1.88 Mb) with at least 26 723 predicted genes for E. ulmoides, the first sequenced genome of the order Garryales, which was obtained using an integrated strategy combining Illumina sequencing, PacBio sequencing, and BioNano mapping. As a sister taxon to lamiids and campanulids, E. ulmoides underwent an ancient genome triplication shared by core eudicots but no further whole-genome duplication in the last ∼125 million years. E. ulmoides exhibits high expression levels and/or gene number expansion for multiple genes involved in stress responses and the biosynthesis of secondary metabolites, which may account for its considerable environmental adaptability. In contrast to the rubber tree (Hevea brasiliensis), which produces cis-polyisoprene, E. ulmoides has evolved to synthesize long-chain trans-polyisoprene via farnesyl diphosphate synthases (FPSs). Moreover, FPS and rubber elongation factor/small rubber particle protein gene families were expanded independently from the H. brasiliensis lineage. These results provide new insights into the biology of E. ulmoides and the origin of polyisoprene biosynthesis.

4-Coumarate: coenzyme A ligase (4CL) is an important rate-limiting enzyme in phenylpropanoid metabolism that plays an important role in the biosynthesis of phenylpropanoid, flavonoid, lignin and other secondary metabolites in plants. However, the family members and functions have not been identified in Eucommia ulmoides (E. ulmoides). In this study, 35 Eu4CLs were identified in the E. ulmoides genome, and the gene structure, conserved domain, evolutionary relationship and expression pattern were comprehensively analyzed. The results show that 35 Eu4CLs were assembled into three subgroups according to the classification in Arabidopsis, where Eu4CLs in the same subgroup had similar gene structures and conserved protein motifs. Putative cis-element analysis of Eu4CL promoter regions uncovered numerous elements related to the response of stress and plant hormones. Expression patterns showed that Eu4CL4/5/13/34 expression levels were positively related to chlorogenic acid content in different periods, which indicate that the synthesis of chlorogenic acid in E. ulmoides was regulated by multiple genes, and the genes regulating the synthesis of chlorogenic acid in different tissues were different. In addition, nine selected Eu4CL genes showed different expression patterns under cold, WeJA (methyl jasmonate), and ethylene by quantitative reverse transcription-PCR (qRT-PCR) assay, suggesting that Eu4CL genes not only play an important role in the synthesis of chlorogenic acid, but also plays an important role in the process of biotic and abiotic stress. Taken together, these findings provide theoretical reference for further exploring the molecular characteristics and biological functions of Eu4CL genes.

Eucommia ulmoides leaves (EUL) contain a variety of natural bioactive compounds including chlorogenic acid, geniposide acid, and aucubin. These bioactive chemicals improve immune function and regulate lipid metabolism. The aim of this study was to investigate the effects of EUL on the biochemical properties of milk. Twenty Holstein dairy cows were randomly allocated to two groups fed a control (CTR, diet without EUL, n = 10) or EUL (diet containing 3% EUL, dry matter, n = 10) diet for 55 d. At the end of the experimental period (d 55), milk samples were collected and analyzed to determine their composition. Though levels of milk fat, protein, lactose, and total milk solids were similar between the groups, small molecules, metabolites, lipids, and cytokines differed. Compared with the CTR group, the EUL group had an improved cluster of differentiation (CD)4/CD8 ratio (P < 0.05) and lower interleukin (IL)-8 and IL-6 content (P < 0.05). Metabolomics analysis identified 14 metabolites including 7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenoic acid (FC = 3.129), adrenic acid (FC = 2.830), and eicosapentaenoic acid (FC=1.685) as having significantly increased in the EUL group (P < 0.05) while 11 metabolites, including indole-2-carboxylic acid (FC = 0.636), cholic acid (FC = 0.430), and creatine (FC = 0.784) had significantly decreased (P < 0.05). Based on a constructed metabolome map, linoleic acid metabolism had the highest impact value for EUL. A total of 21 lipid classes and 1,094 lipid species were detected in the milk by lipidomic analysis, among which 40 differed significantly between the CTR and EUL groups. The present findings showed that the EUL altered milk composition. Correlation analysis showed that 7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenoic acid, adrenic acid, and eicosapentaenoic acid levels were negatively correlated with those of the inflammatory factors IL-6 and IL-8 (P < 0.05), indicating that EUL improved milk quality by reducing inflammatory factors and increasing the CD4/CD8 ratio. Overall, our data demonstrate that EUL had positive effects on milk antioxidant parameters, immune indices, and micro-composition metabolism, thereby improving milk quality.

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.

Studies in multiple species have shown that phospholipid:diacylglycerol acyltransferase (PDAT) and oil bodies are important factors affecting plant oil accumulation. However, little information is available about the PDAT genes and oil bodies in Eucommia ulmoides Oliv. a high-quality oil tree. In this study, the oil bodies in Eucommia ulmoides seeds at different developmental stages were observed by frozen section technology combined with Nile red staining. It was found that there was a significant positive correlation between oil content and oil body number. In addition, seven Eucommia ulmoides PDAT (EuPDAT) genes were cloned from Eucommia ulmoides seeds based on genome data. They were assembled into three subgroups according to their classifications in Arabidopsis. EuPDAT genes in the same subgroup had similar gene structures and conserved motifs. Putative cis-element analysis of EuPDAT gene promoter regions uncovered numerous elements related to stress and plant hormones response. The expression patterns showed that EuPDAT2 and EuPDAT7 expression levels were positively related to oil content in different periods, and the correlation coefficients were 0.78 and 0.91, respectively. Our results suggest that oil body number and EuPDAT genes are important factors affecting oil accumulation in E. ulmoides seeds, and this work provides a theoretical reference for the directional cultivation of new high-oil-content E. ulmoides varieties.

MicroRNAs (miRNAs) play essential roles in regulating various development processes in plants. However, their role in regulating rubber biosynthesis in Eucommia ulmoides is largely unknown. Rubber is mainly distributed in the tissue covering the seed (GZ) rather than the periphery (GB) of the pericarp of E. ulmoides during accumulation in May. To investigate the roles of miRNAs in rubber biosynthesis, we conducted high-throughput small RNA sequencing using GZ and GB collected on 11 May (rapid accumulation) and 11 June (reduced accumulation). In total, 12 and 25 miRNAs were either up- or downregulated in GZ in May (GZ511) compared to GB (GB511) in May, while 27 and 38 miRNAs were either up- or downregulated in GZ in May compared to GZ in June (GZ611), respectively. Functional analyses of differentially expressed (DE−) genes targeted by DE-miRNAs revealed that miRNAs may regulate genes involved in rubber biosynthesis. For instance, when Eu-miR45 expression declined, the expression of its predicted target, small rubber particle protein 1 (EuSRPP1), increased in GZ511 vs. GB511 and GZ511 vs. GZ611, possibly resulting in higher rubber accumulation in GZ511. Additionally, we identified potential lncRNA−miRNA−mRNA networks in rubber biosynthesis. Overall, these results indicate that miRNAs play pivotal roles in regulating rubber biosynthesis via miRNA-target gene pairs and lncRNA−miRNA−mRNA networks in E. ulmoides. Our findings will enhance research on the mechanisms of rubber biosynthesis in plants.