Discovery of New Triterpenoids Extracted from Camellia oleifera Seed Cake and the Molecular Mechanism Underlying Their Antitumor Activity.

Theasaponin derivatives, which are reported to exert antitumor activity, have been widely reported to exist in edible plants, including in the seed cake of Camellia oleifera (C.), which is extensively grown in south of China. The purpose of this study was to isolate new theasaponin derivatives from C. seed cake and explore their potential antitumor activity and their underlying molecular mechanism. In the present study, we first isolated and identified four theasaponin derivatives (compounds 1, 2, 3, and 4) from the total aglycone extract of the seed cake of Camellia oleifera by utilizing a combination of pre-acid-hydrolysis treatment and activity-guided isolation. Among them, compound 1 (C1) and compound 4 (C4) are newly discovered theasaponins that have not been reported before. The structures of these two new compounds were characterized based on comprehensive 1D and 2D NMR spectroscopy and high-resolution mass spectrometry, as well as data reported in the literature. Secondly, the cytotoxicity and antitumor property of the above four purified compounds were evaluated in selected typical tumor cell lines, Huh-7, HepG2, Hela, A549, and SGC7901, and the results showed that the ED50 value of C4 ranges from 1.5 to 11.3 µM, which is comparable to that of cisplatinum (CDDP) in these five cell lines, indicating that C4 has the most powerful antitumor activity among them. Finally, a preliminary mechanistic investigation was performed to uncover the molecular mechanism underlying the antitumor property of C4, and the results suggested that C4 may trigger apoptosis through the Bcl-2/Caspase-3 and JAK2/STAT3 pathways, and stimulate cell proliferation via the NF-κB/iNOS/COX-2 pathway. Moreover, it was surprising to find that C4 can inhibit the Nrf2/HO-1 pathway, which indicates that C4 has the potency to overcome the resistance to cancer drugs. Therefore, C1 and C4 are two newly identified theasaponin derivatives with antitumor activity from the seed cake of Camellia oleifera, and C4 is a promising antitumor candidate not only for its powerful antitumor activity, but also for its ability to function as an Nrf2 inhibitor to enhance the anticancer drug sensitivity.

Hidden in plain sight: Systematic investigation of Leucine-rich repeat containing genes unveil the their regulatory network in response to Fusarium wilt in tung tree.

Leucine-rich repeat-containing genes (LRRs) have been reported to play important roles in responses to diseases. However, we poorly understood the response of LRRs to Fusarium wilt infection in tung tree (Vernicia fordii), which is an important economic tree. Here, 437 LRR-containing proteins containing nine types of LRR domains were identified in V. fordii genome. Phylogenetic analysis suggested that nine types of LRR domains could not be divided into separate classes, implying that these LRR domains had a common origin. Totally, 27 LRRs were related to possible resistance to Fusarium wilt after 2, 8, and 13 days post-inoculation. We further found that Vf06G1605 was up-regulate under Fusarium wilt infection after these three time points, Vf10G1602 and Vf02G1413 were up-regulated at 8, and 13 dpi, while Vf07G2320 was down-regulated at these three time points. The WGCNA and promoter elements suggested that WRKY possibly regulate the responses of LRRs to Fusarium wilt infection. This study highlighted the phylogeny and function of LRRs in V. fordii and provided a systematic analysis of these genes in the V. fordii genome. Our results presented here might clearly illustrate physiological mechanisms of resistance to Fusarium wilt infection and the target of marker-assisted breeding in V. fordii.

Integrative analysis reveals evolutionary patterns and potential functions of SWEET transporters in Euphorbiaceae.

As a macromolecular substance, sucrose contributes to the plant growth and development. SWEET genes, a group of sugar transporters, are a recently found plant gene family and play important roles in sugar efflux, pollen nutrition, nectar secretion, phloem transport, and seed development. The SWEET genes have been identified and characterized in some plants, but the systematic study in tung tree (Vernicia fordii) was limited. Here, we identified 121 SWEETs in five Euphorbiaceae, and could be divided into four classes with 20 different motifs. Multiple sequence alignment revealed seven transmembrane helixes (TMHs) in the SWEET proteins which were created by an internal duplication of an ancestral three-TMHs unit, connected by TMH4. This study provides direct evidence for the first time for internal duplication in Euphorbiaceae. The large-scale duplication events represented the main driving force for SWEET family expansion in Euphorbiaceae. In addition, we determined the key VfSWEETs for sucrose transport from source to sink tissues in V. fordii and proposed a possible sucrose transport model, which would be helpful for understanding the mechanism of sucrose transport in V. fordii. This study provided a new insight into the evolution, expression and structural variations of SWEETs in V. fordii and four other Euphorbiaceae.

New Insights of Salicylic Acid Into Stamen Abortion of Female Flowers in Tung Tree (Vernicia fordii).

Tung tree (Vernicia fordii), an economically important woody oil plant, is a monoecious and diclinous species with male and female flowers on the same inflorescence. The extremely low proportion of female flowers leads to low fruit yield in tung orchards. The female flower normally develops along with stamen abortion; otherwise sterile ovules will be produced. However, little knowledge is known about the molecular basis of the female flower development in tung tree. In this study, integrated analyses of morphological and cytological observations, endogenous phytohormone assay and RNA-seq were conducted to understand the molecular mechanism of the female flower development in tung tree. Cytological observation suggested that the abortion of stamens in female flowers (SFFs) belongs to the type of programmed cell death (PCD), which was caused by tapetum degeneration at microspore mother cell stage. A total of 1,366 differentially expressed genes (DEGs) were identified in female flowers by RNA-seq analysis, of which 279 (20.42%) DEGs were significantly enriched in phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction. Stage-specific transcript identification detected dynamically expressed genes of important transcription regulators in female flowers that may be involved in PCD and floral organ development. Gene expression patterns revealed that 17 anther and pollen development genes and 37 PCD-related genes might be involved in the abortion of SFF. Further analyses of phytohormone levels and co-expression networks suggested that salicylic acid (SA) accumulation could trigger PCD and inhibit the development of SFF in tung tree. This study provides new insights into the role of SA in regulating the abortion of SFF to develop normal female flowers.

Tung Tree (Vernicia fordii) Genome Provides A Resource for Understanding Genome Evolution and Improved Oil Production.

Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil rich in eleostearic acid. Here, we report a high-quality chromosome-scale genome sequence of tung tree. The genome sequence was assembled by combining Illumina short reads, Pacific Biosciences single-molecule real-time long reads, and Hi-C sequencing data. The size of tung tree genome is 1.12 Gb, with 28,422 predicted genes and over 73% repeat sequences. The V. fordii underwent an ancient genome triplication event shared by core eudicots but no further whole-genome duplication in the subsequent ca. 34.55 million years of evolutionary history of the tung tree lineage. Insertion time analysis revealed that repeat-driven genome expansion might have arisen as a result of long-standing long terminal repeat retrotransposon bursts and lack of efficient DNA deletion mechanisms. The genome harbors 88 resistance genes encoding nucleotide-binding sites; 17 of these genes may be involved in early-infection stage of Fusarium wilt resistance. Further, 651 oil-related genes were identified, 88 of which are predicted to be directly involved in tung oil biosynthesis. Relatively few phosphoenolpyruvate carboxykinase genes, and synergistic effects between transcription factors and oil biosynthesis-related genes might contribute to the high oil content of tung seed. The tung tree genome constitutes a valuable resource for understanding genome evolution, as well as for molecular breeding and genetic improvements for oil production.

The complete chloroplast genome sequence of tung tree (Vernicia fordii): Organization and phylogenetic relationships with other angiosperms.

Tung tree (Vernicia fordii) is an economically important tree widely cultivated for industrial oil production in China. To better understand the molecular basis of tung tree chloroplasts, we sequenced and characterized its genome using PacBio RS II sequencing platforms. The chloroplast genome was sequenced with 161,528 bp in length, composed with one pair of inverted repeats (IRs) of 26,819 bp, which were separated by one small single copy (SSC; 18,758 bp) and one large single copy (LSC; 89,132 bp). The genome contains 114 genes, coding for 81 protein, four ribosomal RNAs and 29 transfer RNAs. An expansion with integration of an additional rps19 gene in the IR regions was identified. Compared to the chloroplast genome of Jatropha curcas, a species from the same family, the tung tree chloroplast genome is distinct with 85 single nucleotide polymorphisms (SNPs) and 82 indels. Phylogenetic analysis suggests that V. fordii is a sister species with J. curcas within the Eurosids I. The nucleotide sequence provides vital molecular information for understanding the biology of this important oil tree.

Fatty acid profile and unigene-derived simple sequence repeat markers in tung tree (Vernicia fordii).

Tung tree (Vernicia fordii) provides the sole source of tung oil widely used in industry. Lack of fatty acid composition and molecular markers hinders biochemical, genetic and breeding research. The objectives of this study were to determine fatty acid profiles and develop unigene-derived simple sequence repeat (SSR) markers in tung tree. Fatty acid profiles of 41 accessions showed that the ratio of α-eleostearic acid was increasing continuously with a parallel trend to the amount of tung oil accumulation while the ratios of other fatty acids were decreasing in different stages of the seeds and that α-eleostearic acid (18∶3) consisted of 77% of the total fatty acids in tung oil. Transcriptome sequencing identified 81,805 unigenes from tung cDNA library constructed using seed mRNA and discovered 6,366 SSRs in 5,404 unigenes. The di- and tri-nucleotide microsatellites accounted for 92% of the SSRs with AG/CT and AAG/CTT being the most abundant SSR motifs. Fifteen polymorphic genic-SSR markers were developed from 98 unigene loci tested in 41 cultivated tung accessions by agarose gel and capillary electrophoresis. Genbank database search identified 10 of them putatively coding for functional proteins. Quantitative PCR demonstrated that all 15 polymorphic SSR-associated unigenes were expressed in tung seeds and some of them were highly correlated with oil composition in the seeds. Dendrogram revealed that most of the 41 accessions were clustered according to the geographic region. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, comparative genomics and genetic mapping in tung tree. The lipid profiles in the seeds of 41 tung accessions will be valuable for biochemical and breeding studies.

Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii).

Triacylglycerols (TAG) are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE) are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii), whose seeds are rich in novel TAG with a wide range of industrial applications. The objectives of this study were to identify OLE genes, classify OLE proteins and analyze OLE gene expression in tung trees. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome-wide phylogenetic analysis and multiple sequence alignment demonstrated that the five tung OLE genes represented the five OLE subfamilies and all contained the "proline knot" motif (PX5SPX3P) shared among 65 OLE from 19 tree species, including the sequenced genomes of Prunus persica (peach), Populus trichocarpa (poplar), Ricinus communis (castor bean), Theobroma cacao (cacao) and Vitis vinifera (grapevine). Tung OLE1, OLE2 and OLE3 belong to the S type and OLE4 and OLE5 belong to the SM type of Arabidopsis OLE. TaqMan and SYBR Green qPCR methods were used to study the differential expression of OLE genes in tung tree tissues. Expression results demonstrated that 1) All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2) OLE mRNA levels were much higher in seeds than leaves or flowers; 3) OLE1, OLE2 and OLE3 genes were expressed in tung seeds at much higher levels than OLE4 and OLE5 genes; 4) OLE mRNA levels rapidly increased during seed development; and 5) OLE gene expression was well-coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1-3 probably play major roles in tung oil accumulation and/or oil body development. Therefore, they might be preferred targets for tung oil engineering in transgenic plants.

[Analysis of oil synthesis metabolism pathways based on transcriptome changes in tung oil tree's seeds during three different development stages].

Tung oil tree (Verniciafordii) is one of the important woody oil plants in China. Past researches on tung oil tree mainly focu on the cultivation and conventional breeding while the molecular mechanisms related to tung oil synthesis are still uncovered. We compared transcriptome of tung oil tree's seeds at three different oil synthesis stages using RNA-seq technology and then obtained a lot of differentially expressed Unigenes. Through GO classification and pathway enrichment analysis, all of these differentially expressed Unigenes were classified into 128 metabolism pathways including fatty acid biosynthesis and glycerophospholipid metabolism which are involved in oil synthesis. Some homologous proteins of key enzymes were obtained when the sequences of the Unigenes within these two pathways were aligned against KEGG database. Through analysis of expression profiles of these key enzyme genes during seed's oil synthesis stage, this research not only shed light on elucidation of plant oil synthesis but also provides candidate genes for genetic improvement of tung oil tree thereby increasing the yield per unit area of tung oil tree.