Functional characterization of NBS-LRR genes reveals an NBS-LRR gene that mediates resistance against Fusarium wilt.

BACKGROUND: Most disease resistance (R) genes in plants encode proteins that contain leucine-rich-repeat (LRR) and nucleotide-binding site (NBS) domains, which belong to the NBS-LRR family. The sequenced genomes of Fusarium wilt-susceptible Vernicia fordii and its resistant counterpart, Vernicia montana, offer significant resources for the functional characterization and discovery of novel NBS-LRR genes in tung tree. RESULTS: Here, we identified 239 NBS-LRR genes across two tung tree genomes: 90 in V. fordii and 149 in V. montana. Five VmNBS-LRR paralogous were predicted in V. montana, and 43 orthologous were detected between V. fordii and V. montana. The orthologous gene pair Vf11G0978-Vm019719 exhibited distinct expression patterns in V. fordii and V. montana: Vf11G0978 showed downregulated expression in V. fordii, while its orthologous gene Vm019719 demonstrated upregulated expression in V. montana, indicating that this pair may be responsible for the resistance to Fusarium wilt in V. montana. Vm019719 from V. montana, activated by VmWRKY64, was shown to confer resistance to Fusarium wilt in V. montana by a virus-induced gene silencing (VIGS) experiment. However, in the susceptible V. fordii, its allelic counterpart, Vf11G0978, exhibited an ineffective defense response, attributed to a deletion in the promoter's W-box element. CONCLUSIONS: This study provides the first systematic analysis of NBS-LRR genes in the tung tree and identifies a candidate gene that can be utilized for marker-assisted breeding to control Fusarium wilt in V. fordii.

Genome-Wide Identification of B-Box Gene Family and Candidate Light-Related Member Analysis of Tung Tree (Vernicia fordii).

Light is one of the most important environmental factors for plant growth. In the production process of tung oil tree cultivation, due to the inappropriate growth of shading conditions, the lower branches are often dry and dead, which seriously affects the yield of tung oil trees. However, little is known about the key factors of light-induced tree photomorphogenesis. In this study, a total of 22 VfBBX family members were identified to provide a reference for candidate genes in tung tree seedlings. All members of the VfBBX family have different numbers of highly conserved B-box domains or CCT domains. Phylogenetic evolution clustered the VfBBX genes into four categories, and the highest density of members was on chromosome 6. Interspecific collinearity analysis suggested that there were six pairs of duplicate genes in VfBBX members, but the expression levels of all family members in different growth and development stages of the tung tree were significantly divergent. After different degrees of shading treatment and physiological data determination of tung tree seedlings, the differential expression level and chlorophyll synthesis genes correlation analysis revealed that VfBBX9 was a typical candidate nuclear localization transcription factor that was significantly differentially expressed in light response. This study systematically identified the VfBBX gene family and provided a reference for studying its molecular function, enhanced the theoretical basis for tung tree breeding, and identified excellent varieties.

Triacylglycerol biosynthesis in shaded seeds of tung tree (Vernicia fordii) is regulated in part by Homeodomain Leucine Zipper 21.

Light quantity and quality affect many aspects of plant growth and development. However, few reports have addressed the molecular connections between seed oil accumulation and light conditions, especially dense shade. Shade-avoiding plants can redirect plant resources into extension growth at the expense of leaf and root expansion in an attempt to reach areas containing richer light. Here, we report that tung tree seed oil accumulation is suppressed by dense shade during the rapid oil accumulation phase. Transcriptome analysis confirmed that oil accumulation suppression due to dense shade was attributed to reduced expression of fatty acid and triacylglycerol biosynthesis-related genes. Through weighted gene co-expression network analysis, we identified 32 core transcription factors (TFs) specifically upregulated in densely shaded seeds during the rapid oil accumulation period. Among these, VfHB21, a class I homeodomain leucine zipper TF, was shown to suppress expression of FAD2 and FADX, two key genes related to α-eleostearic acid, by directly binding to HD-ZIP I/II motifs in their respective promoter regions. VfHB21 also binds to similar motifs in the promoters of VfWRI1 and VfDGAT2, two additional key seed lipid regulatory/biosynthetic genes. Functional conservation of HB21 during plant evolution was demonstrated by the fact that AtWRI1, AtSAD1, and AtFAD2 were downregulated in VfHB21-overexpressor lines of transgenic Arabidopsis, with concomitant seed oil reduction, and the fact that AtHB21 expression also was induced by shade. This study reveals some of the regulatory mechanisms that specifically control tung tree seed oil biosynthesis and more broadly regulate plant storage carbon partitioning in response to dense shade conditions.

Molecular mechanism of the extended oil accumulation phase contributing to the high seed oil content for the genotype of tung tree (Vernicia fordii).

BACKGROUND: Oil from seeds of the tung tree (Vernicia fordii) has unique drying properties that are industrially important. We found that the extended oil accumulation period was related to the high seed oil content at maturity among tung tree population. In order to understand the molecular mechanism underlying the high oil content in tung tree seed, Tree H and L were adopted for the further investigation, with seed oil content of about 70 and 45%, respectively. We compared the transcriptomic changes of seed at various times during oil accumulation between the two trees. RESULTS: Transcriptomes analysis revealed that many genes involved in glycolysis, fatty acid synthesis, and tri-acyl glyceride assembly still kept high expression in the late period of seed oil accumulation for Tree H only. Many genes in fatty acid degradation pathway were largely up regulated in the late period of seed oil accumulation for Tree L only. Four transcription factors related to fatty acid biosynthesis had different expression pattern in the seed oil accumulation period for the two trees. WRI1 was down regulated and kept the low expression in the late period of seed oil accumulation for the two trees. PII, LEC1 and LEC1-LIKE extended the high expression in the late period of seed oil accumulation in Tree H only. CONCLUSIONS: The continued accumulation of oil in the late period of seed oil accumulation for Tree H was associated with relatively high expression of the relevant genes in glycolysis, fatty acid synthesis and tri-acyl glyceride assembly. PII, LEC1, and LEC1-LIKE rather than WRI1 should play an important role in the oil continual accumulation in the late period of seed oil accumulation in Tree H. This study provides novel insight into the variation in seed oil content and informs plant breeding strategies to maximize oil yield.

Identification of the major diacylglycerol acyltransferase mRNA in mouse adipocytes and macrophages.

BACKGROUND: Triacylglycerols (TAGs) are the major form of energy storage in eukaryotes. Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of TAG biosynthesis. Mammalian DGATs are classified into DGAT1 and DGAT2 subfamilies. It was unclear which DGAT was the major isoform expressed in animal cells. The objective was to identify the major DGAT mRNA expressed in cultured mouse adipocytes and macrophages and compared it to that expressed in tung tree seeds. METHODS: qPCR evaluated DGAT mRNA levels in mouse 3 T3-L1 adipocytes and RAW264.7 macrophages and tung tree seeds. RESULTS: TaqMan qPCR showed that DGAT2 mRNA levels were 10-30 fold higher than DGAT1 in adipocytes and macrophages, and DGAT mRNA levels in adipocytes were 50-100-fold higher than those in macrophages. In contrast, the anti-inflammatory tristetraprolin/zinc finger protein 36 (TTP/ZFP36) mRNA levels were 2-4-fold higher in macrophages than those in adipocytes and similar to DGAT1 in adipocytes but 100-fold higher than DGAT1 in macrophages. SYBR Green qPCR analyses confirmed TaqMan qPCR results. DGAT2 mRNA as the major DGAT mRNA in the mouse cells was similar to that in tung tree seeds where DGAT2 mRNA levels were 10-20-fold higher than DGAT1 or DGAT3. CONCLUSION: The results demonstrated that DGAT2 mRNA was the major form of DGAT mRNA expressed in mouse adipocytes and macrophages and tung tree seeds.

Proteomic Analysis of Tung Tree (Vernicia fordii) Oilseeds during the Developmental Stages.

The tung tree (Vernicia fordii), a non-model woody plant belonging to the Euphorbiaceae family, is a promising economic plant due to the high content of a novel high-value oil in its seeds. Many metabolic pathways are active during seed development. Oil (triacylglycerols (TAGs)) accumulates in oil bodies distributed in the endosperm cells of tung tree seeds. The relationship between oil bodies and oil content during tung tree seed development was analyzed using ultrastructural observations, which confirmed that oil accumulation was correlated with the volumes and numbers of oil bodies in the endosperm cells during three different developmental stages. For a deeper understanding of seed development, we carried out proteomic analyses. At least 144 proteins were differentially expressed during three different developmental stages. A total of 76 proteins were successfully identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF/MS/MS). These proteins were grouped into 11 classes according to their functions. The major groups of differentially expressed proteins were associated with energy metabolism (25%), fatty acid metabolism (15.79%) and defense (14.47%). These results strongly suggested that a very high percentage of gene expression in seed development is dedicated to the synthesis and accumulation of TAGs.

Identification and characterization of NF-YB family genes in tung tree.

The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.

Phylogenetic Analysis of the PR-4 Gene Family in Euphorbiaceae and Its Expression Profiles in Tung Tree (Vernicia fordii).

Pathogenesis-related protein-4 (PR-4) is generally believed to be involved in physiological processes. However, a comprehensive investigation of this protein in tung tree (Vernicia fordii) has yet to be conducted. In this study, we identified 30 PR-4 genes in the genomes of Euphorbiaceae species and investigated their domain organization, evolution, promoter cis-elements, expression profiles, and expression profiles in the tung tree. Sequence and structural analyses indicated that VF16136 and VF16135 in the tung tree could be classified as belonging to Class II and I, respectively. Phylogenetic and Ka/Ks analyses revealed that Hevea brasiliensis exhibited a significantly expanded number of PR-4 genes. Additionally, the analysis of promoter cis-elements suggested that two VfPR-4 genes may play a role in the response to hormones and biotic and abiotic stress of tung trees. Furthermore, the expression patterns of VfPR-4 genes and their responses to 6-BA, salicylic acid, and silver nitrate in inflorescence buds of tung trees were evaluated using qRT-PCR. Notably, the expression of two VfPR-4 genes was found to be particularly high in leaves and early stages of tung seeds. These results suggest that VF16136 and VF16135 may have significant roles in the development of leaves and seeds in tung trees. Furthermore, these genes were found to be responsive to 6-BA, salicylic acid, and silver nitrate in the development of inflorescence buds. This research provides valuable insights for future investigation into the functions of PR-4 genes in tung trees.

Tung tree stearoyl-acyl carrier protein Δ9 desaturase improves oil content and cold resistance of Arabidopsis and Saccharomyces cerevisiae.

The seed oil of tung tree is rich in a-eleostearic acid (ESA), which endows tung oil with the characteristic of an excellently dry oil. The stearoyl-acyl carrier protein δ9 desaturase (SAD) is a rate-limiting enzyme that converts the stearic acid to the oleic acid, the substrate for the production of the α-ESA. However, the function of the two predicted VfSAD1 and VfSAD2 genes in the tung tree has not been determined. In this study, quantitative real-time PCR (qRT-PCR) analysis showed that VfSAD1 and VfSAD2 were expressed in multiple organs of tung tree but were highly expressed in the seed during the oil rapid accumulation period. Heterologous expression of VfSAD1 and VfSAD2 could promote the production of oleic acid and its derivatives in Arabidopsis thaliana and yeast BY4741, indicating that VfSAD1 and VfSAD2 possess the stearoyl-ACP desaturases function. Furthermore, both VfSAD1 and VfSAD2 could significantly improve seed oil accumulation in Arabidopsis. VfSAD1 could also significantly promote the oil accumulation in the yeast BY4741 strain. In addition, overexpression of VfSAD1 and VfSAD2 enhanced the tolerance of yeast and Arabidopsis seedlings to low temperature stress. This study indicates that the two VfSAD genes play a vital role in the process of oil accumulation and fatty acid biosynthesis in the tung tree seed, and both of them could be used for molecular breeding in tung tree and other oil crops.

Dissection of leucine-rich repeat receptor-like protein kinases: insight into resistance to Fusarium wilt in tung tree.

The tung tree is a woody oil plant native to China and widely distributed in the subtropics. The three main species commonly known as Vernicia are V. fordii, V. montana, and V. cordata. The growth and development of V. fordii are affected by a large number of plant pathogens, such as Fusarium wilt caused by Fusarium sp. In contrast, V. montana shows significant resistance to Fusarium wilt. The leucine-rich repeat receptor-like protein kinase (LRR-RLK) is the largest class of receptor-like kinases associated with plant resistance to Fusarium wilt. Here, we identified 239 VmLRR-RLKs in V. montana, and found that there were characteristic domains of resistance to Fusarium wilt in them. Phylogenetic analysis suggested that the VmLRR-RLKs are divided into 14 subfamilies, indicating that homologous genes in the same group may have similar functions. Chromosomal localization analysis showed that VmLRR-RLKs were unevenly distributed on chromosomes, and segment duplications were the main reason for the expansion of VmLRR-RLK family members. The transcriptome data showed that six orthologous pairs were up-regulated in V. montana in response to Fusarium wilt, while the corresponding orthologous genes showed low or no expression in V. fordii in resistance Fusarium wilt, further indicating the important role of LRR-RLKs in V. montana's resistance to infection by Fusarium spp. Our study provides important reference genes for the future use of molecular breeding to improve oil yield and control of Fusarium wilt in tung tree.

Genome-wide identification and transcriptional profiling of the basic helix-loop-helix gene family in tung tree (Vernicia fordii).

The basic helix-loop-helix (bHLH) transcription factor gene family is one of the largest gene families and is extensively involved in plant growth, development, biotic and abiotic stress responses. Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil rich in eleostearic acid. However, the characteristics of the bHLH gene family in the tung tree genome are still unclear. Hence, VfbHLHs were first searched at a genome-wide level, and their expression levels in various tissues or under low temperature were investigated systematically. In this study, we identified 104 VfbHLHs in the tung tree genome, and these genes were classified into 18 subfamilies according to bHLH domains. Ninety-eight VfbHLHs were mapped to but not evenly distributed on 11 pseudochromosomes. The domain sequences among VfbHLHs were highly conserved, and their conserved residues were also identified. To explore their expression, we performed gene expression profiling using RNA-Seq and RT-qPCR. We identified five, 18 and 28 VfbHLH genes in female flowers, male flowers and seeds, respectively. Furthermore, we found that eight genes (VfbHLH29, VfbHLH31, VfbHLH47, VfbHLH51, VfbHLH57, VfbHLH59, VfbHLH70, VfbHLH72) were significant differential expressed in roots, leaves and petioles under low temperature stress. This study lays the foundation for future studies on bHLH gene cloning, transgenes, and biological mechanisms.

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.

Flower Development and Sex Determination between Male and Female Flowers in Vernicia fordii.

Vernicia fordii is a monoecious and diclinous species with male and female flowers on the same inflorescence. Low female to male flower ratio is one of the main reasons for low yield in this species. However, little is known of its floral development and sex determination. Here, according to the results of scanning electron microscopy and histological analysis, the floral development of V. fordii was divided into 12 stages and the first morphological divergence between the male and female flowers was found to occur at stage 7. The male flowers are always unisexual, but the female flowers present bisexual characteristics, with sterile stamen (staminode) restricted to pre-meiosis of mother sporogenous cells and cell death occurring at later development stages. To further elucidate the molecular mechanism underling sex determination at the divergence stage for male and female flowers, comparative transcriptome analysis was performed. In total, 56,065 unigenes were generated and 608 genes were differentially expressed between male and female flowers, among which 310 and 298 DEGs (differentially expressed genes) showed high expression levels in males and females, respectively. The transcriptome data showed that the sexual dimorphism of female flowers was affected by jasmonic acid, transcription factors, and some genes related to the floral meristem activity. Ten candidate genes showed consistent expression in the qRT-PCR validation and DEGs data. In this study, we provide developmental characterization and transcriptomic information for better understanding of the development of unisexual flowers and the regulatory networks underlying the mechanism of sex determination in V. fordii, which would be helpful in the molecular breeding of V. fordii to improve the yield output.