Cyclocarya Research Articles

Colletotrichum Species Causing Cyclocarya paliurus Anthracnose in Southern China

Cyclocarya paliurus, native to China, is a medicinal and edible plant with important health benefits. Anthracnose is an emerging disease in southern China that causes severe economic losses and poses a great threat to the C. paliurus tea industry. However, to date, the species diversity of pathogens causing C. paliurus anthracnose has remained limited. From 2018 to 2022, a total of 331 Colletotrichum isolates were recovered from symptomatic leaves in eight major C. paliurus planting provinces of southern China. Phylogenetic analyses based on nine loci (ITS, GAPDH, ACT, CHS-1, TUB, CAL, HIS3, GS and ApMat) coupled with phenotypic characteristics revealed that 43 representative isolates belonged to seven known Colletotrichum species, including C. brevisporum, C. fructicola, C. gloeosporioides sensu stricto, C. godetiae, C. nymphaeae, C. plurivorum and C. sojae. Pathogenicity tests demonstrated that all species described above were pathogenic to wounding detached leaves of C. paliurus, with C. fructicola being the most aggressive species. However, C. brevisporum, C. plurivorum and C. sojae were not pathogenic to the intact plant of C. paliurus. These findings reveal the remarkable species diversity involved in C. paliurus anthracnose and will facilitate further studies on implementing effective control of C. paliurus anthracnose in China.

Carboxymethylated and sulfated Cyclocarya paliurus polysaccharides inhibited colon cancer cells growth via PI3K/AKT-MAPKs/NF-κB pathways and immunomodulation

Our previous research revealed that carboxymethylation modification (CM-CP) and sulfation modification (S-CP) could enhance the antitumor activity of Cyclocarya paliurus polysaccharides (CP), however, the antitumor mechanisms of CM-CP and S-CP are not fully clarified and remain largely unexplored. The aims of the present study were to investigate the dual anti-tumor function of CM-CP and S-CP in inhibiting cancer cells and activating macrophages to exert immunomodulation. Results revealed that CM-CP and S-CP preferentially inhibited the proliferation of colon cancer CT-26 cells in dose-dependent and time-dependent ways, without toxicity to normal cell lines. Flow cytometry values further established that CM-CP and S-CP interventions elicited acidification in the cells, arrested the cell cycle in S stage, raised the amount of reactive oxygen species and activated oxidative stress. CM-CP and S-CP treatment elicited the depolarization of mitochondrial membranous potentials and Ca2+ superload, and disrupted the equilibrium of Na+/K+-ATPase and Ca2+-ATPase. Simultaneously, apoptotic proteins expression assays revealed that CM-CP and S-CP promoted CT-26 cell apoptosis through PI3K/AKT-MAPKs/NF-κB signal pathways. In parallel, CM-CP and S-CP dramatically promoted the proliferation and secretory activity (TNF-α and NO) of RAW264.7 cells. Co-culture of cancer cells with CM-CP and S-CP-stimulated macrophage supernatants resulted in more pronounced suppression of cancer cell viability. These findings demonstrated that CM-CP and CM-CP not only inhibit cancer cell growth directly, but also activate macrophages to exert immunomodulatory effects to suppress cancer cells. Furthermore, CM-CP and CM-CP make a contribution to the development of potential functional foods or supplements for the prevention or treatments of colorectal cancer.

Extraction and properties of Cyclocarya paliurus leaf polysaccharide and its phosphorylated derivatives

Chemical modification provides an important idea for advancing and harnessing plant polysaccharides. It is well known that Cyclocarya paliurus (Batalin) Iljinskaja (C. paliurus), is an important industrial crop resource, and polysaccharide stands as one of the compounds displaying a wide range of bioactivities within the leaves of C. paliurus. The study found that C. paliurus polysaccharides (CPP) were subjected to phosphate modification to obtain two phosphorylated polysaccharides (P-CPPL and P-CPPH) with degrees of substitution (DS) of 0.10 and 0.18, correspondingly. The findings indicated that the phosphorylation changed the chemical composition, relative molecular weight, molar ratio of monosaccharide composition and microstructure of CPP, thus changing the thermal stability and bioactivities of CPP. In vitro bioassay results demonstrated that CPP and its phosphorylated derivatives showed antibacterial activity at high concentrations. In addition, phosphorylation changes the water solubility and spatial configuration of CPP, so that P-CPPL and P-CPPH can bind or interact with enzymes more effectively, thus improving the α-glucosidase inhibitory activity in vitro. In vitro cell experiment results demonstrate that CPP and its phosphorylated derivatives can protect IEC-6 cells from oxidative damage by restoring mitochondrial membrane potential levels and modulating the expression and activation of Caspase-3. In summary, phosphorylation provided an essential way of changing the chemical properties of polysaccharides and enhancing their functionalities.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced β-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Insights into the Hormone-Regulating Mechanism of Adventitious Root Formation in Softwood Cuttings of Cyclocarya paliurus and Optimization of the Hormone-Based Formula for Promoting Rooting.

Adventitious root (AR) formation is vital for successful cutting propagation in plants, while the dynamic regulation of phytohormones is viewed as one of the most important factors affecting AR formation. Cyclocarya paliurus, a hard-to-root plant, is faced with the bottleneck of cloning its superior varieties in practice. In this study, ten treatments were designed to figure out the best hormone-based formula for promoting AR formation in softwood cuttings and explore their hormone-regulating mechanisms. Both the rooting process and the rooting parameters of the softwood cuttings were significantly affected by different hormone-based formulas (p < 0.05), while the greatest rooting rate (93%) and root quality index were achieved in the H3 formula (SR3:IR3 = 1:1). Significant differences in the measured phytohormone concentrations, as well as in their ratios, were detected among the cuttings sampled at various AR formation stages (p < 0.05), whereas the dynamics for each phytohormone varied greatly during AR formation. The transcriptome analysis showed 12,028 differentially expressed genes (DEGs) identified during the rooting process of C. paliurus cuttings, while the KEGG enrichment analysis indicated that a total of 20 KEGG terms were significantly enriched in all the comparison samples, with 253 DEGs detected in signal transduction. Furthermore, 19 genes with vital functions in regulating the hormone signaling pathway were identified by means of a WGCNA analysis. Our results not only optimize a hormone-based formula for improving the rooting of C. paliurus cuttings but also provide an insight into the hormonal regulatory network during AR formation in softwood C. paliurus cuttings.

In vitro and in vivo immunomodulatory activity of acetylated polysaccharides from Cyclocarya paliurus leaves

In this study, we aimed to investigate the immunomodulatory effects of polysaccharides from Cyclocarya paliurus leaves after acetylation modification (Ac-CPP0.1) on dendritic cells (DCs) and immunosuppressed mice. In vitro, Ac-CPP0.1 promoted phenotypic and functional maturation of DCs. Specifically, it increased the expression of costimulatory molecules (CD80, CD86, and MHC II) and the secretion of cytokines (TNF-α, IL-6, IL-1β, IL-10, IL-12p70) of DCs. In vivo, Ac-CPP0.1 significantly improved immunosuppression of mice, which was manifested by increased body weight and immune organ index, up-regulated cytokines (IL-4, IL-17, TGF-β3, and TNF-α), and restored short-chain fatty acid (SCFAs) levels of intestinal. The immunoactivation of Ac-CPP0.1 in DCs and in mice is linked to the activation of the TLR4/NF-κB signaling pathway. Furthermore, Ac-CPP0.1 reversed intestinal flora imbalance caused by cyclophosphamide. At the species level, Ac-CPP0.1 increased the abundance of unclassified_Muribaculaceae, unclassified_Desulfovibrio, Bacteroides_acidifaciens and Faecalibaculum_rodentium, decreased the level of Lactobacillus_johnsonii, unclassified_g_Staphylococcus and Staphylococcus_nepalensis. In summary, Ac-CPP0.1 has considerable immunomodulatory potential, which is beneficial to the future utilization and development of Cyclocarya paliurus.

Effects of nanoparticle application on Cyclocarya paliurus growth: Mechanisms underlying the particle- and dose-dependent response

Nanoparticles (NPs) have been considered as potential plant growth stimulants in recent years. In this study, three types of oxide NPs with various concentrations, including ZnO-NPs (0, 50 and 100 mg L−1), SiO2-NPS (0, 300 and 500 mg L−1) and MnO2-NPS (0, 30 and 50 mg L−1), were set up to explore their effects on growth, photosynthetic parameters and gene expressions in Cyclocarya paliurus under outdoor conditions for 120 days. Foliar spraying with higher concentration of SiO2- and MnO2-NPs significantly increasedf the net growth of height and ground diameter C. paliurus by 43.1–26.4% and 28.6–26.3% respectively, via improving photosynthetic and chlorophyll fluorescence parameters, whereas a higher concentration spraying of ZnO-NPs showed a negative impact on the net growth. Transcriptomic analysis revealed that in the presence of SiO2- and MnO2-NPs, 9 genes associated with the activities of photosystem (PS) I and Rubisco were up-regulated, while the 7 PSII-related genes were down-regulated under ZnO-NPs application. Moreover, application of SiO2-NPs also up-regulated the gene expressions related to auxin (16 genes) and cytokinin (8 genes). Our study suggests that the effects of NPs appilation on C. paliurus growth are type- and dose-dependent, and regulated via altering the photosynthesis and hormone signaling at the molecular level. The findings not only provide insights into the regulatory mechanisms of nanoparticles on C. paliurus growth but also serve as references for a potential application of these nanoparticles in woody plants.

Application of Cyclocarya paliurus-Kiwifruit Composite Fermented to Enhance Antioxidant Capacity, Flavor, and Sensory Characteristics of Kiwi Wine.

A new fermentation method for kiwi wine was explored by developing the well-known medicinal and edible plant Cyclocarya paliurus (C. paliurus) to create more value with undersized kiwifruits. In this study, the changes in bioactive substances during the C. paliurus-kiwi winemaking process were analyzed on the basis of response surface optimization results, and the antioxidant capacity, aromatic compounds, and sensory quality of the C. paliurus-kiwi composite wine with kiwi wine and two commercial kiwi wines were compared. The results showed that DPPH radical, OH- radical, and ABTS+ scavenging rates remained at over 60.0%, 90.0%, and 70.0% in C. paliurus-kiwi wine, respectively. The total flavonoid content (TFC) and total polyphenol content (TPC) of C. paliurus-kiwi wine were significantly higher than those of the other three kiwi wines. C. paliurus-kiwi wine received the highest score and detected 43 volatile compounds. Ethyl hexanoate, which showed stronger fruity and sweet aromas, was one of the main aroma components of C. paliurus-kiwi wine and different from commercial wines. This wine has a good flavor with a natural and quality feeling of C. paliurus-kiwifruit extract, low-cost processing, and great market potential.

Potential hypoglycemic and hypolipidemic bioactive constituents in Cyclocarya paliurus leaves explored by affinity ultrafiltration with α‐glucosidase and pancreatic lipase

AbstractCyclocarya paliurus has been widely applied for therapeutic feeding as a traditional Chinese medicine homologous food with high nutritional and medical value. Nevertheless, the specific bioactive compounds liable for hypoglycemic and hypolipidemic properties and underlying mechanisms remain unexplored. In the present study, the in vitro hypoglycemic and hypolipidemic effects of C. paliurus were assessed by α‐glucosidase and pancreatic lipase inhibition assays, and the results indicated that 70% ethanol extract exhibited remarkable α‐glucosidase and pancreatic lipase inhibitory activity with the IC50 value of 3.14 μg/mL and 0.98 μg/mL, respectively. A total of 18 and 15 potential ligand candidates were screened, and the chemical structures were characterized. Furthermore, the docking result showed that chlorogenic acid, quercetin, kaempferol 3‐O‐rhamnoside, and pterocaryoside A contributed to the underlying hypoglycemic and hypolipidemic effects of C. paliurus, further strengthened the bindings of the enzyme‐ligand complex and suppressed the enzyme's activity with the binding energies ranged from −4.00 to −6.31 kcal/mol. The interrelations between the specific bioactive constituents, α‐glucosidase or lipase, and hypoglycemic or hypolipidemic activities were also carified. The present study reveals substantial prospects of C. paliurus to be developed as a natural dietotherapy, disease‐prevention, and healthcare for diabetes and obesity in the near future.

Characterization, antioxidant and antibacterial activities of gelatin-chitosan edible coated films added with Cyclocarya paliurus flavonoids

In this study, gelatin-chitosan (GEL-CS) composite films added with 0.1 %, 0.2 %, and 0.3 % Cyclocarya paliurus flavonoids (CPF) were prepared. Then their appearance properties, mechanical properties, barrier properties, microstructure, thermal stability properties, antioxidant activity, and antibacterial properties were investigated. As compared with GEL-CS film, the GEL-CS films with CPF were darker in color, had higher water vapor barrier, higher elongation at break, and higher thermal stability. Additionally, microstructure analysis with Fourier infrared spectroscopy, scanning electron microscopy, and X-ray diffraction demonstrated that hydrogen bonding was the main force for cross-linking CPF with other membrane substrates. Moreover, the addition of CPF strengthened the antioxidant and antimicrobial properties of the membranes. These results indicated that the CPF addition could endow membranes with more excellent functional properties and bioactivity, accompanied by environmentally friendly and edible features. The GEL-CS-CPF composite film would be a potential and prospective packing material for food preservation applications.

Identification of CpbZIP11 in Cyclocarya paliurus Involved in Environmental Stress Responses

Environmental stresses can disrupt protein structure, resulting in unfolded or misfolded proteins, thereby triggering endoplasmic reticulum (ER) stress. The unfolded protein response (UPR), particularly as activated by Arabidopsis AtbZIP60 gene, is pivotal for counteracting ER stress and ensuring cell survival. The medicinal plant, Cyclocarya paliurus, known for its wealth of beneficial compounds, is threatened by environmental stresses, limiting the exploration of its therapeutic potential. In order to better exploit and utilize its value, it is necessary to understand the signal pathway of environmental stresses. Here, we identify a homolog of AtbZIP60 in C. paliurus, termed CpbZIP11, which can be upregulated by tunicamycin. The conserved double stem-loop structure in its mRNA is spliced under environmental stresses. This splicing event results in a novel CpbZIP11 mRNA variant, leading to the production of a nuclear-localized CpbZIP11 protein with transcriptional activation activity in yeast. We further delve into the study of evolutionary lineage and motif conservation of CpbZIP11 homologs across various plant groups. This research illuminates the stress adaptation mechanisms in C. paliurus and deepens our understanding of the bZIP evolution, which endows versatility for the understanding of this transcription factor.

Transcriptomic and Non-Targeted Metabolomic Analyses Reveal Changes in Metabolic Networks during Leaf Coloration in Cyclocarya paliurus (Batalin) Iljinsk

Secondary metabolites in Cyclocarya paliurus (Batalin) Iljinsk. leaves are beneficial for human health. The synthesis and accumulation of secondary metabolites form a complex process that is influenced by the trade-off between primary and secondary metabolism and by the biosynthetic pathways themselves. In this study, we explored the relationship between secondary metabolite accumulation and the activity of metabolic networks in leaves of C. paliurus. Leaves at three different growth stages were subjected to transcriptomic and non-targeted metabolomic analyses. The results revealed that nitrogen assimilation increased and carbon assimilation decreased as leaves matured, and the patterns of secondary metabolite accumulation and gene expression differed among the leaves at different growth stages. Mature green leaves had higher nitrogen assimilation and lower carbon assimilation, which were correlated with variations in secondary metabolite accumulation. As a major source of carbon and nitrogen, glutamine accumulated in the mature green leaves of C. paliurus. The accumulation of glutamine inhibited phenylalanine biosynthesis by modulating the pentose phosphate pathway but promoted acetyl-CoA biosynthesis through the tricarboxylic acid cycle. These changes led to decreased flavonoid contents and increased triterpenoid contents in mature leaves. These metabolomic and transcriptomic data reveal the differential expression of metabolic regulatory networks during three stages of leaf development and highlight the trade-off between primary and secondary metabolism. Our results provide a comprehensive picture of the metabolic pathways that are active in the leaves of C. paliurus at different growth stages.

Physiological and biochemical changes in leaf abscission of Cyclocarya paliurus stem segments in vitro

Physiological and biochemical changes in leaf abscission of Cyclocarya paliurus stem segments in vitro

Physiological response and molecular regulatory mechanism reveal a positive role of nitric oxide and hydrogen sulfide applications in salt tolerance of Cyclocarya paliurus.

As a multifunctional tree species, Cyclocarya paliurus leaves are rich in bioactive substances with precious healthy values. To meet the huge requirement of C. paliurus leaf production, sites with some environmental stresses would be potential land for developing its plantations due to the limitation of land resources in China. Nitric oxide (NO) and hydrogen sulfide (H2S) are common gas messengers used to alleviate abiotic stress damage, whereas the mechanism of these messengers in regulating salt resistance of C. paliurus still remains unclear. We performed a comprehensive study to reveal the physiological response and molecular regulatory mechanism of C. paliurus seedlings to the application of exogenous NO and H2S under salt stress. The results showed that the application of sodium hydrosulfide (NaHS) and sodium nitroprusside (SNP) not only maintained the photosynthetic capacity and reduced the loss of leaf biomass, but also promoted endogenous NO synthesis and reduced oxidative damage by activating antioxidant enzyme activity and increasing the content of soluble protein and flavonoids. Moreover, transcriptome and metabolome analysis indicated the expression of genes encoding phenylalanine ammonia lyase (PAL), cytochromeP450 (CYP), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) in flavonoid biosynthesis pathway was all up-regulated by the application of NO and H2S. Meanwhile, 15 transcriptional factors (TFs) such as WRKY, ERF, bHLH and HY5 induced by NO were found to regulated the activities of several key enzymes in flavonoid biosynthesis pathway under salt stress, via the constructed co-expression network. Our findings revealed the underlying mechanism of NO and H2S to alleviate salt stress and regulate flavonoid biosynthesis, which provides a theoretical basis for establishing C. paliurus plantations in the salt stress areas.

Genome-Wide Identification and Expression Analysis of Salt Tolerance-Associated WRKY Family Genes in Cyclocarya paliurus

WRKY proteins are key transcription factors involved in a range of physiological processes and environmental adaptations. Yet, little is known about the role WRKY genes play in Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) a significant medicinal plant belonging to the Juglandaceae family. Leveraging the recently released whole-genome sequence of C. paliurus, we conducted an in-depth, genome-wide study on the expression of WRKY genes under environmental stresses. We identified 80 CpWRKY genes, classifying them into distinct groups and subgroups based on their structural and phylogenetic characteristics. Synteny analysis and phylogenetic comparison provided insights into the evolutionary traits of these genes. We further investigated the response of the WRKY gene family to salinity stress in combination with the following specific stressors: Methyl jasmonate (MeJA), sodium hydrosulfide (NaHS), and sodium nitroprusside (SNP), which induce the production of signaling molecules jasmonates (JA), hydrogen sulfide (H2S), and nitric oxide (NO), respectively. Our comprehensive analysis of CpWRKY gene expression patterns under various conditions revealed that combined treatments were more effective in inducing up-regulation of CpWRKY gene expression than individual treatments.

Effects on the Synthesis and Accumulation of Triterpenes in Leaves of Cyclocarya paliurus under MeJA Treatment

Cyclocarya paliurus (Batal.) lljinskaja, the sole and multi-functional tree species of the family Juglandaceae, grows extensively in subtropical areas of China. Species-specific triterpenoids in its leaves have validated health-promoting effects, including hypoglycemic and hypolipidemic activities. To illustrate the effect of MeJA treatment on the accumulation and biosynthesis of triterpenoids in C. paliurus leaves at different stages of maturity, the contents of total triterpenoids and six triterpene compounds, along with the relative expression of three key genes, were detected. The results showed that the contents of triterpenes and expression patterns of the genes significantly differed among the samples. Different treatment times also had diverse effects on triterpenoid accumulation and gene expression. MeJA treatment had positive effects on total triterpenoids, cyclocaric acid B, and cyclocarioside A/B, especially in young leaves. Gene expression was highest in young leaves after 10 days of treatment, indicating that they were the most sensitive to MeJA. This study provided a reference for improving the accumulation of triterpenoids in C. paliurus plantations in the future.

Physiological Response to Low Temperature of Four Genotypes of Cyclocarya paliurus and Their Preliminary Evaluation to Cold Resistance

Cyclocarya paliurus is a versatile tree species with immense potential for development, as it combines edible, medicinal, and ornamental functions. Low temperature is one of the important abiotic factors that affect plant survival and flourishing but their response mechanism to low temperature is not yet clear. In this study, we utilized annual shoots of four genotypes of C. paliurus, namely T2, W10, M31 and S12, as materials. The physiological responses of annual shoots of C. paliurus to low temperature stress were elaborated by determining and comparing indicators related to cold resistance, such as relative electric conductivity, semi-lethal low temperature, malondialdehyde, soluble sugar, soluble protein, proline, superoxide dismutase and peroxidase. The contents of malondialdehyde, proline, soluble protein and peroxidase activities were not only correlated with the treatment temperature but also related to the genotypes. Osmotic substance (soluble sugar, soluble protein and proline) contents and antioxidant enzyme activities (peroxidase and superoxide dismutase) of the four genotypes showed a trend of increasing and then decreasing with the five decreasing temperatures. Furthermore, a comprehensive evaluation of cold resistance was performed by using a combination of principal component analysis and membership function, with the cold resistance ranked as W10 &gt; M31 &gt; S12 &gt; T2. Results from this study would provide some references for extending the plantation areas.

Simulated organic–inorganic nitrogen deposition changes the growth rate, leaf stoichiometry, and phenolic content of Cyclocarya paliurus

Atmospheric nitrogen (N) deposition is composed of both organic nitrogen (ON) and inorganic nitrogen (IN), and these sources of N affect plant growth, the balance of elements, and the accumulation of secondary metabolism substances; however, how these plant traits respond to ON–IN N deposition remains unclear. Here, we explored the effects of N addition with different ON:IN ratios on the growth rate, biomass allocation, leaf N and phosphorus (P) concentration, and leaf phenolic content of Cyclocarya paliurus in a short-term field experiment. Compared with the control, the growth rate (μ) and biomass accumulation of C. paliurus showed a decreasing trend in the 2 years after different N addition treatments. Higher ON:IN ratios not only resulted in higher leaf N content but also significantly increased the leaf P content. In addition, N addition with different ON:IN ratios induced more biomass accumulation allocated to the underground part of C. paliurus, which may be related to the nutrient acquisition strategy. Increased leaf N:P ratios indicated that stoichiometric imbalance occurred and that N and P limitation conditions of C. paliurus changed after different ON–IN N treatments. The growth rate (μ) of C. paliurus showed a trend of first decreasing (14 < leaf N:P ratios < 16) and then increasing (16 < leaf N:P ratios). However, compared with the growth rate, the leaf phenolic content showed an opposite trend to that of leaf stoichiometry, which indicated that leaf stoichiometry significantly affected both the primary growth and secondary metabolism of plants. Our results suggest that simulated N addition with different ON:IN ratios caused stoichiometric imbalance and changed the plant growth rate and accumulation of biomass and secondary metabolism substances. In addition, our study highlights that the type of nutrient limitation and the variation in N deposition components should be considered when predicting the future responses of plant functional traits to N deposition.

Genomic insights into biased allele loss and increased gene numbers after genome duplication in autotetraploid Cyclocarya paliurus

BackgroundAutopolyploidy is a valuable model for studying whole-genome duplication (WGD) without hybridization, yet little is known about the genomic structural and functional changes that occur in autopolyploids after WGD. Cyclocarya paliurus (Juglandaceae) is a natural diploid–autotetraploid species. We generated an allele-aware autotetraploid genome, a chimeric chromosome-level diploid genome, and whole-genome resequencing data for 106 autotetraploid individuals at an average depth of 60 × per individual, along with 12 diploid individuals at an average depth of 90 × per individual.ResultsAutotetraploid C. paliurus had 64 chromosomes clustered into 16 homologous groups, and the majority of homologous chromosomes demonstrated similar chromosome length, gene numbers, and expression. The regions of synteny, structural variation and nonalignment to the diploid genome accounted for 81.3%, 8.8% and 9.9% of the autotetraploid genome, respectively. Our analyses identified 20,626 genes (69.18%) with four alleles and 9191 genes (30.82%) with one, two, or three alleles, suggesting post-polyploid allelic loss. Genes with allelic loss were found to occur more often in proximity to or within structural variations and exhibited a marked overlap with transposable elements. Additionally, such genes showed a reduced tendency to interact with other genes. We also found 102 genes with more than four copies in the autotetraploid genome, and their expression levels were significantly higher than their diploid counterparts. These genes were enriched in enzymes involved in stress response and plant defense, potentially contributing to the evolutionary success of autotetraploids. Our population genomic analyses suggested a single origin of autotetraploids and recent divergence (~ 0.57 Mya) from diploids, with minimal interploidy admixture.ConclusionsOur results indicate the potential for genomic and functional reorganization, which may contribute to evolutionary success in autotetraploid C. paliurus.

Genome assembly of two diploid and one auto-tetraploid Cyclocarya paliurus genomes

Cyclocarya paliurus, an endemic species in the genus Juglandaceae with the character of heterodichogamy, is one of triterpene-rich medicinal plants in China. To uncover the genetic mechanisms behind the special characteristics, we sequenced the genomes of two diploid (protandry, PA-dip and protogyny, PG-dip) and one auto-tetraploid (PA-tetra) C. paliurus genomes. Based on 134.9 (~225x), 75.5 (~125x) and 271.8 Gb (~226x) subreads of PacBio platform sequencing data, we assembled 586.62 Mb (contig N50 = 1.9 Mb), 583.45 Mb (contig N50 = 1.4 Mb), and 2.38 Gb (contig N50 = 430.9 kb) for PA-dip, PG-dip and PA-tetra genome, respectively. Furthermore, 543.53, 553.87, and 2168.65 Mb in PA-dip, PG-dip, and PA-tetra, were respectively anchored to 16, 16, and 64 pseudo-chromosomes using over 65.4 Gb (~109x), 68 Gb (~113x), and 264 (~220x) Hi-C sequencing data. Annotation of PA-dip, PG-dip, and PA-tetra genome assembly identified 34,699, 35,221, and 34,633 protein-coding genes (90,752 gene models) or allele-defined genes, respectively. In addition, 45 accessions from nine locations were re-sequenced, and more than 10 × coverage reads were generated.