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.

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 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.

Integration of transcriptomic and metabolomic analysis unveils the response mechanism of sugar metabolism in Cyclocarya paliurus seedlings subjected to PEG-induced drought stress.

Cyclocarya paliurus (Batal.) Iljinskaja is a multiple function tree species used for functional food and valued timber production. Carbohydrates, especially water-soluble carbohydrates, play an important role in osmotic protection, signal transduction and carbon storage. Under the circumstance of global climate change the abiotic stress would restrict the development of C. paliurus plantation, whereas there is few knowledge on the regulatory mechanisms of sugar metabolism under drought stress in C. paliurus. To investigate the drought response of C. paliurus at molecular level, we conducted an integrated analysis of transcriptomic and metabolomic of C. paliurus at three PEG-induced drought stress levels (0%: control; 15%: moderate drought; 25%: severe drought) in short term. Both moderate and severe drought treatments activated the chemical defense with lowering relative water content, and enhancing the contents of soluble protein, proline and malondialdehyde in the leaves. Meanwhile, alterations in the expression of differentially expressed genes and carbohydrate metabolism profiles were observed among the treatments. Weighted gene co-expression network analysis (WGCNA) showed 3 key modules, 8 structural genes (such as genes encoding beta-fructofuranosidase (INV), sucrose synthase (SUS), raffinose synthase (RS)) and 14 regulatory transcription factors were closely linked to sugar metabolism. Our results provided the foundation to understand the response mechanism of sugar metabolism in C. paliurus under drought stress, and would drive progress in breeding of drought-tolerant varieties and plantation development of the species.

Genome-wide identification of bHLH transcription factors and their response to salt stress in Cyclocarya paliurus.

As a highly valued and multiple function tree species, the leaves of Cyclocarya paliurus are enriched in diverse bioactive substances with healthy function. To meet the requirement for its leaf production and medical use, the land with salt stress would be a potential resource for developing C. paliurus plantations due to the limitation of land resources in China. The basic helix-loop-helix (bHLH) transcription factor protein family, the second largest protein family in plants, has been found to play essential roles in the response to multiple abiotic stresses, especially salt stress. However, the bHLH gene family in C.paliurus has not been investigated. In this study, 159 CpbHLH genes were successfully identified from the whole-genome sequence data, and were classified into 26 subfamilies. Meanwhile, the 159 members were also analyzed from the aspects of protein sequences alignment, evolution, motif prediction, promoter cis-acting elements analysis and DNA binding ability. Based on transcriptome profiling under a hydroponic experiment with four salt concentrations (0%, 0.15%, 0.3%, and 0.45% NaCl), 9 significantly up- or down-regulated genes were screened, while 3 genes associated with salt response were selected in term of the GO annotation results. Totally 12 candidate genes were selected in response to salt stress. Moreover, based on expression analysis of the 12 candidate genes sampled from a pot experiment with three salt concentrations (0%, 0.2% and 0.4% NaCl), CpbHLH36/68/146 were further verified to be involved in the regulation of salt tolerance genes, which is also confirmed by protein interaction network analysis. This study was the first analysis of the transcription factor family at the genome-wide level of C. paliurus, and our findings would not only provide insight into the function of the CpbHLH gene family members involved in salt stress but also drive progress in genetic improvement for the salt tolerance of C. paliurus.

Whole-genome Duplication Reshaped Adaptive Evolution in A Relict Plant Species, Cyclocarya paliurus.

Cyclocarya paliurus is a relict plant species that survived the last glacial period and shows a population expansion recently. Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B. Here, we presented three C. paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly. Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage effects that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids. Resequencing analysis of 45 C. paliurus individuals uncovered two bottlenecks, consistent with the known events of environmental changes, and many selectively swept genes involved in critical biological functions, including plant defense and secondary metabolite biosynthesis. We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes, in particular gibberellin-related genes, associated with the heterodichogamy in C. paliurus species. Our study sheds light on evolutionary history of C. paliurus and provides genomic resources to study the medicinal herbs.

New triterpenoids from the Cyclocarya paliurus (Batalin) Iljinskaja and their anti-fibrotic activity.

Cyclocarya paliurus, a Chinese herbal medicine and new food resource, contains a triterpenic-acid-rich extract that demonstrated ameliorative effect on diabetic nephropathy (DN). A more in-depth discovery of functional components led to the isolation of seven new triterpenoids including two pentacyclic triterpenes, 1α,2α,3ß,23-tetrahydroxyolean-12-en-28-oic acid and 2α,3ß,22α-tirhydroxyurs-12-en-28-oic acid 28-O-ß-D-glucopyranoside, and five tetracyclic triterpenoid glycosides (cypaliurusides N-R), together with twelve known compounds from the leaves of C. paliurus. Their structures were determined using a comprehensive analysis of chemical and spectroscopic data. Partial compounds were assessed for anti-fibrotic activities in high-glucose and TGF-ß1 induced HK-2 cells. Compound 16 remarkably decreased the level of fibronectin with an inhibition rate of 37.1%. Furthermore, 16 effectively alleviated the epithelial-mesenchymal transformation (EMT) process by upregulating E-cadherin expression and downregulating α-SMA expression, and it significantly decreased the level of the transcriptional inhibitors (Snail and Twist) of E-cadherin. The discovery of anti-fibrotic compounds from C. paliurus provides the potential utilization and functional candidates for the DN prevention.

Association Analysis Between Genotype and Environment: Differentiation Between Cyclocarya paliurus Resources That Accumulate Triterpenoids.

Cyclocarya paliurus is mainly distributed in subtropical areas of China. Its leaves are rich in beneficial triterpenoids that have bioactivities against human diseases, including hyperlipemia, diabetes, and hypertension. In this study, data on the genetic diversity, distributing environment, and triterpenoids of C. paliurus samples were collected from a wide area in China. The data covered 316 C. paliurus germplasms collected from 26 distinct populations. Association analysis between genotype and triterpenoids was carried out to describe triterpenoids accumulation pattern. Based on our analyses, we identified the important trend that genotypes with higher triterpenoid contents belonged to a unique genotype subgroup. The results showed that pterocaryoside B and pterocaryoside A significantly vary among the genotypic subgroups. In addition, the different genotypic subgroups showed distinct geographical distributing areas. These findings provide information about the relationship between genetic and environmental factors and how this affects triterpenoids accumulation. This information will be valuable for targeted breeding and for further germplasm selection of C. paliurus resources.

Nitrogen addition modifies the relative gene expression level and accumulation of carbon-based bioactive substances in Cyclocarya paliurus.

In China, lots of Cyclocarya paliurus plantations have been established for tea and functional food production on nitrogen (N)-limited land. The optimum N levels require for biosynthesis and accumulation of carbon-based bioactive substances vary among plant species. This study integrated field trial with hydroponic culture to assess impact of nitrogen addition on accumulation and relative gene expression level of carbon-based secondary metabolites in C. paliurus. N addition significantly influenced not only contents of polyphenols, flavonoids and triterpenoids and relative gene expression levels of their biosynthetic pathway in C. paliurus leaves but also leaf biomass production and the bioactive substance accumulations. An intermediate N addition induced the highest contents of polyphenols, flavonoids and triterpenoids in leaves, but the optimized accumulation of these bioactive substances in the leaves was the trade-off between their contents and leaf biomass production. Correlation analysis showed that related gene expression levels were closely correlated with contents of their leaf corresponding secondary metabolites. Compared with ratios of carbon/N (C/N) and carbon/phosphorus (C/P) in the soil, ratios of C/N and C/P in the leaves were more strongly related to the contents and accumulations of polyphenols, flavonoids and triterpenoids. To obtain higher yields of targeted phytochemicals, the threshold ratios of C/N and C/P in the leaves are recommended for N and P fertilization at similar sites. Overall, our findings would provide the theoretical basis and technical support for manipulating N fertilization in C. paliurus plantations to obtain higher accumulations of targeted bioactive substances.

Identification and Expression Analysis of R2R3-MYB Family Genes Associated with Salt Tolerance in Cyclocarya paliurus.

R2R3-MYB transcription factors are most abundant in the MYB superfamily, while the R2R3-MYB genes play an important role in plant growth and development, especially in response to environmental stress. Cyclocarya paliurus is a multifunction tree species, and the existing resources cannot meet the requirement for its leaf production and medical use. Therefore, lands with some environmental stresses would be potential sites for developing C. paliurus plantations. However, the function of R2R3-MYB genes in C.paliurus in response to environmental stress remains unknown. In this study, to identify the roles of R2R3-MYB genes associated with salt stress response, 153 CpaMYB genes and their corresponding protein sequences were identified from the full-length transcriptome. Based on the comparison with MYB protein sequences of Arabidopsis thaliana, 69 R2R3-MYB proteins in C. paliurus were extracted for further screening combined with conserved functional domains. Furthermore, the MYB family members were analyzed from the aspects of protein sequences alignment, evolution, motif prediction, promoter cis-acting element analysis, and gene differential expression under different salt treatments using both a pot experiment and hydroponic experiment. The results showed that the R2R3-MYB genes of C.paliurus conserved functional domains, whereas four R2R3-MYB genes that might respond to salt stress via regulating plant hormone signals were identified in this study. This work provides a basis for further functional characterization of R2R3-MYB TFs in C. paliurus.

Metabolome and Transcriptome Analyses Unravel the Molecular Regulatory Mechanisms Involved in Photosynthesis of Cyclocarya paliurus under Salt Stress.

Photosynthesis is the primary life process in nature, and how to improve photosynthetic capacity under abiotic stresses is crucial to carbon fixation and plant productivity. As a multi-functional tree species, the leaves of Cyclocarya paliurus possess antihypertensive and hypoglycemic activities. However, the regulatory mechanism involved in the photosynthetic process of C. paliurus exposed to salinity has not yet been elucidated. In this study, the photosynthetic characteristics of C. paliurus seedlings, such as photosynthetic rate (Pn), stomatal conductance (Gs), and electron transfer rate (ETR), were investigated under different salt concentrations, while the metabolome and transcriptome analyses were conducted to unravel its molecular regulatory mechanisms. Salt stress not only significantly affected photosynthetic characteristics of C. paliurus seedlings, but also severely modified the abundance of metabolites (such as fumaric acid, sedoheptulose-7-phosphate, d-fructose-1,6-bisphosphate, and 3-phospho-d-glyceroyl phosphate) involved in central carbon metabolism, and the expression of photosynthetic genes. Through the co-expression network analysis, a total of 27 transcription factors (including ERFs, IDD, DOF, MYB, RAP) were identified to regulate photosynthetic genes under salt stress. Our findings preliminarily clarify the molecular regulatory network involved in the photosynthetic process of C. paliurus under salt stress and would drive progress in improving the photosynthetic capacity and productivity of C. paliurus by molecular technology.

Cyclocarya paliurus triterpenoids attenuate glomerular endothelial injury in the diabetic rats via ROCK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclocarya paliurus (Batal.) Iljinskaja. (C. paliurus) is a distinctive traditional Chinese herb, with remarkable hypoglycemic capacity. Emerging evidence suggested that glomerular endothelial injury is a crucial pathological process of diabetic kidney disease (DKD). Our previous research found that C. paliurus triterpenoids fraction (CPT) has ameliorative effects on DKD. However, whether C. paliurus could counteract the glomerular endothelial injury of DKD is still undefined. AIM OF THE STUDY: We aimed to investigate the effects of CPT on glomerular endothelial function and explore its underlying mechanisms with in vivo and in vitro experiments. MATERIALS AND METHODS: The effects and possible mechanisms of CPT on glomerular endothelial injury in streptozotocin (STZ)-induced diabetic rats and H2O2-challenged primary rat glomerular endothelial cells were successively investigated. RESULTS: In vivo, we found that CPT treatment obviously decreased the levels of blood glucose, microalbumin, BUN and mesangial expansion. Additionally, CPT could ameliorate renal endothelium function by reducing the content of VCAM-1 and ICAM-1, and blocking the loss of glycocalyx. In vitro, CPT could also alleviate H2O2-induced endothelial injury. Mechanistically, CPT remarkably increased the phosphorylation levels of Akt and eNOS, decreased the expression of ROCK and Arg2in vivo and in vitro. Noticeably, the favorable effects mediated by CPT were abolished following ROCK overexpression with plasmid transfection. CONCLUSION: These findings suggested that CPT could be sufficient to protect against glomerular endothelial injury in DKD through regulating ROCK pathway.

3ß,23-Dihydroxy-12-ene-28-ursolic Acid Isolated from Cyclocarya paliurus Alleviates NLRP3 Inflammasome-Mediated Gout via PI3K-AKT-mTOR-Dependent Autophagy.

Gout is regarded as a painful inflammatory arthritis induced by the deposition of monosodium urate crystals in joints and soft tissues. Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-mediated IL-1ß production plays a crucial role in the pathological process of gout. Cyclocarya paliurus (CP) tea was found to have an effect on reducing the blood uric acid level of people with hyperuricemia and gout. However, its medicinal ingredients and mechanism for the treatment of gout are still unclear. Thus, this study was designed to investigate the effects of the active triterpenoids isolated from C. paliurus on gout and explore the underlying mechanism. The results showed that compound 2 (3ß,23-dihydroxy-12-ene-28-ursolic acid) from C. paliurus significantly decreased the protein expression of IL-1ß, caspase-1, pro-IL-1ß, pro-caspase-1, and NLRP3. Furthermore, the production of ROS in the intracellular was reduced after compound 2 treatment. However, ROS agonist rotenone remarkably reversed the inhibitory effect of compound 2 on the protein expression of NLRP3 inflammasome. Additionally, the expression level of LC3 and the ratio of LC3II/LC3I were increased, but the expression level of p62 was suppressed by compound 2 whereas an autophagy inhibitor 3-methyladenine (3-MA) significantly abolished the inhibitory effects of compound 2 on the generation of ROS and the protein expression of NLRP3 inflammasome. Moreover, compound 2 could ameliorate the expression ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR. Interestingly, mTOR activator MHY-1485 could block the promotion effect of compound 2 on autophagy regulation and inhibitory effect of compound 2 on induction of ROS and IL-1ß. In conclusion, these findings suggested that compound 2 may effectively improve NLRP3 inflammasome-mediated gout via PI3K-AKT-mTOR-dependent autophagy and could be further investigated as a potential agent against gout.

Two new triterpenoids from the leaves of Cyclocarya paliurus (Batalin) Iljinskaja.

Two previously undescribed triterpenoids (1-2), along with thirteen known compounds (3-15) were isolated from a CHCl3-soluble extract of the leaves of Cyclocarya paliurus. Their structures were established on the basis of chemical and spectroscopic approaches. These compounds were assessed for their therapeutic effects on diabetic nephropathy (DN)-evoked fibrosis through High-Glucose and transforming growth factor-ß1 (TGF-ß1) challenged HK-2 cells. Among them, compounds 3, 5 and 8 could remarkedly decrease the level of fibronectin to relieve DN with 27.66 ± 2.77%, 6.09 ± 0.57% and 17.74 ± 5.83% inhibition rate at 10 µM, 10 µM and 1 µM, respectively.

RNA in situ hybridization and expression of related genes regulating the accumulation of triterpenoids in Cyclocarya paliurus.

Cyclocarya paliurus (Batal.) Iljinskaja, a woody medicinal species in the Juglandaceae, grows extensively in subtropical areas of China. Triterpenoids in the leaves have health-promoting effects, including hypoglycemic and hypolipidemic activities. To understand triterpenoid biosynthesis, transport and accumulation in C. paliurus during the growing season, gene cloning, gene expression and RNA in situ hybridization of related genes were used, and accumulation was examined in various organs. The complete coding sequences (CDSs) of three genes, CpHMGR, CpDXR and CpSQS, were obtained from GenBank and RACE. RNA in situ hybridization signals of the three genes mainly occurred in the epidermis, palisade tissue, phloem and xylem of leaf, shoot and root, with the signals generally consistent with the accumulation of metabolites in tissues, except in the xylem. Both gene expression and triterpenoid accumulations showed seasonal variations in all organs. However, total triterpenoid content in the leaves was significantly higher than that in the shoots, with the maximum in shoots in August and in leaves in October. According to Pearson correlation analysis, triterpenoid accumulation in the leaves was significantly positively related with the relative expression of CpSQS. However, the relation between gene expression and accumulation was dependent on the role of the gene in the pathway as well as on the plant organ. The results suggested that most of the intermediates catalyzed by CpHMGR and CpDXR in young shoots and roots were used in growth and flowering in the spring, whereas subsequent triterpenoid biosynthesis in the downstream catalyzed by CpSQS mainly occurred in the leaves by using transferred and in situ intermediates as substrates. Thus, this study provides a reference to improve triterpenoid accumulation in future C. paliurus plantations.

Genotypic variations in 107 poplar clones grown on a short-term waterlogging site: Long-term (1992-2015) data on survival rate, growth performance and branching traits.

The dataset reported data of survival rate, growth performance, branching architecture derived from 107 different poplar clones, including 104 introduced poplar clones (Populus deltoides) and 3 local extended poplar clones, measured within a long-term field experiment in lowland plantations in China. After 24 growing seasons from 1992 to 2015, the suvival rate for all the 107 clones, and tree height, diameter at breast-height (1.3 m), and tree volume of each tree were measured and calculated for all the 107 clones, in total of 301 alive trees (one to 9 trees for each clone), in three replicated plots. Subsequently, a total of 17 potential clones were selected by approximately 15% selection intensity, and 17 sample trees (one mean tree for each clone) were harvested for the investigation of the branching number, branching angle and base diameter of first-order branches. For the interpretation of the results of this experiment the readers are referred to Ref. [1]. The data presented in this article will aid selection of superior poplar clones for study and future applications in the similar lowland sites. The data on the suvival rate and growth performance of 107 poplar clones can help farmers and breeders to designing optimal schemes to increase timber yield and log assortment in poplar plantations. Raw data on tree structure parameters and branching traits can be used to evaluate the different performance of the clones, testing their different spacing and rotation requirement, and also designing innovative plantation schemes.

Stem Canker on Cyclocarya paliurus Is Caused by Botryosphaeria dothidea.

Cyclocarya paliurus, an important endangered plant in China, has considerable medicinal, timber, and horticultural value. However, little is known about diseases that affect its health. In recent years, stem canker diseases on C. paliurus have been observed frequently in newly established nurseries in Jiangsu Province, China. Symptomatic trees showed elliptical, sunken lesions on the bark, with internal discoloration, leading to enlarging cankers with delineated margins. Pathogenicity tests with fungi isolated from symptomatic samples reproduced typical canker symptoms on both detached branches and potted plants of C. paliurus. Moreover, conidia from pycnidia of isolate ZB-23 could also cause stem canker on C. paliurus. Through combined morphological observation and DNA sequences of ITS region, ß-tubulin, and translation elongation factor 1-α genes, the pathogen was identified as Botryosphaeria dothidea. Multigene maximum likelihood and maximum parsimony phylogenetic analyses further supported the identification of the pathogen. To our knowledge, this is the first report of B. dothidea causing stem canker on C. paliurus in China.

Etiology of Cyclocarya paliurus Anthracnose in Jiangsu Province, China.

Cyclocarya paliurus is an extremely valuable and multifunctional tree species whose leaves have traditionally been used in used in medicine or as a medicinal tea in China. In recent years, anthracnose has been frequently observed on young leaves of C. paliurus in several nurseries located in Jiangsu Province, resulting in great yield and quality losses. To date, no information is available about the prevalence of C. paliurus anthracnose in China. The main purpose of the present study was to characterize the etiology of C. paliurus anthracnose. Phylogenetic analysis of the eight-loci concatenated dataset revealed that all 44 single-spore Colletotrichum isolates belonged to three species in the Colletotrichum gloeosporioides species complex, namely, Colletotrichum aenigma, Colletotrichum fructicola, and C. gloeosporioides sensu stricto. Phenotypic features, including the colony appearance and the morphology of conidia, appressoria, and ascospores, were consistent with the phylogenetic grouping. Virulence tests validated that the three Colletotrichum species could cause typical symptoms of anthracnose on C. paliurus leaves, similar to those observed in the field. The optimum mycelial growth temperature ranged from 25 to 30°C for all representative isolates, while C. gloeosporioides s. s. isolates exhibited greater tolerance to high temperature (40°C). Fungicide sensitivity assays indicated that all three Colletotrichum species were sensitive to tetramycin, which may be a potential alternative for the management of C. paliurus anthracnose. To our knowledge, this study provides the first report of C. aenigma, C. fructicola, and C. gloeosporioides s. s. causing C. paliurus anthracnose in China as well as in the world.

Localization and dynamic change of saponins in Cyclocarya paliurus (Batal.) Iljinskaja.

Cyclocarya paliurus is a unique tree species of that grows in southern China. The tree contains distinctive saponins in the leaf that has hypoglycemic and hypolipidemic effects. It was aimed to detect localization of saponins and suitable time of harvest for medicinal uses. Histochemical, cytochemical localization and UV-spectrophotometry were carried out in C. paliurus plant. We found that in all organs, the saponins were primarily located in the parenchyma cells and the highest saponins accumulation was in the palisade tissue in leaves. Cytochemical localization results indicated that saponins were mainly distributed in the chloroplast, vesicle, and plasmalemma. On average, the total saponins content in leaves (20.57 mg·g-1) was two and three times greater than in root (10.19 mg·g-1) and shoot (6.20 mg·g-1), respectively. Moreover, the saponins content in the leaf and root exhibited fluctuations, which were highest in September. Considering saponins levels and biomass, we conclude that harvesting all leaves in September is an economical and effective strategy for medicinal use in C. paliurus.

Morphological Characterization of Flower Buds Development and Related Gene Expression Profiling at Bud Break Stage in Heterodichogamous Cyclocarya paliurus (Batal.) lljinskaja.

Cyclocarya paliurus (Batal.) Iljinskaja, a unique species growing in southern China, is a multi-function tree species with medicinal, healthcare, material, and ornamental values. So far, sexual reproduction is the main method for extensive cultivation of C. paliurus plantations, but this is limited by low seed plumpness resulted from the character of heterodichogamy. Phenological observations have revealed the asynchronism of flower development in this species. However, its molecular mechanism remains largely unknown. To reveal molecular mechanism of heterodichogamy in C. paliurus, transcriptome of female (F) and male (M) buds from two mating types (protandry, PA; protogyny, PG) at bud break stage were sequenced using Illumina Hiseq 4000 platform. The expression patterns of both 32 genes related to flowering and 58 differentially expressed transcription factors (DETFs) selected from 6 families were divided four groups (PG-F, PG-M, PA-F, and PA-M) into two categories: first flowers (PG-F and PA-M) and later flowers (PA-F and PG-M). The results indicated that genes related to plant hormones (IAA, ABA, and GA) synthesis and response, glucose metabolism, and transcription factors (especially in MIKC family) played significant roles in regulating asynchronism of male and female flowers in the same mating type. The expression of DETFs showed two patterns. One contained DETFs up-regulated in first flowers in comparison to later flowers, and the other was the reverse. Nine genes related to flowering were selected for qRT-PCR to confirm the accuracy of RNA-seq, and generally, the RPKM values of these genes were consistent with the result of qRT-PCR. The results of this work could improve our understanding in asynchronism of floral development within one mating type in C. paliurus at transcriptional level, as well as lay a foundation for further study in heterodichogamous plants.