PtWRKY2, a WRKY transcription factor from Pinellia ternata confers heat tolerance in Arabidopsis.

High temperatures are a major stress factor that limit the growth of Pinellia ternata. WRKY proteins widely distribute in plants with the important roles in plant growth and stress responses. However, WRKY genes have not been identified in P. ternata thus far. In this study, five PtWRKYs with four functional subgroups were identified in P. ternata. One group III WRKY transcription factor, PtWRKY2, was strongly induced by high temperatures, whereas the other four PtWRKYs were suppressed. Analysis of transcription factor characteristics revealed that PtWRKY2 localized to the nucleus and specifically bound to W-box elements without transcriptional activation activity. Overexpression of PtWRKY2 increased the heat tolerance of Arabidopsis, as shown by the higher percentage of seed germination and survival rate, and the longer root length of transgenic lines under high temperatures compared to the wild-type. Moreover, PtWRKY2 overexpression significantly decreased reactive oxygen species accumulation by increasing the catalase, superoxide dismutase, and peroxidase activities. Furthermore, the selected heat shock-associated genes, including five transcription factors (HSFA1A, HSFA7A, bZIP28, DREB2A, and DREB2B), two heat shock proteins (HSP70 and HSP17.4), and three antioxidant enzymes (POD34, CAT1, and SOD1), were all upregulated in transgenic Arabidopsis. The study identifies that PtWRKY2 functions as a key transcriptional regulator in the heat tolerance of P. ternata, which might provide new insights into the genetic improvement of P. ternata.

Endogenous Hormone Levels and Transcriptomic Analysis Reveal the Mechanisms of Bulbil Initiation in Pinellia ternata.

Pinellia ternata is a medicinal plant that has important pharmacological value, and the bulbils serve as the primary reproductive organ; however, the mechanisms underlying bulbil initiation remain unclear. Here, we characterized bulbil development via histological, transcriptomic, and targeted metabolomic analyses to unearth the intricate relationship between hormones, genes, and bulbil development. The results show that the bulbils initiate growth from the leaf axillary meristem (AM). In this stage, jasmonic acid (JA), abscisic acid (ABA), isopentenyl adenosine (IPA), and salicylic acid (SA) were highly enriched, while indole-3-acetic acid (IAA), zeatin, methyl jasmonate (MeJA), and 5-dexoxystrigol (5-DS) were notably decreased. Through OPLS-DA analysis, SA has emerged as the most crucial factor in initiating and positively regulating bulbil formation. Furthermore, a strong association between IPA and SA was observed during bulbil initiation. The transcriptional changes in IPT (Isopentenyltransferase), CRE1 (Cytokinin Response 1), A-ARR (Type-A Arabidopsis Response Regulator), B-ARR (Type-B Arabidopsis Response Regulator), AUX1 (Auxin Resistant 1), ARF (Auxin Response Factor), AUX/IAA (Auxin/Indole-3-acetic acid), GH3 (Gretchen Hagen 3), SAUR (Small Auxin Up RNA), GA2ox (Gibberellin 2-oxidase), GA20ox (Gibberellin 20-oxidase), AOS (Allene oxide synthase), AOC (Allene oxide cyclase), OPR (Oxophytodienoate Reductase), JMT (JA carboxy l Methyltransferase), COI1 (Coronatine Insensitive 1), JAZ (Jasmonate ZIM-domain), MYC2 (Myelocytomatosis 2), D27 (DWARF27), SMAX (Suppressor of MAX2), PAL (Phenylalanine Ammonia-Lyase), ICS (Isochorismate Synthase), NPR1 (Non-expressor of Pathogenesis-related Genes1), TGA (TGACG Sequence-specific Binding), PR-1 (Pathogenesis-related), MCSU (Molybdenium Cofactor Sulfurase), PP2C (Protein Phosphatase 2C), and SnRK (Sucrose Non-fermenting-related Protein Kinase 2) were highly correlated with hormone concentrations, indicating that bulbil initiation is coordinately controlled by multiple phytohormones. Notably, eight TFs (transcription factors) that regulate AM initiation have been identified as pivotal regulators of bulbil formation. Among these, WUS (WUSCHEL), CLV (CLAVATA), ATH1 (Arabidopsis Thaliana Homeobox Gene 1), and RAX (Regulator of Axillary meristems) have been observed to exhibit elevated expression levels. Conversely, LEAFY demonstrated contrasting expression patterns. The intricate expression profiles of these TFs are closely associated with the upregulated expression of KNOX(KNOTTED-like homeobox), suggesting a intricate regulatory network underlying the complex process of bulbil initiation. This study offers a profound understanding of the bulbil initiation process and could potentially aid in refining molecular breeding techniques specific to P. ternata.

Integrated analysis of transcriptome and miRNAome reveals the heat stress response of Pinellia ternata seedlings.

Pinellia ternata (Thunb.) Briet., a valuable herb native to China, is susceptible to the "sprout tumble" phenomenon because of high temperatures, resulting in a significant yield reduction. However, the molecular regulatory mechanisms underlying the response of P. ternata to heat stress are not well understood. In this study, we integrated transcriptome and miRNAome sequencing to identify heat-response genes, microRNAs (miRNAs), and key miRNA-target pairs in P. ternata that differed between heat-stress and room-temperature conditions. Transcriptome analysis revealed extensive reprogramming of 4,960 genes across various categories, predominantly associated with cellular and metabolic processes, responses to stimuli, biological regulation, cell parts, organelles, membranes, and catalytic and binding activities. miRNAome sequencing identified 1,597 known/conserved miRNAs that were differentially expressed between the two test conditions. According to the analysis, genes and miRNAs associated with the regulation of transcription, DNA template, transcription factor activity, and sequence-specific DNA binding pathways may play a major role in the resistance to heat stress in P. ternata. Integrated analysis of the transcriptome and miRNAome expression data revealed 41 high-confidence miRNA-mRNA pairs, forming 25 modules. MYB-like proteins and calcium-responsive transcription coactivators may play an integral role in heat-stress resistance in P. ternata. Additionally, the candidate genes and miRNAs were subjected to quantitative real-time polymerase chain reaction to validate their expression patterns. These results offer a foundation for future studies exploring the mechanisms and critical genes involved in heat-stress resistance in P. ternata.

Transcriptomic and metabolomic profiling provide insight into the role of sugars and hormones in leaf senescence of Pinellia ternata.

KEY MESSAGE: The interaction network and pathway map uncover the potential crosstalk between sugar and hormone metabolisms as a possible reason for leaf senescence in P. ternata. Pinellia ternata, an environmentally sensitive medicinal plant, undergoes leaf senescence twice a year, affecting its development and yield. Understanding the potential mechanism that delays leaf senescence could theoretically decrease yield losses. In this study, a typical senescent population model was constructed, and an integrated analysis of transcriptomic and metabolomic profiles of P. ternata was conducted using two early leaf senescence populations and two stay-green populations. The result showed that two key gene modules were associated with leaf senescence which were mainly enriched in sugar and hormone signaling pathways, respectively. A network constructed by unigenes and metabolisms related to the obtained two pathways revealed that several compounds such as D-arabitol and 2MeScZR have a higher significance ranking. In addition, a total of 130 hub genes in this network were categorized into 3 classes based on connectivity. Among them, 34 hub genes were further analyzed through a pathway map, the potential crosstalk between sugar and hormone metabolisms might be an underlying reason of leaf senescence in P. ternata. These findings address the knowledge gap regarding leaf senescence in P. ternata, providing candidate germplasms for molecular breeding and laying theoretical basis for the realization of finely regulated cultivation in future.

Comprehensive in silico characterization of NAC transcription factor family of Pinellia ternata and functional analysis of PtNAC66 under high-temperature tolerance in transgenic Arabidopsis thaliana.

Pinellia ternata, a valuable Chinese herb, suffers yield reduction due to "sprout tumble" under high temperatures. However, the mechanisms underlying its high-temperature stress remain poorly understood. NAM, ATAF1/2, and CUC2 (NAC) transcription factors regulate plant tissue growth and abiotic stress. Hence, there has been no comprehensive research conducted on NAC transcription factors in P. ternata. We identified 98 PtNAC genes unevenly distributed across 13 chromosomes, grouped into 15 families via phylogenetic analysis. Gene expression analysis revealed diverse expression patterns of PtNAC genes in different tissue types. Further studies revealed that PtNAC5/7/17/35/43/47/57/66/86 genes were highly expressed in various tissues of P. ternata and induced by heat stress, among which PtNAC66 was up-regulated at the highest folds induced by heat temperature. PtNAC66 is a nuclear protein that can selectively bind to the cis-responsive region NACRS but lacks the ability to activate transcription in yeast. For further research, PtNAC66 was cloned and transgenic Arabidopsis was obtained. PtNAC66 overexpression increased high-temperature tolerance compared to wild-type plants. Transcriptome profiling demonstrated that overexpression of PtNAC66 led to significant modification of genes responsible for regulating binding, catalytic activity, transcription regulator activity and transporter activity response genes. Additionally, PtNAC66 was found to bind the promoters of CYP707A3, MYB102 and NAC055, respectively, and inhibited their expression, affecting the high-temperature stress response in Arabidopsis. Our research established the foundation for functional studies of PtNAC genes in response to high-temperature forcing by characterizing the P. ternata NAC gene family and examining the biological role of PtNAC66 in plant high-temperature tolerance.

Assembly and comparative analysis of the complete mitochondrial genome of Pinellia ternata.

Pinellia ternata is an important natural medicinal herb in China. However, it is susceptible to withering when exposed to high temperatures during growth, which limits its tuber production. Mitochondria usually function in stress response. The P . ternata mitochondrial (mt) genome has yet to be explored. Therefore, we integrated PacBio and Illumina sequencing reads to assemble and annotate the mt genome of P . ternata . The circular mt genome of P . ternata is 876 608bp in length and contains 38 protein-coding genes (PCGs), 20 tRNA genes and three rRNA genes. Codon usage, sequence repeats, RNA editing and gene migration from chloroplast (cp) to mt were also examined. Phylogenetic analysis based on the mt genomes of P . ternata and 36 other taxa revealed the taxonomic and evolutionary status of P . ternata . Furthermore, we investigated the mt genome size and GC content by comparing P . ternata with the other 35 species. An evaluation of non-synonymous substitutions and synonymous substitutions indicated that most PCGs in the mt genome underwent negative selection. Our results provide comprehensive information on the P . ternata mt genome, which may facilitate future research on the high-temperature response of P . ternata and provide new molecular insights on the Araceae family.

Transcriptome Analysis Reveals Long Non-Coding RNAs Involved in Shade-Induced Growth Promotion in Pinellia ternata.

BACKGROUND: High temperature and drought environments are important limiting factors for Pinellia ternata growth, whereas shading can promote growth by relieving these stresses. However, the mechanism of growth promotion by shading in P. ternata is unknown. Long non-coding RNAs (lncRNAs) play important roles in the plant's growth and environmental response, but few analyses of lncRNAs in P. ternata have been reported. METHODS: We performed lncRNAs analysis of P. ternata in response to shading using RNA-seq data from our previous studies. A total of 13,927 lncRNAs were identified, and 145 differentially expressed lncRNAs (DELs) were obtained from the comparisons of 5 days shade (D5S) vs. 5 days of natural light (D5CK), 20 days of shade (D20S) vs. 20 days of natural light (D20CK), D20S vs. D5S, and D20CK vs. D5CK. Of these, 119 DELs (82.07%) were generated from the D20S vs. D20CK comparison. RESULTS: Gene ontology (GO) analysis indicated that the reactive oxygen (ROS) metabolism and programmed cell death (PCD) processes might regulate shade-induced growth promotion. The "signal transduction" and "environmental adaptation" in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for lncRNA-mRNA regulatory network construction and showed that the lncRNAs might mediate P. ternata growth by regulating ROS accumulation and light signals. CONCLUSIONS: This study explores lncRNAs' functions and regulatory mechanisms related to P. ternata growth and lays a foundation for further research on P. ternata.

mRNA-Seq and miRNA-Seq Analyses Provide Insights into the Mechanism of Pinellia ternata Bulbil Initiation Induced by Phytohormones.

Pinellia ternata (Thunb.) Breit (abbreviated as P. ternata) is a plant with an important medicinal value whose yield is restricted by many factors, such as low reproductive efficiency and continuous cropping obstacles. As an essential breeding material for P. ternata growth and production, the bulbils have significant advantages such as a high survival rate and short breeding cycles. However, the location effect, influencing factors, and molecular mechanism of bulbil occurrence and formation have not been fully explored. In this study, exogenously applied phytohormones were used to induce in vitro petiole of P. ternata to produce bulbil structure. Transcriptome sequencing of mRNA and miRNA were performed in the induced petiole (TCp) and the induced bulbil (TCb). Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for the identification of key genes and pathways involved in bulbil development. A total of 58,019 differentially expressed genes (DEGs) were identified. The GO and KEGG analysis indicated that DEGs were mainly enriched in plant hormone signal transduction and the starch and sucrose metabolism pathway. The expression profiles of miR167a, miR171a, and miR156a during bulbil induction were verified by qRT-PCR, indicating that these three miRNAs and their target genes may be involved in the process of bulbil induction and play an important role. However, further molecular biological experiments are required to confirm the functions of the identified bulbil development-related miRNAs and targets.

Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

OBJECTIVE: In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). RESULTS: The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20 g·l-1 cellulase and 15 g·l-1 macerozyme for 6 h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. CONCLUSIONS: We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

Transcriptome Profiling Reveals Differential Gene Expression during the Process of Microtuber Formation in Pinellia ternata.

Using petiole material as explants and directly inducing the formation of microtubers without going through the callus stage is an essential way to rapidly expand scarce medical plants such as Pinellia ternata. However, the early molecular mechanism underlying the formation of the microtuber is largely elusive. Here, we conducted cytology and dynamic transcriptome analyses of inchoate microtubers in Pinellia explants and identified 1092 differentially expressed genes after their cultivation in vitro for 0, 5, and 15 days. Compared with 0 day, the number and size of the microtuber cells were larger at 5 and 15 days of culture. Detailed categorization revealed that the differentially expressed genes were mainly related to responses to stimulus, biological regulation, organelles, membranes, transcription factor activity, and protein binding. Further analysis revealed that the microtuber at different incubation days exhibited quite a difference in both hormone signaling pathway transduction and the regulation pattern of transcription factors. Therefore, this study contributes to a better understanding of the early molecular regulation during the formation of the microtuber and provides new insights for the study of the rapid expansion of P. ternata and other medical plants.

Integrative Analysis of Metabolomic and Transcriptomic Data Reveals the Mechanism of Color Formation in Corms of Pinellia ternata.

Pinellia ternata (Thunb.) Breit. (P. ternata) is a very important plant that is commonly used in traditional Chinese medicine. Its corms can be used as medicine and function to alleviate cough, headache, and phlegm. The epidermis of P. ternata corms is often light yellow to yellow in color; however, within the range of P. ternata found in JingZhou City in Hubei Province, China, there is a form of P. ternata in which the epidermis of the corm is red. We found that the total flavonoid content of red P. ternata corms is significantly higher than that of yellow P. ternata corms. The objective of this study was to understand the molecular mechanisms behind the difference in epidermal color between the two forms of P. ternata. The results showed that a high content of anthocyanidin was responsible for the red epidermal color in P. ternata, and 15 metabolites, including cyanidin-3-O-rutinoside-5-O-glucoside, cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoside, were screened as potential color markers in P. ternata through metabolomic analysis. Based on an analysis of the transcriptome, seven genes, including PtCHS1, PtCHS2, PtCHI1, PtDFR5, PtANS, PtUPD-GT2, and PtUPD-GT3, were found to have important effects on the biosynthesis of anthocyanins in the P. ternata corm epidermis. Furthermore, two transcription factors (TFs), bHLH1 and bHLH2, may have regulatory functions in the biosynthesis of anthocyanins in red P. ternata corms. Using an integrative analysis of the metabolomic and transcriptomic data, we identified five genes, PtCHI, PtDFR2, PtUPD-GT1, PtUPD-GT2, and PtUPD-GT3, that may play important roles in the presence of the red epidermis color in P. ternata corms.

Physiological and transcriptional responses to heat stress in a typical phenotype of Pinellia ternata.

Pinellia ternata is an important medicinal plant, and its growth and development are easily threatened by high temperature. In this study, comprehensive research on physiological, cytological and transcriptional responses to different levels of heat stress were conducted on a typical phenotype of P. ternata. First, P. ternata exhibited tolerance to the increased temperature, which was supported by normal growing leaves, as well as decreased and sustained photosynthetic parameters. Severe stress aggravated the damages, and P. ternata displayed an obvious leaf senescence phenotype, with significantly increased SOD and POD activities (46% and 213%). In addition, mesophyll cells were seriously damaged, chloroplast thylakoid was fuzzy, grana lamellae and stroma lamellae were obviously broken, and grana thylakoids were stacked, resulting in a dramatically declined photosynthetic rate (74.6%). Moreover, a total of 16 808 genes were significantly differential expressed during this process, most of which were involved in photosynthesis, transmembrane transporter activity and plastid metabolism. The number of differentially expressed transcription factors in MYB and bHLH families was the largest, indicating that these genes might participate in heat stress response in P. ternata. These findings provide insight into the response to high temperature and facilitate the standardized cultivation of P. ternata.

Integrated analysis of metabolome and transcriptome reveals key candidate genes involved in flavonoid biosynthesis in Pinellia ternata under heat stress.

Pinellia ternata (Thunb.) Breit. is an important traditional Chinese medicinal herb and very sensitive to high temperatures. To gain a better understanding of flavonoid biosynthesis under heat stress in P. ternata, we performed integrated analyses of metabolome and transcriptome data. P. ternata plants were subjected to a temperature of 38 °C, and samples were collected after 10 d of treatment. A total of 502 differential accumulated metabolites and 5040 different expressed transcripts were identified, with flavonoid biosynthesis predominantly enriched. Integrated metabolomics and transcriptome analysis showed that high temperature treatment upregulated the expression of CYP73A and downregulated the expression of other genes (such as HCT, CCoAOMT, DFR1, DFR2), which might inhibit the biosynthesis of the downstream metabolome, including such metabolites as chlorogenic acid, pelargonidin, cyanidin, and (-)-epigallocatechin in the flavonoid biosynthesis pathway. The transcription expression levels of these genes were validated by real-time PCR. Our results provide valuable insights into flavonoid composition and accumulation patterns and the candidate genes participating in the flavonoid biosynthesis pathways under heat stress in P. ternata.

Transcriptome analysis reveals important regulatory genes and pathways for tuber color variation in Pinellia ternata (Thunb.) Breit.

During the growth of Pinellia ternata (Thunb.) Breit. (P. ternata), the violet-red skin was occasionally produced spontaneously under natural cultivation. However, the specific mechanism leading to the color change is still unclear. This study performed transcriptomes in violet-red and pale-yellow skin and their peeled tubers of P. ternata, and the total flavonoids and anthocyanin contents were also determined. The results showed that the majority of genes involved in anthocyanin production were considerably increased in the violet-red skin of P. ternata tuber compared to the pale-yellow skin. Especially, phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) showed a remarkable increase in gene expression levels. Notably, shikimate O-hydroxycinnamoyltransferase (HCT), naringenin 3-dioxygenase (F3H), flavanone 4-reductase (DFR), and anthocyanidin synthase (ANS) were explicitly expressed in violet-red skin of P. ternata tuber, while undetectable in pale-yellow skin. The upregulation of these genes may explain the accumulation of anthocyanins, which forms the violet-red skin of P. ternata tuber. The transcription factors, including C2H2, bZIP, ERF, GATA, bHLH, C3H, NAC, MYB-related, and MYB families, might trigger the skin color change in P. ternata. The entire anthocyanin content in the violet-red skin of P. ternata tuber was 71.10 µg/g, and pale-yellow skin was 7.74 µg/g. According to phenotypic and transcriptome results, the elevated expression levels of genes linked to the synthesis of anthocyanins considerably contributed to the violet-red skin alterations in P. ternata tuber. This study provides a new understanding of the formation of the violet-red skin, lays a theoretical foundation for the cultivation of unique varieties of P. ternata, and provides transcriptome data for further study of the differences between different colors of P. ternata.

Comprehensive evaluation of Pinellia ternata germplasm resources based on phenotypic trait classification / 中国中药杂志

The evaluation of germplasm resources is the prerequisite for the development, utilization, and conservation of Chinese medicinal resources. The selection of excellent germplasm is the key to the breeding and orderly production of Pinellia ternata. In this study, 21 germplasm materials of P. ternata from major production areas in China were collected and analyzed for population diversity after phenotypic preliminary screening. The results have revealed that the P. ternata population has abundant phenotypic variation, and the phenotypic changes could be divided into five phenotypes in terms of organ trait variation. Further analysis of variation in 20 quantitative traits of the population revealed that the coefficient of variation for adenosine content(339.05%) was the largest, while the coefficient of variation for the underground plant height(16.35%) was the smallest. Correlation analysis showed that there was a strong correlation among various traits, with 52 pairs of traits showing highly significant correlation(P<0.01) and 19 pairs of traits showing a significant correlation(P<0.05). The 21 germplasms in the test could be classified into three major clusters by cluster analysis, with Cluster Ⅱ having the highest number and content of nucleosides, making it suitable for the selection and breeding of P. ternata varieties with high content of nucleosides. The yield in Cluster Ⅲ was higher than that in other groups, making it suitable for the selection and breeding of P. ternata varieties with a high yield. All trait indicators could be simplified into five principal component factors through principal component analysis, and the cumulative contribution rate was up to 86.04%. Further, comprehensive analysis using membership function and stepwise regression analysis identified nine traits, such as plant height, main leaf length, and underground plant height as characteristic indicators for the comprehensive evaluation of germplasm resources of P. ternata. BX007, BX008, and BX005 were identified as germplasms with both high yield and high uridine content, with BX007 having the highest uridine content of 479.51 μg·g~(-1). It belonged to the germplasm of P. ternata with double bulbils and could be cultivated as a potential good variety. Based on the phenotypic classification of P. ternata, systematic resource evaluation was carried out in this study, which could lay a foundation for the excavation of genetic resources and the breeding of new varieties of P. ternata.

Physiological and transcriptional responses to heat stress in a typical phenotype of Pinellia ternata / 中国天然药物

Pinellia ternata is an important medicinal plant, and its growth and development are easily threatened by high temperature. In this study, comprehensive research on physiological, cytological and transcriptional responses to different levels of heat stress were conducted on a typical phenotype of P. ternata. First, P. ternata exhibited tolerance to the increased temperature, which was supported by normal growing leaves, as well as decreased and sustained photosynthetic parameters. Severe stress aggravated the damages, and P. ternata displayed an obvious leaf senescence phenotype, with significantly increased SOD and POD activities (46% and 213%). In addition, mesophyll cells were seriously damaged, chloroplast thylakoid was fuzzy, grana lamellae and stroma lamellae were obviously broken, and grana thylakoids were stacked, resulting in a dramatically declined photosynthetic rate (74.6%). Moreover, a total of 16 808 genes were significantly differential expressed during this process, most of which were involved in photosynthesis, transmembrane transporter activity and plastid metabolism. The number of differentially expressed transcription factors in MYB and bHLH families was the largest, indicating that these genes might participate in heat stress response in P. ternata. These findings provide insight into the response to high temperature and facilitate the standardized cultivation of P. ternata.

[Comprehensive evaluation of Pinellia ternata germplasm resources based on phenotypic trait classification].

The evaluation of germplasm resources is the prerequisite for the development, utilization, and conservation of Chinese medicinal resources. The selection of excellent germplasm is the key to the breeding and orderly production of Pinellia ternata. In this study, 21 germplasm materials of P. ternata from major production areas in China were collected and analyzed for population diversity after phenotypic preliminary screening. The results have revealed that the P. ternata population has abundant phenotypic variation, and the phenotypic changes could be divided into five phenotypes in terms of organ trait variation. Further analysis of variation in 20 quantitative traits of the population revealed that the coefficient of variation for adenosine content(339.05%) was the largest, while the coefficient of variation for the underground plant height(16.35%) was the smallest. Correlation analysis showed that there was a strong correlation among various traits, with 52 pairs of traits showing highly significant correlation(P<0.01) and 19 pairs of traits showing a significant correlation(P<0.05). The 21 germplasms in the test could be classified into three major clusters by cluster analysis, with Cluster Ⅱ having the highest number and content of nucleosides, making it suitable for the selection and breeding of P. ternata varieties with high content of nucleosides. The yield in Cluster Ⅲ was higher than that in other groups, making it suitable for the selection and breeding of P. ternata varieties with a high yield. All trait indicators could be simplified into five principal component factors through principal component analysis, and the cumulative contribution rate was up to 86.04%. Further, comprehensive analysis using membership function and stepwise regression analysis identified nine traits, such as plant height, main leaf length, and underground plant height as characteristic indicators for the comprehensive evaluation of germplasm resources of P. ternata. BX007, BX008, and BX005 were identified as germplasms with both high yield and high uridine content, with BX007 having the highest uridine content of 479.51 µg·g~(-1). It belonged to the germplasm of P. ternata with double bulbils and could be cultivated as a potential good variety. Based on the phenotypic classification of P. ternata, systematic resource evaluation was carried out in this study, which could lay a foundation for the excavation of genetic resources and the breeding of new varieties of P. ternata.

[Identification, biological characteristics, and control of pathogen causing southern blight of Pinellia ternata].

In summer in 2020, Pinellia ternata in many planting areas in Hubei suffered from serious southern blight, as manifested by the yellowing and wilted leaves and rotten tubers. This study aims to identify the pathogen, clarify the biological characteristics of the pathogen, and screen fungicides. To be specific, the pathogen was isolated, purified, and identified, and the pathogenicity was detected according to the Koch's postulates. Moreover, the biological characteristics of the pathogen were analyzed. Furthermore, PDA plates and seedlings were used to determine the most effective fungicides. The results showed that the mycelia of the pathogen were white and villous with silk luster, which produced a large number of white to black brown sclerotia. The pathogen was identified as Athelia rolfsii by morphological observation and molecular identification based on LSU and TEF gene sequences. The optimum growth conditions for A. rolfsii were 30 ℃ and pH 5-8, and the optimum conditions for the germination of sclerotia were 25 ℃ and pH 7-9. Bacillus subtilis, difenoconazole, and flusilazole were identified as effective fungicides with PDA, and their half maximal effective concentration(EC_(50)) was all less than 5 mg·L~(-1). The effective fungicides screened with the seedlings were hymexazol and difenoconazole. Based on the screening experiments, difenoconazole can be used as the main agent for the prevention and treatment of southern blight.

Large-scale analysis of protein crotonylation reveals its diverse functions in Pinellia ternata.

BACKGROUND: Pinellia ternata is an important traditional medicine in China, and its growth is regulated by the transcriptome or proteome. Lysine crotonylation, a newly identified and important type of posttranslational modification, plays a key role in many aspects of cell metabolism. However, little is known about its functions in Pinellia ternata. RESULTS: In this study, we generated a global crotonylome analysis of Pinellia ternata and examined its overlap with lysine succinylation. A total of 2106 crotonylated sites matched on 1006 proteins overlapping in three independent tests were identified, and we found three specific amino acids surrounding crotonylation sites in Pinellia ternata: KcrF, K***Y**Kcr and Kcr****R. Gene Ontology (GO) and KEGG pathway enrichment analyses showed that two crucial alkaloid biosynthesis-related enzymes and many stress-related proteins were also highly crotonylated. Furthermore, several enzymes participating in carbohydrate metabolism pathways were found to exhibit both lysine crotonylation and succinylation modifications. CONCLUSIONS: These results indicate that lysine crotonylation performs important functions in many biological processes in Pinellia ternata, especially in the biosynthesis of alkaloids, and some metabolic pathways are simultaneously regulated by lysine crotonylation and succinylation.

Transcriptome Analysis Reveals an Essential Role of Exogenous Brassinolide on the Alkaloid Biosynthesis Pathway in Pinellia Ternata.

Pinellia ternata (Thunb.) Druce is a traditional medicinal plant containing a variety of alkaloids, which are important active ingredients. Brassinolide (BR) is a plant hormone that regulates plant response to environmental stress and promotes the accumulation of secondary metabolites in plants. However, the regulatory mechanism of BR-induced alkaloid accumulation in P. ternata is not clear. In this study, we investigated the effects of BR and BR biosynthesis inhibitor (propiconazole, Pcz) treatments on alkaloid biosynthesis in the bulbil of P. ternata. The results showed that total alkaloid content and bulbil yield was enhanced by 90.87% and 29.67% under BR treatment, respectively, compared to the control. We identified 818 (476 up-regulated and 342 down-regulated) and 697 (389 up-regulated and 308 down-regulated) DEGs in the BR-treated and Pcz-treated groups, respectively. Through this annotated data and the Kyoto encyclopedia of genes and genomes (KEGG), the expression patterns of unigenes involved in the ephedrine alkaloid, tropane, piperidine, pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis were observed under BR and Pcz treatments. We identified 11, 8, 2, and 13 unigenes in the ephedrine alkaloid, tropane, piperidine, and pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis, respectively. The expression levels of these unigenes were increased by BR treatment and were decreased by Pcz treatment, compared to the control. The results provided molecular insight into the study of the molecular mechanism of BR-promoted alkaloid biosynthesis.