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 / 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.

Variation analysis of genomic methylation induced by high temperature stress in Pinellia ternata / 中国中药杂志

Pinellia ternata belongs to the Araceae family and is a medicinal herb. The tuber is the medicinal organ with antitussive, antiemetic and anti-tumor activities. It is easy to encounter high temperature environment during the growth periods, leading to decrease of tuber production. At present, the mechanism of response to high temperature stress in P. ternata is still unknown. DNA methylation plays a vital role in plant protection against adversity stress as a way of epigenetic regulation. In this study, P. ternata was used as material for treatment of high temperature stress at 0 h, 6 h and 80 h, and methylation sensitive amplification polymorphism(MSAP) analysis was conducted on the changes of DNA methylation in its genome. The results showed that 20 pairs of MSAP primers were selected from 100 MSAP primers with multiple clear and uniform bands, and 353, 355 and 342 loci were amplified from materials of P. ternata treated in the high temperature stress 0 h, 6 h and 80 h, respectively. Cytosine methylation levels of CCGG context in the above materials were characterized as 60.91%, 44.79% and 44.74%, respectively. And the full methylation ratios were 16.71%, 22.25% and 29.24, respectively. It demonstrated that high temperature stress significantly induced the down-regulation of DNA methylation level and up-regulation of the full methylation rate in P. ternata genome. This study provides a preliminary theoretical reference for analyzing the mechanism of P. ternata responding to high temperature stress from the epigenetic perspective.

cDNA cloning and expression analysis of a mannose-binding lectin from Pinellia pedatisecta.

Pinellia pedatisecta agglutinin (PPA)is a very basic protein that accumulates in the tuber of P.pedatisecta .PPA is a hetero-tetramer protein of 40 kDa,composed of two polypeptide chains A (about 12 kDa)and two polypeptides chains B (about 12 kDa).The full-length cDNA of PPA was cloned from P.pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF)encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide.The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity,PPA-DOM 1 (polypeptides A)and PPA-DOM 2 (polypeptides B).PPA shared varying identities,ranging from 40% to 85%,with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix,spathe and tuber.Cloning of the ppa gene not only provides a basis for further investigation of its structure,expression and regulatory mechanism,but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

Genomic cloning and characterization of a PPA gene encoding a mannose-binding lectin from Pinellia pedatisecta

A gene encoding a mannose-binding lectin, Pinellia pedatisecta agglutinin (PPA), was isolated from leaves of Pinellia pedatisecta using genomic walker technology. The ppa contained an 1140-bp 5'-upstream region, a 771-bp open reading frame (ORF) and an 829-bp 3'-downstream region. The ORF encoded a precursor polypeptide of 256 amino acid residues with a 24-amino acid signal peptide. There were one putative TATA box and six possible CAAT boxes lying in the 5'-upstream region of ppa. The ppa showed significant similarity at the nucleic acid level with genes encoding mannose-binding lectins from other Araceae species such as Pinellia ternata, Arisaema heterophyllum, Colocasia esculenta and Arum maculatum. At the amino acid level, PPA also shared varying homology (ranging from 40% to 85%) with mannose-binding lectins from other plant species, such as those from Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. The cloning of the ppa gene not only provides a basis for further investigation of PPA's structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into tobacco and rice in the future