Integrated Metabolites and Transcriptomics at Different Growth Stages Reveal Polysaccharide and Flavonoid Biosynthesis in Cynomorium songaricum.

Cynomorium songaricum is a perennial parasitic herb, and its stem is widely used as a traditional Chinese medicine, which largely relies on bioactive compounds (e.g., polysaccharides, flavonoids, and triterpenes). To date, although the optimum harvest time of stems has been demonstrated at the unearthed stage (namely the early flowering stage, EFS), the accumulation mechanism of polysaccharides and flavonoids during growth stages is still limited. In this study, the physiological characteristics (stem fresh weight, contents of soluble sugar and flavonoids, and antioxidant capacity) at four different growth stages (germination stage (GS), vegetative growth stage (VGS), EFS, and flowering stage (FS)) were determined, transcriptomics were analyzed by illumina sequencing, and expression levels of key genes were validated by qRT-PCR at the GS, VGS, and EFS. The results show that the stem biomass, soluble sugar and total flavonoids contents, and antioxidant capacity peaked at EFS compared with GS, VGS, and FS. A total of 6098 and 13,023 differentially expressed genes (DEGs) were observed at VGS and EFS vs. GS, respectively, with 367 genes co-expressed. Based on their biological functions, 109 genes were directly involved in polysaccharide and flavonoid biosynthesis as well as growth and development. The expression levels of key genes involved in polysaccharides (e.g., GLCs, XTHs and PMEs), flavonoids (e.g., 4CLLs, CYPs and UGTs), growth and development (e.g., AC58, TCPs and AP1), hormones biosynthesis and signaling (e.g., YUC8, AIPT and ACO1), and transcription factors (e.g., MYBs, bHLHs and WRKYs) were in accordance with changes of physiological characteristics. The combinational analysis of metabolites with transcriptomics provides insight into the mechanism of polysaccharide and flavonoid biosynthesis in C. songaricum during growth stages.

Species and geographic specificity between endophytic fungi and host supported by parasitic Cynomorium songaricum and its host Nitraria tangutorum distributed in desert.

This study was aimed to investigate whether host plant species and lifestyles, and environmental conditions in the desert affect endophytic fungi composition. Endophytic fungal communities from parasitic plant Cynomorium songaricum and its host Nitraria tangutorum were investigated from three sites including Tonggu Naoer, Xilin Gaole, and Guazhou in Tengger and Badain Jaran Deserts in China using the next-generation sequencing of a ribosomal RNA gene region. Similarity and difference in endophytic fungal composition from different geographic locations were evaluated through multivariate statistical analysis. It showed that plant genetics was a deciding factor affecting endophytic fungal composition even when C. songaricum and N. tangutorum grow together tightly. Not only that, the fungal composition was also greatly affected by the local environment and rainfall. However, the distribution and richness of fungal species indicated that the geographical distance exerted little influence on characterizing the fungal composition. Overall, the findings suggested that plant species, parasitic or non-parasitic lifestyles of the plant, and local environment strongly affected the number and diversity of the endophytic fungal species, which may provide valuable insights into the microbe ecology, symbiosis specificity, and the tripartite relationship among parasitic plant, host, and endophytic fungi, especially under desert environment.

Sequential horizontal gene transfers from different hosts in a widespread Eurasian parasitic plant, Cynomorium coccineum.

PREMISE: Parasitic plants with large geographic ranges, and different hosts in parts of their range, may acquire horizontally transferred genes (HGTs), which might sometimes leave a footprint of gradual host and range expansion. Cynomorium coccineum, the only member of the Saxifragales family Cynomoriaceae, is a root holoparasite that occurs in water-stressed habitats from western China to the Canary Islands. It parasitizes at least 10 angiosperm families from different orders, some of them only in parts of its range. This parasite therefore offers an opportunity to trace HGTs as long as parasite-host pairs can be obtained and sequenced. METHODS: By sequencing mitochondrial, plastid, and nuclear loci from parasite-host pairs from throughout the parasite's range and with prior information from completely assembled mitochondrial and plastid genomes, we detected 10 HGTs of five mitochondrial genes. RESULTS: The 10 HGTs appear to have occurred sequentially as C. coccineum expanded from East to West. Molecular-clock models yield Cynomorium stem ages between 66 and 156 Myr, with relaxed clocks converging on 66-67 Myr. Chinese Sapindales, probably Nitraria, were the first source of transferred genes, followed by Iranian and Mediterranean Caryophyllales. The most recently acquired gene appears to come from a Tamarix host in the Iberian Peninsula. CONCLUSIONS: Data on HGTs that have accumulated over the past 15 years, along with this discovery of multiple HGTs within a single widespread species, underline the need for more whole-genome data from parasite-host pairs to investigate whether and how transferred copies coexist with, or replace, native functional genes.

Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales.

Cynomoriaceae, one of the last unplaced families of flowering plants, comprise one or two species or subspecies of root parasites that occur from the Mediterranean to the Gobi Desert. Using Illumina sequencing, we assembled the mitochondrial and plastid genomes as well as some nuclear genes of a Cynomorium specimen from Italy. Selected genes were also obtained by Sanger sequencing from individuals collected in China and Iran, resulting in matrices of 33 mitochondrial, 6 nuclear, and 14 plastid genes and rDNAs enlarged to include a representative angiosperm taxon sampling based on data available in GenBank. We also compiled a new geographic map to discern possible discontinuities in the parasites' occurrence. Cynomorium has large genomes of 13.70-13.61 (Italy) to 13.95-13.76 pg (China). Its mitochondrial genome consists of up to 49 circular subgenomes and has an overall gene content similar to that of photosynthetic angiosperms, while its plastome retains only 27 of the normally 116 genes. Nuclear, plastid and mitochondrial phylogenies place Cynomoriaceae in Saxifragales, and we found evidence for several horizontal gene transfers from different hosts, as well as intracellular gene transfers.

[Molecular identification of Cynomorii herba using ITS2 DNA barcoding].

OBJECTIVE: To identify the Cynomorii Herba and its analogues species using DNA barcoding technique. METHOD: Total genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2 (ITS2) regions were amplified using polymerase chain reaction (PCR), and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter (K2P) distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) trees. RESULT: The ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification. CONCLUSION: The ITS2 region is an efficient barcode for identification of Cynomorii Herba, which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.

Cynomorium songaricum induces spermatogenesis with glial cell-derived neurotrophic factor (GDNF) enhancement in rat testes.

AIM OF THE STUDY: Cynomorium songaricum Ruprecht has been used in traditional Korean medicine to treat male infertility, including sexual dysfunction, by improving kidney function. Glial cell-derived neurotrophic factor (GDNF) produced by Sertoli cells induces the proliferation of undifferentiated spermatogonia. We investigated the effects of Cynomorium songaricum on sperm parameters and GDNF expression in rat testes. MATERIALS AND METHODS: Sperm analysis, RT-PCR, and Western blotting assays were performed after administration of CS to 8-week-old male Wistar rats for 56 consecutive days (1.0g/kg/day, p.o.), the period of sperm formation in the rat. RESULTS AND CONCLUSIONS: The CS-treated animals showed significant increases in epididymal sperm count and absolute testes weights compared to the control group. CS also increased the expression of GDNF at both the mRNA and protein levels. These results suggest that CS may improve male fertility by enhancing spermatogenesis and GDNF expression.

Extensive intraindividual variation in plastid rDNA sequences from the holoparasite Cynomorium coccineum (Cynomoriaceae).

Ribosomal genes are considered to have a high degree of sequence conservation between species and also at higher taxonomic levels. In this paper we document a case where a single individual of Cynomorium coccineum (Cynomoriaceae), a nonphotosynthetic holoparasitic plant, contains highly divergent plastid ribosomal genes. PCR amplification a nearly complete ribosomal DNA cistron was performed using genomic DNA, the products cloned, and the 23S rDNA genes were sequenced from 19 colonies. Of these, five distinct types were identified. Fifteen of the sequences were nearly identical (11 or fewer differences) and these were designated Type I. The remaining types (II-V) were each represented by a single clone and differed from Type I by 93 to 255 changes. Compared with green vascular plants, we found that there are more substitutional differences in the 23S rDNA sequences within a single individual of Cynomorium than among all sequenced photosynthetic vascular plants. Several trends of molecular evolution observed in 16S rDNA from other holoparasitic angiosperms and heterotrophic green algae have been also observed in Cynomorium 23S rDNA. Higher-order structures were constructed for representatives of the five clone types, and in all cases these possessed complete complements of the major structural elements present in functional plastid 23S rRNAs. These data indicate that such molecules may be subject to purifying selection, thus providing indirect evidence that they have retained some degree of functionality. This intraindividual polymorphism is probably a case of plastid heteroplasmy but translocation of ribosomal cistrons to the nucleus or mitochondria has not been tested and therefore cannot be ruled out.