A Paeonia ostii caffeoyl-CoA O-methyltransferase confers drought stress tolerance by promoting lignin synthesis and ROS scavenging.

Paeonia ostii is an emerging woody oil crop, but drought severely inhibits its growth and promotion in arid or semiarid areas, and little is known about the mechanism governing this inhibition. In this study, the full-length cDNA of a caffeoyl-CoA O-methyltransferase gene (CCoAOMT) from P. ostii was isolated, and determined to be comprised of 987 bp. PoCCoAOMT encoded a 247-amino acid protein, which was located in the nucleus and cytosol. Significantly higher PoCCoAOMT transcription was detected in P. ostii treated with drought stress. Subsequently, the constitutive overexpression of PoCCoAOMT in tobacco significantly conferred drought stress tolerance. Under drought stress, transgenic lines exhibited lower reactive oxygen species (ROS) accumulation, and higher antioxidant enzyme activities and photosynthesis. Moreover, the expression levels of senescence-associated genes were significantly downregulated, whereas the expression levels of lignin biosynthetic genes and PoCCoAOMT were significantly upregulated in transgenic lines. Similarly, transgenic lines produced significantly higher lignin, especially guaiacyl-lignin. These results suggest that PoCCoAOMT is a vital gene in promoting lignin synthesis and ROS scavenging to confer drought stress tolerance in P. ostii.

Characterization of the glycerol-3-phosphate acyltransferase gene and its real-time expression under cold stress in Paeonia lactiflora Pall.

Elucidating the cold tolerance mechanism of Paeonia lactiflora, which is one of the most valuable ornamental and medicinal plants in Asia, fundamentally impacts its breeding and production. The glycerol-3-phosphate acyltransferase (GPAT) gene plays a pivotal role in cold resistance in a variety of plant species. Here, we cloned the P. lactiflora GPAT gene, determined its expression pattern, and tested its role in cold resistance. We obtained the full-length P. lactiflora GPAT gene using tissue-cultured seedlings and real-time polymerase chain reaction and rapid amplification of cDNA ends analyses. We named this gene PlGPAT in P. lactiflora. Phylogenetic analysis indicates that the PlGPAT gene is closely related with the GPAT genes in core eudicots. The phylogenetic tree containing 31 angiosperm species based on GPAT protein sequences is largely consistent with the known phylogeny in flowering plants. We conducted a time-course PlGPAT expression analysis and demonstrated that PlGPAT expression is correlated with low-temperature stress. Our results suggest that the PlGPAT gene plays an important role in regulating cold resistance in P. lactiflora.

Fatty acid desaturase 3 (PsFAD3) from Paeonia suffruticosa reveals high α-linolenic acid accumulation.

α-linolenic acid (ALA) deficiency and a skewed ω6: ω3 fatty acid ratio in the diet are thought to be a major cause for the high incidence of cardiovascular, inflammatory, and autoimmune diseases. Recent years, tree peony (Paeonia suffruticosa Andr.) with the high proportion of ALA (more than 45% in seed oil) is widely concerned. However, the underlying accumulation mechanism of the ALA in tree peony seeds remains unknown. In this study, comparative transcriptome analysis was performed between two cultivars ('Saiguifei' and 'Jingshenhuanfa') with different ALA contents. The analysis of the metabolic enzymes associated with ALA biosynthesis and temporal accumulation patterns of unsaturated fatty acids demonstrated the importance of microsomal ω-3 fatty acid desaturase 3 (FAD3). Moreover, PsFAD3 gene was identified from tree peony seeds, which was located in endoplasmic reticulum and the expression levels of PsFAD3 were consistent with ALA accumulation patterns in seeds. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated PsFAD3 protein could catalyze ALA synthesis. These results indicated that PsFAD3 was involved in the synthesis of ALA in seeds and could be exploited by the genetic breeding of new cultivars with high ALA content in tree peony as well as other potential crops.

Cloning, Characterization, and Expression Analysis of Three FAD8 Genes Encoding a Fatty Acid Desaturase from Seeds of Paeonia ostii.

The FAD8 gene catalyzes the conversion of diene fatty acids to triene fatty acids and is a key enzyme that determines the synthesis of alpha-linolenic acid. In this study, the full-length cDNAs of FAD8-1, FAD8-2, and FAD8-3 are cloned from Paeonia ostii T. Hong & J. X. Zhang and named as PoFAD8-1, PoFAD8-2, and PoFAD8-3. Their open reading frame is 1203 bp, 1152 bp, and 1353 bp which encoded 400, 371, and 450 amino acids. The molecular weights of the amino acids are 46 kDa, 43 kDa, and 51 kDa while the isoelectric points are 7.34, 8.74, and 9.23, respectively. Bioinformatics analysis shows that all three genes are hydrophobic-hydrophobic, PoFAD8-1 has three transmembrane domains, and PoFAD8-2 and PoFAD8-3 have two transmembrane domains. Multiple series alignment and phylogenetic analysis revealed that PoFAD8-1 and PoFAD8-2 are closely related while PoFAD8-3 is more closely related to Paeonia delavayi. Subcellular localization results showed that PoFAD8-1 was located on the ER membrane and PoFAD8-2 and PoFAD8-3 were located on the chloroplast membrane. The relative expression level of PoFAD8-1 in seeds is very high. PoFAD8-2 expressed more in the ovary than the other two genes. PoFAD8-3 was highly expressed in roots, stems, leaves, petals, and ovaries.

PrLPAAT4, a Putative Lysophosphatidic Acid Acyltransferase from Paeonia rockii, Plays an Important Role in Seed Fatty Acid Biosynthesis.

Lysophosphatidic acid acyltransferases (LPAATs) are essential for the acylation of lysophosphatidic acid (LPA) and the synthesis of phosphatidic acid (PA), a key intermediate in the synthesis of membrane phospholipids and storage lipids. Here, a putative lysophosphatidic acid acyltransferase gene, designated PrLPAAT4, was isolated from seed unsaturated fatty acid (UFA)-rich P. rockii. The complete PrLPAAT4 cDNA contained a 1116-bp open reading frame (ORF), encoding a 42.9 kDa protein with 371 amino acid residues. Bioinformatic analysis indicates that PrLPAAT4 is a plasma membrane protein belonging to acyl-CoA:1-acylglycerol-sn-3-phosphate acyltranferases (AGPAT) family. PrLPAAT4 shared high sequence similarity with its homologs from Citrus clementina, Populus trichocarpa, Manihot esculenta, and Ricinus communis. In Arabidopsis, overexpression of PrLPAAT4 resulted in a significant increase in the content of oleic acid (OA) and total fatty acids (FAs) in seeds. AtDGAT1, AtGPAT9, and AtOleosin, involved in TAG assembly, were upregulated in PrLPAAT4-overexpressing lines. These results indicated that PrLPAAT4 functions may be as a positive regulator in seed FA biosynthesis.

Cloning, Characterization, and Expression Analysis of a Gene Encoding a Putative Lysophosphatidic Acid Acyltransferase from Seeds of Paeonia rockii.

Tree peony (Paeonia section Moutan DC.) is an excellent woody oil crop, and the cloning and functional analysis of genes related to fatty acid (FA) metabolism from this organism has not been reported. Lysophosphatidic acid acyltransferase (LPAAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA), catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG) biosynthesis. This project reports a putative lysophosphatidic acid acyltransferase gene PrLPAAT1 isolated from Paeonia rockii. Our data indicated that PrLPAAT1 has 1047 nucleotides and encodes a putative 38.8 kDa protein with 348 amino acid residues. Bioinformatic analysis demonstrated that PrLPAAT1 contains two transmembrane domains (TMDs). Subcellular localization analysis confirmed that PrLPAAT1 is a plasma membrane protein. Phylogenetic analysis revealed that PrLPAAT1 shared 74.3 and 65.5% amino acid sequence identities with the LPAAT1 sequences from columbine and grape, respectively. PrLPAAT1 belongs to AGPAT family, and may have acyltransferase activity. PrLPAAT1 was ubiquitously expressed in diverse tissues, and PrLPAAT1 expression was higher in the flower and developing seed. PrLPAAT1 is probably an important component in the FA accumulation process, especially during the early stages of seed development. PrLPAAT1 overexpression using a seed-specific promoter increased total FA content and the main FA accumulation in Arabidopsis transgenic plants.

Characterization of a monoterpene synthase from Paeonia lactiflora producing α-pinene as its single product.

OBJECTIVES: To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin. RESULTS: Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product. CONCLUSIONS: This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

Changes in soil microbial functional diversity and biochemical characteristics of tree peony with amendment of sewage sludge compost.

A greenhouse experiment was conducted to investigate the impact of sewage sludge compost application on functional diversity of soil microbial communities, based on carbon source utilization, and biochemical characteristics of tree peony (Paeonia suffruticosa). Functional diversity was estimated with incubations in Biolog EcoPlates and well color development was used as the functional trait for carbon source utilization. The average well color development and Shannon index based on the carbon source utilization pattern in Biolog EcoPlates significantly increased with the increasing sludge compost application in the range of 0-45%, with a decreasing trend above 45%. Principal component analysis of carbon source utilization pattern showed that sludge compost application stimulated the utilization rate of D-cellobiose and α-D-lactose, while the utilization rate of ß-methyl-D-glucoside, L-asparagine, L-serine, α-cyclodextrin, γ-hydroxybutyric acid, and itaconic acid gradually increased up to a sludge compost amendment dosage of 45% and then decreased above 45%. The chlorophyll content, antioxidase (superoxide dismutase, catalase, and peroxidase) activities, plant height, flower diameter, and flower numbers per plant of tree peony increased significantly with sludge compost dosage, reaching a peak value at 45 %, and then decreased with the exception that activity of superoxide dismutase and catalase did not vary significantly.

Years-identification mathematical model of Paeonia lactiflora Pall. based on the allometric-scaling.

The fixed number years-identification of Chinese material medicinal was a difficulty in the process of the traditional Chinese pharmacology, the phenomenon was found in our study of telomerase that the speed of the vascular cambium outside expanding growth fluctuated in a definite value. Based on it, we put forward a hypothesis that the radial length of the vascular cambium to expand (Δb)and the radius of cross section (Δr) are constant in every activity cycle if the external environment factors unchanged every year. Therefore, We defined that the proportion (k) of Δr and Δb is constant (Δr/Δb = k). Then, each of Δb and Δr fluctuating in a fixed value in every year, because of the different rainfall, temperature, and sunniness every year. The hypothesis was proved correct within the domain of definition range, through the extrapolation of mathematical method. Hence, the telomerase experimental results just become effective evidence on mechanism. The conclusion we obtained include the following three: the telomerase experimental show that the activity gradually decreased in the Paeonia overall taproot, while, the it does not seem significantly change in the parts of the cambium cells with increasing age; Microscopy studies and mathematical models exploration gave us an identification method which can determine the growth years of Chinese medicinal materials (Refers to a kind of herbs exclusive which contains taproot), and, for example, we can use the mathematical model y = 0.02x(-0.5) (P = (0.08)/(k(2)nΔb)-(0.0016)/(Δb(2)n(2)k(2))) to identify the years of the Paeonia lactiflora Pall; the power function of allometric-scaling explored at microscopic cellular-level firstly. Ginseng, for example, more definitive proved a mathematical relationship of the allometric scaling in the taproot of plants.

[Enzyme activities in Paeonia ostii rhizosphere and non-rhizosphere soil of Tongling copper mining].

The study on the calatase, polyphenol-oxidase, invertase, urease and phosphatase activities in Paeonia ostii rhizosphere and non-rhizosphere soil of Tongling copper mining showed that all test enzyme activities were higher in rhizosphere than in non-rhizosphere soil. Soil calatase, urease and phosphatase were sensitive to heavy metals pollution, and their activities could be used as the indicators of heavy metals' joint pollution. The effects of rhizosphere environment on the soil enzyme activities were in the sequence of phosphatase > urease > calatase > invertase > polyphenol-oxidase, and the affecting rate was 131.562%, 92.492%, 87.557%, 59.673% and 34.076%, respectively. The test enzyme activities were negatively correlated with soil heavy metals pollution, and the correlation coefficients were all higher than -0.898, suggesting the inhibitory effects of heavy metals' joint pollution on soil enzyme activities. P. ostii could effectively improve soil environment, and thus, enhance the activities of soil enzymes.