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Summary Cucumber mosaic virus (CMV) is a highly prevalent viral pathogen causing substantial damage to the bulb and cut‐flower production of Lilium spp. Here, we performed an Illumina RNA sequencing (RNA‐Seq) study on the leaf tissues of a virus‐resistant species Lilium regale inoculated with mock control and CMV. A total of 1346 differentially expressed genes (DEGs) were identified in the leaves of L. regale upon CMV inoculation, which contained 34 up‐regulated and 40 down‐regulated DEGs that encode putative transcription factors (TFs). One up‐regulated TF, LrNAC35, belonging to the NAM/ATAF/CUC (NAC) superfamily, was selected for further functional characterization. Aside from CMV, lily mottle virus and lily symptomless virus infections provoked a striking increase in LrNAC35 transcripts in both resistant and susceptible Lilium species. The treatments with low temperature and several stress‐related hormones activated LrNAC35 expression, contrary to its reduced expression under salt stress. Ectopic overexpression of LrNAC35 in petunia (Petunia hybrida) resulted in reduced susceptibility to CMV and Tobacco mosaic virus infections, and enhanced accumulation of lignin in the cell walls. Four lignin biosynthetic genes, including PhC4H, Ph4CL, PhHCT and PhCCR, were found to be up‐regulated in CMV‐infected petunia lines overexpressing LrNAC35. In vivo promoter‐binding tests showed that LrNAC35 specifically regulated the expression of Ph4CL. Taken together, our results suggest a positive role of transcriptome‐derived LrNAC35 in transcriptional modulation of host defence against viral attack.

Tree peony (Paeonia section Moutan DC.) has recently been recognised as an important woody oil plant of high economic value, with its seeds containing high levels of α-linolenic acids (ALA,...

Tree peony has been widely planted as an excellent oil crop with high proportion of α-linolenic acid (ALA) in its seeds. It was reported that pollen sources have clear effects on fatty acid (FA) composition of its kernels. Nevertheless, the molecular basis of this novel phenomenon is poorly understood. In this study, four Paeonia suffruticosa cultivars whose pollen caused significant differences in ALA content of Paeonia rockii were selected as pollen donors and grouped under high and low ALA groups. After pollination, total FAs content, composition and expression profiles of 10 genes involved in the FA and triacylglycerol (TAG) biosynthetic pathways were investigated during seed development. During the rapid oil accumulation in seeds, three desaturase genes (SAD, FAD2, FAD3) showed much higher expression in high ALA group, and their differential expression profiles corresponded well with the variations in ALA content among different pollen sources. Overall, pollen sources could affect the expression level of genes associated with fatty acid modification and subsequently affect the composition of FA in the kernels.

The effects of different pollen sources on fruit and seed characteristics of Paeonia ostii ‘Feng Dan’ were investigated using analysis of three different pollination treatments and pollen sources from fifteen cultivars in two successive years. The results showed that self-pollination decreased fruit setting, the number of seeds per fruit, seed volume, seed and kernel weights, and the linoleic acid (LA) concentration in the seed oil, but increased the concentration of oleic acid (OA) compared with cross-pollination. Among those pollen donors, P. suffruticosa ‘Yinhong Qiaodui’ produced the highest fruit set (87.5%); the lowest fruit set was obtained with P. suffruticosa ‘Mo Run Jue Lun’ (44.33%). The most seeds per fruit were achieved by P. suffruticosa ‘Mochi Jin Hui’. P. suffruticosa ‘Dahong Baozhu’ produced the largest fruit, which contained larger and heaviest seeds. The oil extraction ratio (26% to 31.6%) and the concentration of three major unsaturated fatty acids (UFAs) in seed oil also significantly differed among pollen sources. The content of OA, LA, and α-linolenic acid (ALA) ranged from 13.82 to 24.79, 12.09 to 21.84, and 23.50 to 38.64 g/100 g crude oil, respectively. Overall, pollen source has clear effects on seed yield and even on fatty acid (FA) composition of seed oil in tree peony.

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.

The L. regale ATP-binding cassette transporter gene, LrABCF1 belonging to GCN subfamily, functions as a positive regulator of plant defense against Cucumber mosaic virus, Tobacco rattle virus , and Botrytis cinerea in petunia. ATP-binding cassette (ABC) transporters are essential for membrane translocation in diverse biological processes, such as plant development and defense response. Here, a general control non-derepressible (GCN)-type ABC transporter gene, designated LrABCF1, was identified from Cucumber mosaic virus (CMV)-induced cDNA library of L. regale. LrABCF1 was up-regulated upon inoculation with CMV and Lily mottle virus (LMoV). Salicylic acid (SA) and ethylene (ET) application and treatments with abiotic stresses such as cold, high salinity, and wounding increased the transcript abundances of LrABCF1. Constitutive overexpression of LrABCF1 in petunia (Petunia × hybrida) resulted in an impairment of plant growth and development. LrABCF1 overexpression conferred reduced susceptibility to CMV, Tobacco rattle virus (TRV), and B. cinerea infection in transgenic petunia plants, accompanying by elevated transcripts of PhGCN2 and a few defense-related genes in SA-signaling pathway. Our data indicate that LrABCF1 positively modulates viral and fungal resistance.

Highlight PhERF2, an ethylene-responsive element binding factor, acts as a positive transcriptional regulator in antiviral RNA silencing and is essential for efficient silencing of genes in plants., Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2. Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.

Stamen from Paeonia ostii 'Fengdan Bai' and Paeonia rockii is rich in phenolic compounds and popularly used as tea materials with various pharmaceutical functions. In order to investigate whether stamen from other tree peony cultivars could be used as a natural antioxidant, the quality of stamen from thirty-five cultivars collected from the same garden was evaluated based on their phenolic composition and content by high-performance liquid chromatography analysis and in vitro antioxidant properties coupled with comprehensive chemometrics analysis. The results revealed that phenolic contents and antioxidant capacities of tree peony stamen were unique and cultivar dependent. Stamen from 'Zi Erqiao' exhibited the highest total phenolic and flavonol content, and strongest antioxidant activities, while that of 'Fengdan Bai' and P. rockii were at below-average levels among test samples. Further, thirty-seven cultivars of tree peony were divided into three major groups with a significant difference in total metabolites content and antioxidant properties, which were mainly contributed by six phytochemical compounds. Among these, naringin and benzoylpaeoniflorin were found to be critical chemical markers for the identification of tree peony stamen with high quality by chemometric analysis. Moreover, correlation analysis suggested that stamen from the earlier flowering cultivars with the hidden pistil, double petal, shorter thrum, more carpel and volume were possibly of higher quality. Together, cultivars with stamen enriched in phenolics and antioxidants properties, and their relevant critical phenotypic and phytochemical traits were screened out. This study would benefit the rapid identification of tree peony stamen with high quality and provide a valuable reference for its development and utilization as functional foods and pharmaceutical resources.

The increasing demand for healthy edible oil has generated the need to identify promising oil crops. Tree peony (Paeonia section Moutan DC.) is a woody oil crop with α-linolenic acid (ALA) contributing for 45% of the total fatty acid (FA) content in seeds. Molecular and genetic differences that contribute to varied FA content and composition among the wild peony species are, however, poorly understood. Analyses of FA content and composition during seed development in three tree peony species (Paeonia rockii, P. potaninii, and P. lutea) showed varied FA content among them with highest in P. rockii, followed by P. potaninii, and P. lutea. Total FA content among these species increased with seed development and reached its maximum in its final stage. Seed FA composition analysis of the three species also revealed that ALA (C18:3) was the most abundant, followed by oleic (C18:1) and linoleic (C18:2) acids. Additionally, quantitative real-time RT-PCR analyses of 10 key seed oil synthesis genes in the three tree peony species revealed that FAD3, FAD2, β-PDHC, LPAAT, and Oleosin gene expression levels positively correlate with total FA content and rate of accumulation. Specifically, the abundance of FAD3 transcripts in P. rockii compared with P. potaninii, and P. lutea suggests that FAD3 might play an important role in synthesis of ALA via phosphatidylcholine-derived pathway. Overall, comparative analyses of FA content and composition in three different peony species revealed a correlation between efficient lipid accumulation and lipid gene expression during seed development. Further characterization and metabolic engineering of these key genes from peonies will allow for subsequent improvement of tree peony oil quality and production.

Paeonia qiui is a wild species of tree peony. P. qiui has good ornamental value owing to its leaf color change in spring. So far, the molecular mechanism of leaf color change in P. qiui is unclear. This study analyzes the anthocyanin level and transcriptome of two different color stages in P. qiui leaf. The purplish-red leaf stage is rich in anthocyanin, while the green leaf has very low levels of anthocyanin. Transcriptome analysis reveals that 6678 differentially-expressed genes (DEGs) are up-regulated, and 14,667 are down-regulated in the purplish-red leaf. Among these DEGs, 40 MYB (v-myb avian myeloblastosis viral oncogene homolog) genes, 40 bHLH (MYC-like basic helix-loop-helix) genes, and 15 WD40 (WD-repeat protein) genes were found. Based on phylogenetic and alignment analysis with the deduced amino acid sequences with known transcription factors, Unigene0024459 (MYB1) is likely the R2R3-MYB that promotes anthocyanin biosynthesis; Unigene0050761 (MYB2) is likely the R2R3-MYB that represses anthocyanin biosynthesis; Unigene0005081 (bHLH1) and Unigene0006146 (WD40-1) are likely the bHLH and WD40 that participate in regulating anthocyanin biosynthesis. Additionally, quantitative RT-PCR results confirmed the transcriptome analyses for key genes.

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.

Eukaryotic translation elongation factors are implicated in protein synthesis across different living organisms, but their biological functions in the pathogenesis of cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) infections are poorly understood. Here, we isolated and characterized a cDNA clone, LreEF1A4, encoding the alpha subunit of elongation factor 1, from a CMV-elicited suppression subtractive hybridization library of Lilium regale. The infection tests using CMV remarkably increased transcript abundance of LreEF1A4, however, it also led to inconsistent expression profiles of three other LreEF1A homologs (LreEF1A1&ndash, 3). Protein modelling analysis revealed that the amino acid substitutions among four LreEF1As may not affect their enzymatic functions. LreEF1A4 was ectopically overexpressed in petunia (Petunia hybrida), and transgenic plants exhibited delayed leaf and flower senescence, concomitant with increased transcription of photosynthesis-related genes and reduced expression of senescence-associated genes, respectively. A compromised resistance to CMV and TRV infections was found in transgenic petunia plants overexpressing LreEF1A4, whereas its overexpression resulted in an enhanced tolerance to salt and drought stresses. Taken together, our data demonstrate that LreEF1A4 functions as a positive regulator in viral multiplication and plant adaption to high salinity and dehydration.

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii is presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized for Agrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more complete Agrobacterium penetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter gene PoPDS encoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenous PoPDS transcripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

Tree peony (Paeonia suffruticosa Andrews) is an important traditional flower in China. Besides its beautiful flower, the leaf of tree peony has also good ornamental value owing to its leaf color change in spring. So far, the molecular mechanism of leaf color change in tree peony is unclear. In this study, the pigment level and transcriptome of three different color stages of tree peony leaf were analyzed. The purplish red leaf was rich in anthocyanin, while yellowish green leaf was rich in chlorophyll and carotenoid. Transcriptome analysis revealed that 4302 differentially expressed genes (DEGs) were upregulated, and 4225 were downregulated in the purplish red leaf vs. yellowish green leaf. Among these DEGs, eight genes were predicted to participate in anthocyanin biosynthesis, eight genes were predicted involved in porphyrin and chlorophyll metabolism, and 10 genes were predicted to participate in carotenoid metabolism. In addition, 27 MYBs, 20 bHLHs, 36 WD40 genes were also identified from DEGs. Anthocyanidin synthase (ANS) is the key gene that controls the anthocyanin level in tree peony leaf. Protochlorophyllide oxido-reductase (POR) is the key gene which regulated the chlorophyll content in tree peony leaf.

In the present study, we quantitatively measured five major fatty acids (FA) in seed oil using gas chromatography/mass spectrometry (GC/MS) and examined four phenotypic characteristics of the seeds from 19 populations from nine wild tree peony species native to China. The results showed that the unsaturated FAs contents were dominant, of which α-linolenic acid (ALA), linoleic acid, and oleic acid (OA) contents ranged from 14.84 to 42.54 g/100 g, 7.33 to 19.66 g/100 g, and 15.07 - 35.31 g/100 g crude oil, respectively. The phenotypic seed characteristics, such as thousand seed weight (244.01 - 1772.91 g), seed volume (91.31 - 1000.79 mm3 ), weight rate of kernel and coat (1.29 - 3.62) and oil extraction ratio (20.32 - 34.69%), dramatically varied. Based on the contents of the five FAs, the nine species were classified into two groups. The species belonging to subsection Vaginatae were arranged in cluster I and were characterized by high ALA content. Cluster II, consistent with subsection Delavayanae, had a high OA content. From horizontal and vertical perspectives, the natural distribution areas of these two groups were different, reflecting differences in the FA contents and phenotypic seed characteristics. In conclusion, the FAs composition could be used as a chemotaxonomic marker for tree peony species.

The aim of this study was to investigate the essential oil (EO) compositions and antioxidant activities from petals of three wild tree peony species (Paeonia delavayi, P. lutea and P. rockii) and eleven P. suffruticosa cultivars from different cultivar groups. The EOs yields varied from 0.63% to 1.25% (v/v) among samples when using supercritical CO2 extraction. One hundred and sixty-three components were detected by GC-MS; and among them, linalool oxide, Z-5-dodecen-1-yl acetate, nonadecane, Z-5-nonadecene, heneicosane, phytol, and linoleic acid ethyl ester were dominant. According to hierarchical cluster analysis, principal component analysis and correspondence analysis, P. lutea, P. delavayi, and ‘High Noon’ were clustered in a group described as having a refreshing herbal-like note due to high rates of phytol and linalool oxide. Notably, P. lutea and P. delavayi also had strong DPPH and ABTS radical scavenging activities. These results suggest that P. lutea and P. delavayi are the most promising candidates as useful sources of fragrances and natural antioxidants. This article is protected by copyright. All rights reserved.

An ocs element binding factor, PhOBF1, positively regulates antiviral RNA silencing mediated by salicylic acid biosynthesis and is crucial for efficient gene silencing in petunia., Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway.