Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony.

Cut flower quality is severely restrained by stem bending due to low stem strength. Melatonin has been shown to function in many aspects of plant growth and development, yet whether it can enhance stem strength, and the corresponding underlying mechanisms remain unclear. We investigated the role of melatonin in enhancement of stem strength in herbaceous peony (Paeonia lactiflora Pall.) by applying exogenous melatonin and changing endogenous melatonin biosynthesis. Endogenous melatonin content positively correlated with lignin content and stem strength in various P. lactiflora cultivars. Supplementation with exogenous melatonin significantly enhanced stem strength by increasing lignin content and the S/G lignin compositional ratio, up-regulating lignin biosynthetic gene expression. Moreover, overexpression of TRYPTOPHAN DECARBOXYLASE GENE (TDC) responsible for the first committed step of melatonin biosynthesis in tobacco, significantly increased endogenous melatonin, which further increased the S/G ratio and stem strength. In contrast, silencing PlTDC in P. lactiflora decreased endogenous melatonin, the S/G ratio and stem strength. Finally, manipulating the expression of CAFFEIC ACID O-METHYLTRANSFERASE GENE (COMT1), which is involved in both melatonin and lignin biosynthesis, showed even greater effects on melatonin, the S/G ratio and stem strength. Our results suggest that melatonin has a positive regulatory effect on P. lactiflora stem strength.

Analysis and Functional Verification of PoWRI1 Gene Associated with Oil Accumulation Process in Paeonia ostii.

The plant transcription factor WRINKLED1 (WRI1), a member of AP2/EREBP, is involved in the regulation of glycolysis and the expression of genes related to the de novo synthesis of fatty acids in plastids. In this study, the key regulator of seed oil synthesis and accumulation transcription factor gene PoWRI1 was identified and cloned, having a complete open reading frame of 1269 bp and encoding 422 amino acids. Subcellular localization analysis showed that PoWRI1 is located at the nucleus. After the expression vector of PoWRI1 was constructed and transformed into wild-type Arabidopsis thaliana, it was found that the overexpression of PoWRI1 increased the expression level of downstream target genes such as BCCP2, KAS1, and PKP-ß1. As a result, the seeds of transgenic plants became larger, the oil content increased significantly, and the unsaturated fatty acid content increased, which provide a scientific theoretical basis for the subsequent use of genetic engineering methods to improve the fatty acid composition and content of plant seeds.

Identification of genes associated with the biosynthesis of unsaturated fatty acid and oil accumulation in herbaceous peony 'Hangshao' (Paeonia lactiflora 'Hangshao') seeds based on transcriptome analysis.

BACKGROUND: Paeonia lactiflora 'Hangshao' is widely cultivated in China as a traditional Chinese medicine 'Radix Paeoniae Alba'. Due to the abundant unsaturated fatty acids in its seed, it can also be regarded as a new oilseed plant. However, the process of the biosynthesis of unsaturated fatty acids in it has remained unknown. Therefore, transcriptome analysis is helpful to better understand the underlying molecular mechanisms. RESULTS: Five main fatty acids were detected, including stearic acid, palmitic acid, oleic acid, linoleic acid and α-linolenic acid, and their absolute contents first increased and then decreased during seed development. A total of 150,156 unigenes were obtained by transcriptome sequencing. There were 15,005 unigenes annotated in the seven functional databases, including NR, NT, GO, KOG, KEGG, Swiss-Prot and InterPro. Based on the KEGG database, 1766 unigenes were annotated in the lipid metabolism. There were 4635, 12,304, and 18,291 DEGs in Group I (60 vs 30 DAF), Group II (90 vs 60 DAF) and Group III (90 vs 30 DAF), respectively. A total of 1480 DEGs were detected in the intersection of the three groups. In 14 KEGG pathways of lipid metabolism, 503 DEGs were found, belonging to 111 enzymes. We screened out 123 DEGs involved in fatty acid biosynthesis (39 DEGs), fatty acid elongation (33 DEGs), biosynthesis of unsaturated fatty acid (24 DEGs), TAG assembly (17 DEGs) and lipid storage (10 DEGs). Furthermore, qRT-PCR was used to analyze the expression patterns of 16 genes, including BBCP, BC, MCAT, KASIII, KASII, FATA, FATB, KCR, SAD, FAD2, FAD3, FAD7, GPAT, DGAT, OLE and CLO, most of which showed the highest expression at 45 DAF, except for DGAT, OLE and CLO, which showed the highest expression at 75 DAF. CONCLUSIONS: We predicted that MCAT, KASIII, FATA, SAD, FAD2, FAD3, DGAT and OLE were the key genes in the unsaturated fatty acid biosynthesis and oil accumulation in herbaceous peony seed. This study provides the first comprehensive genomic resources characterizing herbaceous peony seed gene expression at the transcriptional level. These data lay the foundation for elucidating the molecular mechanisms of fatty acid biosynthesis and oil accumulation for herbaceous peony.

Age-associated methylation change of CHI promoter in herbaceous peony (Paeonia lactiflora Pall).

Chalcone isomerase gene (CHI) is a key gene that regulates the formation of yellow traits in petals. To reveal transcriptional regulatory mechanisms of CHI gene in petals of Paeonia lactiflora, we investigated the CHI expression using qPCR, the pigment content by HPLC, and methylation levels using BSP+Miseq sequencing in 'Huangjinlun' variety during different developmental stages including flower-bud stage (S1), initiating bloom (S2), bloom stage (S3), and withering stage (S4). Results showed that the expression level of CHI gene at S2 stage was significantly higher than that at other stages (P<0.05), and at S4 stage was extremely significantly lower than other stages (P<0.01). Besides, total anthocyanin, anthoxanthin, and flavonoid contents in petals presented a similar trend with CHI expression during developmental stages. A total of 16 CpG sites varying methylation levels were detected in CHI gene core promoter region, of which the methylation levels at mC-4 and mC-16 sites were extremely significantly negatively correlated with CHI mRNA expression (P<0.01). mC-16 site is located in the binding region of C/EBPα transcription factor, suggesting that methylation at the mC-16 site may inhibit the binding of C/EBPα to CHI promoter DNA, thereby regulating the tissue-specific expression of CHI gene. Our study revealed the expression pattern of CHI gene in petal tissues of P. lactiflora at different developmental stages, which is related to promoter methylation. Moreover, the important transcription regulation element-C/EBPα was identified, providing theoretical reference for in-depth study on the function of CHI gene in P. lactiflora.

Daminozide reduces red color intensity in herbaceous peony (Paeonia lactiflora Pall.) flowers by inhibiting the expression of flavonoid biosynthetic genes.

Daminozide is a plant growth retardant that inhibits 2-oxoglutarate-dependent dioxygenase activity in the gibberellin biosynthesis pathway, but few studies have focused on its effect on the flavonoid biosynthesis pathway involved in flower color. In the present study, the effect of daminozide on the regulation of flower color was investigated. An herbaceous peony (Paeonia lactiflora Pall.) cultivar 'Fenzhu Pan' was used as the material and treated with daminozide. Color parameters of petals were measured; flavonoid composition and content as well as flavonoid biosynthetic gene expression were monitored. The results showed that daminozide treatment caused less red coloration in herbaceous peony. The content of total anthocyanins was decreased by 23%, and the content of total anthoxanthins was increased by 8%; thus, it appeared that less red coloration in daminozide-treated flowers was associated with restricted anthocyanin accumulation. Furthermore, the gene expression patterns of flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase and anthocyanidin synthase were lower after daminozide treatment, which could decrease anthocyanin accumulation and lead to less red coloration in flowers. These findings would improve our understanding of daminozide regulation of flower color in herbaceous peony.

Nano-silver modifies the vase life of cut herbaceous peony (Paeonia lactiflora Pall.) flowers.

Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-grade cut flower because of higher ornamental value. However, its short flowering time severely restricts the production and application of cut P. lactiflora flowers. In this study, nano-silver (NS) was applied to prolong the vase life of cut P. lactiflora flowers. Under the NS treatment, related physiological indices including relative electrical conductivity (REC), malondialdehyde (MDA), superoxide anion free radical (O2·-), hydrogen peroxide (H2O2) and free proline contents, and protective enzyme activities including superoxide dismutase (SOD), peroxidase (POD) and ascorbic acid peroxidase (APX) all increased in cut P. lactiflora flowers except soluble protein. Meanwhile, NS treatment increased relative water uptake (RWU) and Ag+ distribution. Moreover, the observation of microstructures indicated that the stem-ends without NS treatment were blocked by microbes which were identified as Alternaria sp. and Phoma sp., and NS effectively inhibited their growth by antibacterial efficacy observation. Additionally, three aquaporin genes (AQPs) including two plasma membrane intrinsic protein genes (PlPIP1;2, PlPIP2;1) and one NOD26-like intrinsic protein gene (PlNIP) were isolated, PlPIP1;2, and PlPIP2;1 that were induced by NS treatment took common effects on maintaining the water balance of cut P. lactiflora flowers. Consequently, the vase life of cut P. lactiflora flowers was prolonged and flower fresh weight together with flower diameter was well kept because of these above factors. These results would provide a theoretical basis for prolonging the vase life and improving the ornamental quality of cut P. lactiflora flowers with NS application.

Effect of calcium sprays on mechanical strength and cell wall fractions of herbaceous peony (Paeonia lactiflora pall.) inflorescence stems.

Calcium is an essential element and imparts significant structural rigidity to the plant cell walls, which provide the main mechanical support to the entire plant. In order to increase the mechanical strength of the inflorescence stems of herbaceous peony, the stems are treated with calcium chloride. The results shows that preharvest sprays with 4% (w/v) calcium chloride three times after bud emergence are the best at strengthening "Da Fugui" peonies' stems. Calcium sprays increased the concentrations of endogenous calcium, total pectin content as well as cell wall fractions in herbaceous peonies stems, and significantly increased the contents of them in the top segment. Correlation analysis showed that the breaking force of the top segment of peonies' stems was positively correlated with the ratio of water insoluble pectin to water soluble pectin (R = 0.673) as well as lignin contents (R = 0.926) after calcium applications.

Cloning of phytoene desaturase and expression analysis of carotenogenic genes in persimmon (Diospyros kaki L.) fruits.

Persimmon is a commercially important fruit crop, and the fruit is rich in different kinds of bioactive compounds, among which carotenoids contribute significantly to its color and nutritional value. In this study, the cDNA of phytoene desaturase gene (PDS) was isolated by rapid amplification of cDNA ends (RACE) technique. Sequence analysis indicated that the full-length cDNA of PDS was 2064 bp, encoding 586 amino acids and containing one open reading frame (ORF) of 1761 bp. Homology analysis showed that DkPDS, which had been submitted in GenBank with accession number GU112527, shared high similarities of 80-86% with PDS cloned from other plants. Prediction of deduced proteins showed that there was no signal peptide and transmembrane topological structure in DkPDS. It was a hydrophilic and stable protein, and located in chloroplast. To examine the specific expression patterns of carotenogenic genes we had cloned from persimmon, including phytoene synthase (DkPSY), DkPDS, ζ-carotene desaturase (DkZDS), lycopene ß-cyclase (DkLCYB) and ß-carotene hydroxylase (DkBCH), real-time quantitative PCR (Q-PCR) was performed in flesh at five different developmental stages. The results revealed that the expression levels of DkPSY, DkPDS and DkZDS gradually increased. Nevertheless, the expression level of DkLCYB was very low and maintained relatively stable. The expression level of DkBCH was also at a low level from stage 1 to 4, and then reached the maximum at stage 5. In addition, the expression level of DkZDS was higher than that of other genes. Carotenoid detection demonstrated that both ß-cryptoxanthin and total carotenoids increased with fruit development, and zeaxanthin had little change, but with a sudden increase in final stage.