PoWRKY69-PoVQ11 module positively regulates drought tolerance by accumulating fructose in Paeonia ostii.

Drought is a detrimental environmental factor that restricts plant growth and threatens food security throughout the world. WRKY transcription factors play vital roles in abiotic stress response. However, the roles of IIe subgroup members from WRKY transcription factor family in soluble sugar mediated drought response are largely elusive. In this study, we identified a drought-responsive IIe subgroup WRKY transcription factor, PoWRKY69, from Paeonia ostii. PoWRKY69 functioned as a positive regulator in response to drought stress with nucleus expression and transcriptional activation activity. Silencing of PoWRKY69 increased plants sensitivity to drought stress, whereas conversely, overexpression of PoWRKY69 enhanced drought tolerance in plants. As revealed by yeast one-hybrid, electrophoretic mobility shift assay, and luciferase reporter assays, PoWRKY69 could directly bind to the W-box element of fructose-1,6-bisphosphate aldolase 5 (PoFBA5) promoter, contributing to a cascade regulatory network to activate PoFBA5 expression. Furthermore, virus-induced gene silencing and overexpression assays demonstrated that PoFBA5 functioned positively in response to drought stress by accumulating fructose to alleviate membrane lipid peroxidation and activate antioxidant defense system, these changes resulted in reactive oxygen species scavenging. According to yeast two-hybrid, bimolecular fluorescence complementation, and firefly luciferase complementation imaging assays, valine-glutamine 11 (PoVQ11) physically interacted with PoWRKY69 and led to an enhanced activation of PoWRKY69 on PoFBA5 promoter activity. This study broadens our understanding of WRKY69-VQ11 module regulated fructose accumulation in response to drought stress and provides feasible molecular measures to create novel drought-tolerant germplasm of P. ostii.

PoWRKY71 is involved in Paeonia ostii resistance to drought stress by directly regulating light-harvesting chlorophyll a/b-binding 151 gene.

Although the functions of WRKY transcription factors in drought resistance are well known, their regulatory mechanisms in response to drought by stabilising photosynthesis remain unclear. Here, a differentially expressed PoWRKY71 gene that was highly expressed in drought-treated Paeonia ostii leaves was identified through transcriptome analysis. PoWRKY71 positively responded to drought stress with significantly enhanced expression patterns and overexpressing PoWRKY71 in tobacco greatly improved plant tolerance to drought stress, whereas silencing PoWRKY71 in P. ostii resulted in a drought-intolerant phenotype. Furthermore, lower chlorophyll contents, photosynthesis, and inhibited expression of photosynthesis-related light-harvesting chlorophyll a/b-binding 151 (CAB151) gene were found in PoWRKY71-silenced P. ostii. Meanwhile, a homologous system indicated that drought treatment increased PoCAB151 promoter activity. Interactive assays revealed that PoWRKY71 directly bound on the W-box element of PoCAB151 promoter and activated its transcription. In addition, PoCAB151 overexpressing plants demonstrated increased drought tolerance, together with significantly higher chlorophyll contents and photosynthesis, whereas these indices were dramatically lower in PoCAB151-silenced P. ostii. The above results indicated that PoWRKY71 activated the expression of PoCAB151, thus stabilising photosynthesis via regulating chloroplast homeostasis and chlorophyll content in P. ostii under drought stress. This study reveals a novel drought-resistance mechanism in plants and provides a feasible strategy for improving plant drought resistance via stabilising photosynthesis.

Selection and verification of reliable internal reference genes in stem development of herbaceous peony (Paeonia lactiflora Pall.).

Herbaceous peony (Paeonia lactiflora Pall.) has emerged in the cut flower market due to its beautiful appearance. The bending flower stems caused by a lack of mechanical strength is the main problem restricting the development of the cut P. lactiflora industry. So it is of great worth to reveal the basis of stem development changes in P. lactiflora to improve its cut flower quality. Quantitative research on gene expression characteristics can provide clues for understanding their biological functions, and the screening of relatively stable expression genes is a prerequisite for the quantitative study of gene expression characteristics. Thus, it is necessary to find appropriate genes during stem development so as to analyze the qRT‒PCR results. In this study, 10 genes were screened, and these genes expressed stably in stems of different stem strengths at three different developmental stages. Then, their expressions were evaluated by RefFinder, BestKeeper, NormFinder, and GeNorm programs. The results demonstrated that γ-tubulin (γ-TUB) was the most suitable gene, followed by α-tubulin (α-TUB) and ß-D-glucosidase (ß-GUS), whereas histone H3 (His) was the least suitable gene. Besides, the temporal and spatial expression characteristics of PlCOMT1, the key gene concerned with the synthesis of cell wall fillers in P. lactiflora, were also used to evaluate the suitability of genes. Consequently, γ-TUB and α-TUB are the two best combinations during stem development, and their combination can be used for the stem development of P. lactiflora. These findings will provide a reference for the selection of genes related to stem development and the study of molecular mechanisms related to stem development in P. lactiflora. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01325-5.

An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony.

Stem strength is an important agronomic trait affecting plant lodging, and plays an essential role in the quality and yield of plants. Thickened secondary cell walls in stems provide mechanical strength that allows plants to stand upright, but the regulatory mechanism of secondary cell wall thickening and stem strength in cut flowers remains unclear. In this study, first, a total of 11 non-redundant Paeonia lactiflora R2R3-MYBs related to stem strength were identified and isolated from cut-flower herbaceous peony, among which PlMYB43, PlMYB83 and PlMYB103 were the most upregulated differentially expressed genes. Then, the expression characteristics revealed that these three R2R3-MYBs were specifically expressed in stems and acted as transcriptional activators. Next, biological function verification showed that these P. lactiflora R2R3-MYBs positively regulated stem strength, secondary cell wall thickness and lignin deposition. Furthermore, yeast-one-hybrid and dual luciferase reporter assays demonstrated that they could bind to the promoter of caffeic acid O-methyltransferase gene (PlCOMT2) and/or laccase gene (PlLAC4), two key genes involved in lignin biosynthesis. In addition, the function of PlLAC4 in increasing lignin deposition was confirmed by virus-induced gene silencing and overexpression. Moreover, PlMYB83 could also act as a transcriptional activator of PlMYB43. The findings of the study propose a regulatory network of R2R3-MYBs modulating lignin biosynthesis and secondary cell wall thickening for improving stem lodging resistance, and provide a resource for molecular genetic engineering breeding of cut flowers.

Tree peony PsMYB44 negatively regulates petal blotch distribution by inhibiting dihydroflavonol-4-reductase gene expression.

BACKGROUND AND AIMS: The tree peony (Paeonia suffruticosa Andr.) has been widely cultivated as a field plant, and petal blotch is one of its important traits, which not only promotes proliferation but also confers high ornamental value. However, the regulatory network controlling blotch formation remains elusive owing to the functional differences and limited conservation of transcriptional regulators in dicots. METHODS: We performed phylogenetic analysis to identify MYB44-like transcription factors in P. suffruticosa blotched cultivar 'High noon' petals. A candidate MYB44-like transcription factor, PsMYB44, was analysed via expression pattern analysis, subcellular localization, target gene identification, gene silencing in P. suffruticosa petals and heterologous overexpression in tobacco. KEY RESULTS: A blotch formation-related MYB44-like transcription factor, PsMYB44, was cloned. The C-terminal of the PsMYB44 amino acid sequence had a complete C2 motif that affects anthocyanin biosynthesis, and PsMYB44 was clustered in the MYB44-like transcriptional repressor branch. PsMYB44 was located in the nucleus, and its spatial and temporal expression patterns were negatively correlated with blotch formation. Furthermore, a yeast one-hybrid assay showed that PsMYB44 could target the promoter of the late anthocyanin biosynthesis-related dihydroflavonol-4-reductase (DFR) gene, and a dual-luciferase assay demonstrated that PsMYB44 could repress PsDFR promoter activity. On the one hand, overexpression of PsMYB44 significantly faded the red colour of tobacco flowers and decreased the anthocyanin content by 42.3 % by downregulating the expression level of the tobacco NtDFR gene. On the other hand, PsMYB44-silenced P. suffruticosa petals had a redder blotch colour, which was attributed to the fact that silencing PsMYB44 redirected metabolic flux to the anthocyanin biosynthesis branch, thereby promoting more anthocyanin accumulation in the petal base. CONCLUSION: These results demonstrated that PsMYB44 negatively regulated the biosynthesis of anthocyanin by directly binding to the PsDFR promoter and subsequently inhibiting blotch formation, which helped to elucidate the molecular regulatory network of anthocyanin-mediated blotch formation in plants.

The herbaceous peony transcription factor WRKY41a promotes secondary cell wall thickening to enhance stem strength.

Stem bending or lodging caused by insufficient stem strength is an important limiting factor for plant production. Secondary cell walls play a crucial role in plant stem strength, but whether WRKY transcription factors can positively modulate secondary cell wall thickness are remain unknown. Here, we characterized a WRKY transcription factor PlWRKY41a from herbaceous peony (Paeonia lactiflora), which was highly expressed in stems. PlWRKY41a functioned as a nucleus-localized transcriptional activator and enhanced stem strength by positively modulating secondary cell wall thickness. Moreover, PlWRKY41a bound to the promoter of the XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE4 (PlXTH4) and activated the expression of PlXTH4. PlXTH4-overexpressing tobacco (Nicotiana tabacum) had thicker secondary cell walls, resulting in enhanced stem strength, while PlXTH4-silenced P. lactiflora had thinner secondary cell walls, showing decreased stem strength. Additionally, PlWRKY41a directly interacted with PlMYB43 to form a protein complex, and their interaction induced the expression of PlXTH4. These data support that the PlMYB43-PlWRKY41a protein complex can directly activate the expression of PlXTH4 to enhance stem strength by modulating secondary cell wall thickness in P. lactiflora. The results will enhance our understanding of the formation mechanism of stem strength and provide a candidate gene to improve stem straightness in plants.

Analysis and Functional Verification of PlPM19L Gene Associated with Drought-Resistance in Paeonia lactiflora Pall.

The herbaceous peony (Paeonia lactiflora Pall.) is widely cultivated as an ornamental, medicinal and edible plant in China. Drought stress can seriously affect the growth of herbaceous peony and reduce its quality. In our previous research, a significantly differentially expressed gene, PM19L, was obtained in herbaceous peony under drought stress based on transcriptome analysis, but little is known about its function. In this study, the first PM19L that was isolated in herbaceous peony was comprised of 910 bp, and was designated as PlPM19L (OP480984). It had a complete open reading frame of 537 bp and encoded a 178-amino acid protein with a molecular weight of 18.95 kDa, which was located in the membrane. When PlPM19L was transferred into tobacco, the transgenic plants had enhanced tolerance to drought stress, potentially due to the increase in the abscisic acid (ABA) content and the reduction in the level of hydrogen peroxide (H2O2). In addition, the enhanced ability to scavenge H2O2 under drought stress led to improvements in the enzyme activity and the potential photosynthetic capacity. These results combined suggest that PlPM19L is a key factor to conferring drought stress tolerance in herbaceous peony and provide a scientific theoretical basis for the following improvement in the drought resistance of herbaceous peony and other plants through genetic engineering technology.

Herbaceous peony PlACLB2 positively regulates red petal formation by promoting anthocyanin accumulation.

ATP-citrate lyase (ACL) gene catalyzes the formation of acetyl-CoA to provide intermediate precursors for many secondary metabolites, and also plays an important role in anthocyanin biosynthesis of plants. Herbaceous peony (Paeonia lactiflora Pall.) is an international cut flower known for its rich flower colors, however, the function of the ACL gene in flower color regulation is still unclear. Here, double-colored P. lactiflora 'Hebao Jinlian' were used to study the molecular mechanism of red petal, and acetyl-CoA and anthocyanin biosynthesis related PlACLB2, PlCHS, PlDFR, PlANS, and PlbHLH1 genes were initially found to highly expressed in the red outer-petals. The expression pattern of PlACLB2 was consistent with the spatial accumulation of anthocyanins. The correlation analysis of PlACLB2 expression pattern, acetyl-CoA content, and anthocyanin accumulation revealed that PlACLB2 was positively correlated with the acetyl-CoA and anthocyanin contents with correlation coefficients of 0.82 and 0.80. Moreover, multiple sequence alignment identified two typical conserved domains in PlACLB2, and phylogenetic analysis clustered PlACLB2 into the ACLB clade. PlACLB2 was localized in the nucleus and cytoplasm. On the one hand, silencing PlACLB2 in P. lactiflora red outer-petal resulted in lighter petal color and decreased acetyl-CoA accumulation, and quantitative analysis detected that PlACLB2-silenced petals lost more anthocyanins than the control groups with a decrease of 31.0%, and the main pigment component cyanidin-3,5-O-diglucoside was reduced by 31.9%. On the other hand, overexpression of PlACLB2 significantly promoted red coloration, acetyl-CoA content, and anthocyanin accumulation in tobacco flowers. These results demonstrated that PlACLB2 promoted anthocyanin accumulation by increasing the abundance of its precursor substrate acetyl-CoA, thereby regulating the formation of the red petals in P. lactiflora.

Correction: Zhao et al. Integration of Transcriptome, Proteome, and Metabolome Provides Insights into How Calcium Enhances the Mechanical Strength of Herbaceous Peony Inflorescence Stems. Cells 2019, 8, 102.

The authors wish to make the following changes to their paper [...].

Tree Peony R2R3-MYB Transcription Factor PsMYB30 Promotes Petal Blotch Formation by Activating the Transcription of the Anthocyanin Synthase Gene.

Petal blotches are commonly observed in many angiosperm families and not only influence plant-pollinator interactions but also confer high ornamental value. Tree peony (Paeonia suffruticosa Andr.) is an important cut flower worldwide, but few studies have focused on its blotch formation. In this study, anthocyanins were found to be the pigment basis for blotch formation of P. suffruticosa, and peonidin-3,5-di-O-glucoside (Pn3G5G) was the most important component of anthocyanins, while the dihydroflavonol-4-reductase gene was the key factor contributing to blotch formation. Then, the R2R3-myeloblastosis (MYB) transcription factor PsMYB30 belonging to subgroup 1 was proven as a positive anthocyanin regulator with transcriptional activation and nuclear expression. Furthermore, silencing PsMYB30 in P. suffruticosa petals reduced blotch size by 37.9%, faded blotch color and decreased anthocyanin and Pn3G5G content by 23.6% and 32.9%, respectively. Overexpressing PsMYB30 increased anthocyanin content by 14.5-fold in tobacco petals. In addition, yeast one-hybrid assays, dual-luciferase assays and electrophoretic mobility shift assays confirmed that PsMYB30 could bind to the promoter of the anthocyanin synthase (ANS) gene and enhance its expression. Altogether, a novel MYB transcription factor, PsMYB30, was identified to promote petal blotch formation by activating the expression of PsANS involved in anthocyanin biosynthesis, which provide new insights for petal blotch formation in plants.

Herbaceous peony AP2/ERF transcription factor binds the promoter of the tryptophan decarboxylase gene to enhance high-temperature stress tolerance.

Global warming has multifarious adverse effects on plant growth and productivity. Nonetheless, the effects of endogenous phytomelatonin on the high-temperature resistance of plants and the underlying genetic mechanisms remain unclear. Here, herbaceous peony (Paeonia lactiflora Pall.) tryptophan decarboxylase (TDC) gene involved in phytomelatonin biosynthesis was shown to respond to high-temperature stress at the transcriptional level, and its transcript level was positively correlated with phytomelatonin production. Moreover, overexpression of PlTDC enhanced phytomelatonin production and high-temperature stress tolerance in transgenic tobacco, while silencing PlTDC expression decreased these parameters in P. lactiflora. In addition, a 2402 bp promoter fragment of PlTDC was isolated, and DNA pull-down assay revealed that one APETALA2/ethylene-responsive element-binding factor (AP2/ERF) transcription factor, PlTOE3, could specifically activate the PlTDC promoter, which was further verified by yeast one-hybrid assay and luciferase reporter assay. PlTOE3 was a nucleus-localized protein, and its transcript level responded to high-temperature stress. Additionally, transgenic tobacco overexpressing PlTOE3 showed enhanced phytomelatonin production and high-temperature stress tolerance, while silencing PlTDC expression obtained the opposite results. These results illustrated that PlTOE3 bound the PlTDC promoter to enhance high-temperature stress tolerance by increasing phytomelatonin production in P. lactiflora.

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.

CsiLAC4 modulates boron flow in Arabidopsis and Citrus via high-boron-dependent lignification of cell walls.

The mechanisms underlying plant tolerance to boron (B) excess are far from fully understood. Here we characterized the role of the miR397-CsiLAC4/CsiLAC17 (from Citrus sinensis) module in regulation of B flow. Live-cell imaging techniques were used in localization studies. A tobacco transient expression system tested modulations of CsiLAC4 and CsiLAC17 by miR397. Transgenic Arabidopsis were generated to analyze the biological functions of CsiLAC4 and CsiLAC17. CsiLAC4's role in xylem lignification was determined by mRNA hybridization and cytochemistry. In situ B distribution was analyzed by laser ablation inductively coupled plasma mass spectrometry. CsiLAC4 and CsiLAC17 are predominantly localized in the apoplast of tobacco epidermal cells. Overexpression of CsiLAC4 in Arabidopsis improves the plants' tolerance to boric acid excess by triggering high-B-dependent lignification of the vascular system's cell wall and reducing free B content in roots and shoots. In Citrus, CsiLAC4 is expressed explicitly in the xylem parenchyma and is modulated by B-responsive miR397. Upregulation of CsiLAC4 in Citrus results in lignification of the xylem cell walls, restricting B flow from xylem vessels to the phloem. CsiLAC4 contributes to plant tolerance to boric acid excess via high-B-dependent lignification of cell walls, which set up a 'physical barrier' preventing B flow.

Multi-walled carbon nanotubes prevent high temperature-induced damage by activating the ascorbate-glutathione cycle in Paeonia ostii T. Hong et J. X. Zhang.

Multi-walled carbon nanotubes (MWCNTs) are considered important nanomaterials with rapidly growing applications. They are inevitably released into the environment, which has attracted considerable attention for their potential threats to ecosystems. In this study, Paeonia ostii T. Hong et J. X. Zhang was exposed to MWCNTs at different concentrations under high temperature. The results showed that high temperature-induced P. ostii damage was prevented by MWCNTs, and 200 mg/L was the most effective concentration. First, MWCNTs prevented increases in reactive oxygen species, relative electrical conductivity and free proline content, and reduced decreases in SPAD, chlorophyll and carotenoid contents. Moreover, the ascorbate-glutathione (ASA-GSH) cycle was activated in response to the MWCNTs treatments, whereas the superoxide dismutase and catalase activities were inhibited. And the MWCNTs treatments also resulted in higher photosynthesis and more intact anatomical structures. Furthermore, the metabolome also confirmed that the ASA-GSH cycle played a critical role in P. ostii high-temperature tolerance, and other biological processes also responded to the MWCNTs treatments. Additionally, the genes involved in the P. ostii ASA-GSH cycle were highly expressed in response to the MWCNTs treatments. These results elucidated the beneficial role of MWCNTs in P. ostii growth under high temperature.

R2R3-MYB Transcription Factor PlMYB108 Confers Drought Tolerance in Herbaceous Peony (Paeonia lactiflora Pall.).

The MYB transcription factor (TF) is crucial for plant growth, development, and response to abiotic stress, but it is rarely reported in the herbaceous peony (Paeonia lactiflora Pall.). Here, an MYB TF gene was isolated, and based on our prior mRNA data from P. lactiflora samples, it was treated with drought stress (DS). Its complete cDNA structure was 1314 bp, which encoded 291 amino acids (aa). Furthermore, using sequence alignment analysis, we demonstrated that PlMYB108 was an R2R3-MYB TF. We also revealed that PlMYB108 was primarily localized in the nucleus. Its levels rose during DS, and it was positively correlated with drought tolerance (DT) in P. lactiflora. In addition, when PlMYB108 was overexpressed in tobacco plants, the flavonoid content, antioxidant enzyme activities, and photosynthesis were markedly elevated. Hence, the transgenic plants had stronger DT with a higher leaf water content and lower H2O2 accumulation compared to the wild-type (WT) plants. Based on these results, PlMYB108 is a vital gene that serves to increase flavonoid accumulation, reactive oxygen species (ROS), scavenging capacity, and photosynthesis to confer DT. The results would provide a genetic resource for molecular breeding to enhance plant DT.

WRKY Transcription Factor Response to High-Temperature Stress.

Plant growth and development are closely related to the environment, and high-temperature stress is an important environmental factor that affects these processes. WRKY transcription factors (TFs) play important roles in plant responses to high-temperature stress. WRKY TFs can bind to the W-box cis-acting elements of target gene promoters, thereby regulating the expression of multiple types of target genes and participating in multiple signaling pathways in plants. A number of studies have shown the important biological functions and working mechanisms of WRKY TFs in plant responses to high temperature. However, there are few reviews that summarize the research progress on this topic. To fully understand the role of WRKY TFs in the response to high temperature, this paper reviews the structure and regulatory mechanism of WRKY TFs, as well as the related signaling pathways that regulate plant growth under high-temperature stress, which have been described in recent years, and this paper provides references for the further exploration of the molecular mechanisms underlying plant tolerance to high temperature.

Silicon enhances stem strength by promoting lignin accumulation in herbaceous peony (Paeonia lactiflora Pall.).

Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-end cut flower, but stem bending caused by low stem strength severely decreases its quality. To enhance stem strength, the regulatory effects of exogenous silicon were investigated in P. lactiflora. The results showed that silicon application enhanced stem strength by increasing the thickness of secondary cell walls and the layers of thickened secondary cells. Moreover, more lignin accumulated, particularly G-lignin and S-lignin, and the activities of lignin biosynthetic enzymes increased with silicon application. In addition, based on transcriptome analysis, silicon application induced the expression of genes participating in lignin biosynthesis pathway. Among them, hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase gene (HCT1) was isolated from P. lactiflora and found to be mainly localized in the cytoplasm of cells. Overexpression of PlHCT1 increased the layers of thickened secondary cells and lignin accumulation in tobacco, resulting in enhanced stem strength and demonstrably straight stems. Finally, silicon content, lignin content and PlHCT1 expression in P. lactiflora cultivars with high stem strengths were totally higher than those in cultivars with low stem strengths. These results indicated that silicon application enhanced stem strength by promoting lignin accumulation in P. lactiflora, which has prospects for stem quality improvement in general.

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.

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.