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Stem strength plays a crucial role in the growth and development of plants, as well as in their flowering and fruiting. It not only impacts the lodging resistance of crops, but also influences the ornamental value of ornamental plants. Stem development is closely linked to stem strength; however, the roles of the SPL transcription factors in the stem development of herbaceous peony ( Paeonia lactiflora Pall.) are not yet fully elucidated. In this study, we obtained and cloned the full-length sequence of PlSPL14 , encoding 1085 amino acids. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression level of PlSPL14 gradually increased with the stem development of P. lactiflora and was significantly expressed in vascular bundles. Subsequently, utilizing the techniques of virus-induced gene silencing (VIGS) and heterologous overexpression in tobacco ( Nicotiana tabacum L.), it was determined that PlSPL14 -silenced P. lactiflora had a thinner xylem thickness, a decreased stem diameter, and weakened stem strength, while PlSPL14 -overexpressing tobacco resulted in a thicker xylem thickness, an increased stem diameter, and enhanced stem strength. Further screening of the interacting proteins of PlSPL14 using a yeast two-hybrid (Y2H) assay revealed an interactive relationship between PlSPL14 and PlSLR1 protein, which acts as a negative regulator of gibberellin (GA). Additionally, the expression level of PlSLR1 gradually decreased during the stem development of P. lactiflora . The above results suggest that PlSPL14 may play a positive regulatory role in stem development and act in the xylem, making it a potential candidate gene for enhancing stem straightness in plants.

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 Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2022 John Wiley & Sons Ltd.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Graphene oxide (GO) is considered to be an emerging environmental pollutant with its inevitable release into the environment. Thus, its potential environmental risks and biosafety are receiving increased attention. In this study, Paeonia ostii was exposed to GO under drought stress. The results demonstrated that GO prevented soil water from evaporating due to its hydrophilic oxygen-containing functional groups and did not change the soil pH. Moreover, GO treatment resulted in lower increases in reactive oxygen species, relative electrical conductivity and free proline content, and greater increases in the antioxidant enzyme activities of P. ostii under drought stress compared with those in the control. And under drought stress, higher photosynthesis, more intact mesophyll cells and organelles and open stomata were found in P. ostii under GO treatment. Furthermore, GO treatment induced greater changes in the expression patterns of genes required for lignin biosynthesis, photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms, and glyoxylate and dicarboxylate metabolism. Additionally, GO did not accumulate in P. ostii due to the soil environment and the electrostatic repulsion between GO and the roots. GO did not have toxic effects on P. ostii and was an effective soil water retention agent; therefore, it could be economically beneficial for the production of plants under drought stress.

Weak stem mechanical strength severely restrains cut flowers quality and stem weakness can be alleviated by calcium (Ca) treatment, but the mechanisms underlying Ca-mediated enhancement of stem mechanical strength remain largely unknown. In this study, we performed a comparative transcriptomic, proteomic, and metabolomic analysis of herbaceous peony ( Paeonia lactiflora Pall.) inflorescence stems treated with nanometer Ca carbonate (Nano-CaCO₃). In total, 2643 differentially expressed genes (DEGs) and 892 differentially expressed proteins (DEPs) were detected between the Control and nano-CaCO₃ treatment. Among the 892 DEPs, 152 were coregulated at both the proteomic and transcriptomic levels, and 24 DEPs related to the secondary cell wall were involved in signal transduction, energy metabolism, carbohydrate metabolism and lignin biosynthesis, most of which were upregulated after nano-CaCO₃ treatment during the development of inflorescence stems. Among these four pathways, numerous differentially expressed metabolites (DEMs) related to lignin biosynthesis were identified. Furthermore, structural observations revealed the thickening of the sclerenchyma cell walls, and the main wall constitutive component lignin accumulated significantly in response to nano-CaCO₃ treatment, thereby indicating that Ca can enhance the mechanical strength of the inflorescence stems by increasing the lignin accumulation. These results provided insights into how Ca treatment enhances the mechanical strength of inflorescence stems in P. lactiflora .

The molecular mechanisms controlling petal shape in a herbaceous peony, Paeonia lactiflora Pall., a popular high-grade cut flower worldwide, remain unclear. Here, we selected inner and outer petals from P. lactiflora 'ZiFengyu' with an anemone type as the study object. Using transcriptome sequencing and isobaric tags for relative and absolute quantitation proteome, 979 differentially expressed genes and 266 differentially expressed proteins were detected within the inner and outer petals. Of these, the present study identified a key gene APETALA2 that regulates flower shape development. In addition, we obtained a 1935 bp full-length cDNA sequence of APETALA2 by rapid amplification of cDNA ends amplification. Through further validation using quantitative real-time polymerase chain reaction and Western blot analysis, APETALA2 showed a markedly higher expression in outer than that in inner petals. Therefore, the present study indicates that the increased expression of APETALA2 contributes to the formation of petals in P. lactiflora ., (© 2018 The Author(s).)