Cloning and functional verification of the CmHSP17.9 gene from chrysanthemum.

Small molecular heat shock proteins (sHSPs) belong to the HSP family of molecular chaperones. Under high-temperature stress, they can prevent the aggregation of irreversible proteins and maintain the folding of denatured proteins to enhance heat resistance. In this study, the CmHSP17.9-1 and CmHSP17.9-2 genes, which were cloned from chrysanthemum (Chrysanthemum×morifolium 'Jinba') by homologous cloning, had a complete open reading frame of 480 bp each, encoding 159 amino acids. The protein subcellular localization analysis showed that CmHSP17.9-1 and CmHSP17.9-2 were located in the cytoplasm and mostly aggregated in granules, especially around the nucleus. Real-time quantitative PCR (qRT-PCR) analysis showed that the relative expression level of the CmHSP17.9-1 and CmHSP17.9-2 genes was highest in the terminal buds of the chrysanthemum, followed by the leaves. CmHSP17.9-1 and CmHSP17.9-2 overex-pression vectors were constructed and used to transform the chrysanthemum; overexpression of these genes led to the chrysanthemum phenotypes being less affected by high-temperature, and the antioxidant capacity was enhanced. The results showed that chrysanthemum with overex-pression of the CmHSP17.9-1 and CmHSP17.9-2 genes had stronger tolerance than the wild type chrysanthemum after high-temperature treatment or some degree of heat exercise, and overex-pression of the CmHSP17.9-1 gene led to stronger heat resistance than that of the CmHSP17.9-2 gene, providing an important theoretical basis for the subsequent molecular breeding and pro-duction applications of chrysanthemum.

Induction of volatile organic compounds in chrysanthemum plants following infection by Rhizoctonia solani.

This study investigated the effects of Rhizoctonia solani J.G. Kühn infestation on the volatile organic compound (VOC) emissions and biochemical composition of ten cultivars of chrysanthemum (Chrysanthemum × morifolium /Ramat./ Hemsl.) to bring new insights for future disease management strategies and the development of resistant chrysanthemum cultivars. The chrysanthemum plants were propagated vegetatively and cultivated in a greenhouse under semi-controlled conditions. VOCs emitted by the plants were collected using a specialized system and analyzed by gas chromatography/mass spectrometry. Biochemical analyses of the leaves were performed, including the extraction and quantification of chlorophylls, carotenoids, and phenolic compounds. The emission of VOCs varied among the cultivars, with some cultivars producing a wider range of VOCs compared to others. The analysis of the VOC emissions from control plants revealed differences in both their quality and quantity among the tested cultivars. R. solani infection influenced the VOC emissions, with different cultivars exhibiting varying responses to the infection. Statistical analyses confirmed the significant effects of cultivar, collection time, and their interaction on the VOCs. Correlation analyses revealed positive relationships between certain pairs of VOCs. The results show significant differences in the biochemical composition among the cultivars, with variations in chlorophyll, carotenoids, and phenolic compounds content. Interestingly, R. solani soil and leaf infestation decreased the content of carotenoids in chrysanthemums. Plants subjected to soil infestation were characterized with the highest content of phenolics. This study unveils alterations in the volatile and biochemical responses of chrysanthemum plants to R. solani infestation, which can contribute to the development of strategies for disease management and the improvement of chrysanthemum cultivars with enhanced resistance to R. solani.

CmDOF18 positively regulates salinity tolerance in Chrysanthemum morifolium by activating the oxidoreductase system.

BACKGROUND: Chrysanthemum, one of the four major cut flowers all over the world, is very sensitive to salinity during cultivation. DNA binding with one finger (DOF) transcription factors play important roles in biological processes in plants. The response mechanism of CmDOF18 from chrysanthemum to salt stress remains unclear. RESULTS: In this study, CmDOF18 was cloned from Chrysanthemum morifolium, and its expression was induced by salinity stress. The gene encodes a 291-amino acid protein with a typical DOF domain. CmDOF18 was localized to the nucleus in onion epidermal cells and showed transcriptional activation in yeast. CmDOF18 transgenic plants were generated to identify the role of this gene in resistance to salinity treatment. Chrysanthemum plants overexpressing CmDOF18 were more resistant to salinity stress than wild-type plants. Under salinity stress, the malondialdehyde content and leaf electrolyte conductivity in CmDOF18-overexpressing transgenic plants were lower than those in wild-type plants, while the proline content, chlorophyll content, superoxide dismutase activity and peroxidase activity were higher than those in wild-type plants. The opposite findings were observed in gene-silenced plants compared with wild-type plants. The gene expression levels of oxidoreductase increased in CmDOF18-overexpressing transgenic plants but decreased in CmDOF18-SRDX gene-silenced transgenic plants. CONCLUSION: In summary, we analyzed the function of CmDOF18 from chrysanthemum, which may regulate salinity stress in plants, possibly due to its role in the regulation of oxidoreductase.

Comparative transcriptome analysis provides molecular insights into heterosis of waterlogging tolerance in Chrysanthemum indicum.

BACKGROUND: Heterosis breeding is one of the most important breeding methods for chrysanthemum. To date, the genetic mechanisms of heterosis for waterlogging tolerance in chrysanthemum are still unclear. This study aims to analyze the expression profiles and potential heterosis-related genes of two hybrid lines and their parents with extreme differences in waterlogging tolerance under control and waterlogging stress conditions by RNA-seq. RESULTS: A population of 140 F1 progeny derived from Chrysanthemum indicum (Nanchang) (waterlogging-tolerant) and Chrysanthemum indicum (Nanjing) (waterlogging-sensitive) was used to characterize the extent of genetic variation in terms of seven waterlogging tolerance-related traits across two years. Lines 98 and 95, respectively displaying positive and negative overdominance heterosis for the waterlogging tolerance traits together with their parents under control and waterlogging stress conditions, were used for RNA-seq. In consequence, the maximal number of differentially expressed genes (DEGs) occurred in line 98. Gene ontology (GO) enrichment analysis revealed multiple stress-related biological processes for the common up-regulated genes. Line 98 had a significant increase in non-additive genes under waterlogging stress, with transgressive up-regulation and paternal-expression dominant patterns being the major gene expression profiles. Further, GO analysis identified 55 and 95 transgressive up-regulation genes that overlapped with the up-regulated genes shared by two parents in terms of responses to stress and stimulus, respectively. 6,640 genes in total displaying maternal-expression dominance patterns were observed in line 95. In addition, 16 key candidate genes, including SAP12, DOX1, and ERF017 which might be of significant importance for the formation of waterlogging tolerance heterosis in line 98, were highlighted. CONCLUSION: The current study provides a comprehensive overview of the root transcriptomes among F1 hybrids and their parents under waterlogging stress. These findings lay the foundation for further studies on molecular mechanisms underlying chrysanthemum heterosis on waterlogging tolerance.

Chrysanthemum morifolium as a traditional herb: A review of historical development, classification, phytochemistry, pharmacology and application.

ETHNOPHARMACOLOGICAL RELEVANCE: In recent years, Chinese herbal medicine has gained more and more recognition in disease prevention and control due to its low toxicity and comprehensive treatment. C. morifolium (Chrysanthemum morifolium Ramat.), as the medicine food homology plant with the bioactivity of anti-oxidation, anti-inflammatory, neuroprotection and cardiovascular protection, has important therapeutic effects and health benefits for colds, inflammation, cardiovascular diseases and various chronic diseases. AIM OF THE STUDY: By reviewing the historical development, classification and distribution of germplasm resources, phytochemistry, pharmacology, and modern application of C. morifolium, the paper provides a reliable basis for the further research and application of chrysanthemum as therapeutic agents and functional additives. MATERIALS AND METHODS: The literature and information about C. morifolium published in the last ten years were collected from various platforms, including Google Scholar, PubMed, ScienceDirect, Web of Science and China Knowledge Network. RESULTS: A comprehensive analysis confirmed that C. morifolium originated in China, and it went through the development process from food and tea to medicine for more than 3000 years. During this period, different cultivars emerged through several breeding techniques and were distributed throughout the world. Moreover, A variety of chemical components such as flavonoids, phenolic acids, volatile oils, and terpenes in chrysanthemum have been proven they possess various pharmacology of anti-inflammatory, anti-oxidant, and prevention of chronic diseases by regulating inflammatory cytokines, oxidative stress responses and signaling pathways, which are the essential conditions to play a role in TCM, nutraceuticals and diet. CONCLUSION: This paper provides a comprehensive review of historical development, classification, phytochemistry, pharmacology, and modern application of C. morifolium. However, future studies should continue to focus on the bioactive compounds and the synergistic mechanism of the "multi-component, multi-target, and multi-pathway" of chrysanthemum, and it is necessary to develop more innovative products with therapeutic effects.

The A subunit of vacuolar H+-ATPase gene (CmVHA-A) plays opposite roles in plant growth and drought tolerance of chrysanthemum under different growing conditions.

As the most prominent proton pumps in plants, vacuolar H+-ATPases (VHAs) comprise multiple subunits that are important for physiological processes and stress tolerance in plants. However, few studies on the roles of subunit genes of VHAs in chrysanthemum have been reported to date. In this study, the gene of A subunit of V-ATPase in chrysanthemum (CmVHA-A) was cloned and identified. CmVHA-A was conserved with VHA-A proteins from other plants. Expression analysis showed that CmVHA-A was highly expressed in most tissues of chrysanthemum except for the flower bud, and was readily induced by polyethylene glycol (PEG) treatment. Functional analysis demonstrated that CmVHA-A exerted a negative influence on the growth and development of shoot and root of chrysanthemum under normal conditions. RNA-sequencing (RNA-seq) analysis revealed the possible explanations for phenotypic differences between transgenic and wild-type (WT) plants. Under drought conditions, CmVHA-A positively affected the drought tolerance of chrysanthemum by enhancing antioxidase activity and alleviating photosynthetic disruption. Overall, CmVHA-A plays opposite roles in plant growth and drought tolerance of chrysanthemums under different growing conditions.

[Identification and expression of genome of uridine diphosphate glycosyltransferase (UGT) gene family from Chrysanthemum indicum].

Uridine diphosphate glycosyltransferase(UGT) is involved in the glycosylation of a variety of secondary metabolites in plants and plays an important role in plant growth and development and regulation of secondary metabolism. Based on the genome of a diploid Chrysanthemum indicum, the UGT gene family from Ch. indicum was identified by bioinformatics methods, and the physical and chemical properties, subcellular localization prediction, conserved motif, phylogeny, chromosome location, gene structure, and gene replication events of UGT protein were analyzed. Transcriptome and real-time fluorescence quantitative polymerase chain reaction(PCR) were used to analyze the expression pattern of the UGT gene in flowers and leaves of Ch. indicum. Quasi-targeted metabolomics was used to analyze the differential metabolites in flowers and leaves. The results showed that a total of 279 UGT genes were identified in the Ch. indicum genome. Phylogenetic analysis showed that these UGT genes were divided into 8 subfamilies. Members of the same subfamily were distributed in clusters on the chromosomes. Tandem duplications were the main driver of the expansion of the UGT gene family from Ch. indicum. Structural domain analysis showed that 262 UGT genes had complete plant secondary metabolism signal sequences(PSPG box). The analysis of cis-acting elements indicated that light-responsive elements were the most ubiquitous elements in the promoter regions of UGT gene family members. Quasi-targeted metabolome analysis of floral and leaf tissue revealed that most of the flavonoid metabolites, including luteolin-7-O-glucoside and kaempferol-7-O-glucoside, had higher accumulation in flowers. Comparative transcriptome analysis of flower and leaf tissue showed that there were 72 differentially expressed UGT genes, of which 29 genes were up-regulated in flowers, and 43 genes were up-regulated in leaves. Correlation network and phylogenetic analysis showed that CindChr9G00614970.1, CindChr2G00092510.1, and CindChr2G00092490.1 may be involved in the synthesis of 7-O-flavonoid glycosides in Ch. indicum, and real-time fluorescence quantitative PCR analysis further confirmed the reliability of transcriptome data. The results of this study are helpful to understand the function of the UGT gene family from Ch. indicum and provide data reference and theoretical basis for further study on the molecular regulation mechanism of flavonoid glycosides synthesis in Ch. indicum.

Influence of low-cost Thai leucoxene minerals on the growth, bioactive compounds, and antibacterial activities of Chrysanthemum indium L. cuttings in in vitro culture.

The effects of low-cost Thai leucoxene mineral (LM) at different concentrations (10, 20, 30, 40, 50, and 60 mg/L) on the growth and antibacterial properties of Chrysanthemum indium L. cuttings under in vitro were evaluated. The primary chemical composition of LM was approximately 86% titanium dioxide (TiO2), as determined by dispersive X-ray spectroscopy. The crystalline structure, shape, and size were investigated by X-ray diffraction and scanning electron microscopy. LM at 40 and 50 mg/L significantly increased plant height, leaf number, node number, and fresh and dry weight. These growth-promoting properties were accompanied by improved chlorophyll and carotenoid contents and antioxidant enzyme activities and reduced malondialdehyde levels. Additionally, LM treatment at 40 and 50 mg/L had positive effects on antibacterial activity, as indicated by the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. The high levels of phenolic compounds in the plants contributed to the MIC and MBC values. In conclusion, these findings provide evidence for the effectiveness of LM in enhancing the growth of Chrysanthemum plants in in vitro culture and improving their antibacterial abilities.

Simultaneously efficient dissolution and structural modification of chrysanthemum pectin: Targeting at proliferation of Bacteroides.

Pectic polysaccharide is a bioactive ingredient in Chrysanthemum morifolium Ramat. 'Hangbaiju' (CMH), but the high proportion of HG domain limited its use as a prebiotic. In this study, hot water, cellulase-assisted, medium-temperature alkali, and deep eutectic solvent extraction strategies were firstly used to extract pectin from CMH (CMHP). CMHP obtained by cellulase-assisted extraction had high purity and strong ability to promote the proliferation of Bacteroides and mixed probiotics. However, 4 extraction strategies led to general high proportion of HG domain in CMHPs. To further enhance the dissolution and prebiotic potential of CMHP, pectinase was used alone and combined with cellulase. The key factor for the optimal extraction was enzymolysis by cellulase and pectinase in a mass ratio of 3:1 at 1 % (w/w) dosage. The optimal CMHP had high yield (15.15 %), high content of total sugar, and Bacteroides proliferative activity superior to inulin, which was probably due to the cooperation of complex enzyme on the destruction of cell wall and pectin structural modification for raised RG-I domain (80.30 %) with relatively high degree of branching and moderate HG domain. This study provided a green strategy for extraction of RG-I enriched prebiotic pectin from plants.

A GASA Protein Family Gene, CmGEG, Inhibits Petal Growth in Chrysanthemum.

The diversity in the petal morphology of chrysanthemums makes this species an excellent model for investigating the regulation mechanisms of petal size. However, our understanding of the molecular regulation of petal growth in chrysanthemums remains limited. The GASA (gibberellic acid [GA]-stimulated Arabidopsis) protein plays a significant role in various aspects of plant growth and development. Previous studies have indicated that GEG (a gerbera homolog of the gibberellin-stimulated transcript 1 [GAST1] from tomato) is involved in regulating ray petal growth by inhibiting cell expansion in gerberas. In this study, we successfully cloned the GASA family gene from chrysanthemums, naming it CmGEG, which shares 81.4% homology with GEG. Our spatiotemporal expression analysis revealed that CmGEG is expressed in all tissues, with the highest expression levels observed in the ray florets, particularly during the later stages of development. Through transformation experiments, we demonstrated that CmGEG inhibits petal elongation in chrysanthemums. Further observations indicated that CmGEG restricts cell elongation in the top, middle, and basal regions of the petals. To investigate the relationship between CmGEG and GA in petal growth, we conducted a hormone treatment assay using detached chrysanthemum petals. Our results showed that GA promotes petal elongation while downregulating CmGEG expression. In conclusion, the constrained growth of chrysanthemum petals may be attributed to the inhibition of cell elongation by CmGEG, a process regulated by GA.

Multi-locus genome-wide association studies reveal the dynamic genetic architecture of flowering time in chrysanthemum.

KEY MESSAGE: The dynamic genetic architecture of flowering time in chrysanthemum was elucidated by GWAS. Thirty-six known genes and 14 candidate genes were identified around the stable QTNs and QEIs, among which ERF-1 was highlighted. Flowering time (FT) adaptation is one of the major breeding goals in chrysanthemum, a multipurpose ornamental plant. In order to reveal the dynamic genetic architecture of FT in chrysanthemum, phenotype investigation of ten FT-related traits was conducted on 169 entries in 2 environments. The broad-sense heritability of five non-conditional FT traits, i.e., budding (FBD), visible coloring (VC), early opening (EO), full-bloom (OF) and decay period (DP), ranged from 56.93 to 84.26%, which were higher than that of the five derived conditional FT traits (38.51-75.13%). The phenotypic variation coefficients of OF_EO and DP_OF were relatively large ranging from 30.59 to 36.17%. Based on 375,865 SNPs, the compressed variance component mixed linear model 3VmrMLM was applied for a multi-locus genome-wide association study (GWAS). As a result, 313 quantitative trait nucleotides (QTNs) were identified for the non-conditional FT traits in single-environment analysis, while 119 QTNs and 67 QTN-by-environment interactions (QEIs) were identified in multi-environment analysis. As for the conditional traits, 343 QTNs were detected in single-environment analysis, and 119 QTNs and 83 QEIs were identified in multi- environment analysis. Among the genes around stable QTNs and QEIs, 36 were orthologs of known FT genes in Arabidopsis and other plants; 14 candidates were mined by combining the transcriptomics data and functional annotation, including ERF-1, ACA10, and FOP1. Furthermore, the haplotype analysis of ERF-1 revealed six elite accessions with extreme FBD. Our findings contribute to the understanding of dynamic genetic architecture of FT and provide valuable resources for future chrysanthemum molecular breeding programs.

Propyl gallate induces cell death in human pulmonary fibroblast through increasing reactive oxygen species levels and depleting glutathione.

Propyl gallate (PG) exhibits an anti-growth effect on various cell types. The present study investigated the impact of PG on the levels of reactive oxygen species (ROS) and glutathione (GSH) in primary human pulmonary fibroblast (HPF) cells. Moreover, the effects of N-acetyl cysteine (NAC, an antioxidant), L-buthionine sulfoximine (BSO, a GSH synthesis inhibitor), and small interfering RNA (siRNAs) against various antioxidant genes on ROS and GSH levels and cell death were examined in PG-treated HPF cells. PG (100-800 µM) increased the levels of total ROS and O2·- at early time points of 30-180 min and 24 h, whereas PG (800-1600 µM) increased GSH-depleted cell number at 24 h and reduced GSH levels at 30-180 min. PG downregulated the activity of superoxide dismutase (SOD) and upregulated the activity of catalase in HPF cells. Treatment with 800 µM PG increased the number of apoptotic cells and cells that lost mitochondrial membrane potential (MMP; ΔΨm). NAC treatment attenuated HPF cell death and MMP (ΔΨm) loss induced by PG, accompanied by a decrease in GSH depletion, whereas BSO exacerbated the cell death and MMP (ΔΨm) loss without altering ROS and GSH depletion levels. Furthermore, siRNA against SOD1, SOD2, or catalase attenuated cell death in PG-treated HPF cells, whereas siRNA against GSH peroxidase enhanced cell death. In conclusion, PG induced cell death in HPF cells by increasing ROS levels and depleting GSH. NAC was found to decrease HPF cell death induced by PG, while BSO enhanced cell death. The findings shed light on how manipulating the antioxidant system influence the cytotoxic effects of PG in HPF cells.

Evaluation of Environmental Factor Effects on the Polyphenol and Flavonoid Content in the Leaves of Chrysanthemum indicum L. and Its Habitat Suitability Prediction Mapping.

The leaves of Chrysanthemum indicum L. are known to have various bioactive compounds; however, industrial use is extremely limited. To overcome this situation by producing high-quality leaves with high bioactive content, this study examined the environmental factors affecting the phytochemical content and antioxidant activity using C. indicum leaves collected from 22 sites in Kochi Prefecture, Japan. Total phenolic and flavonoid content in the dry leaves ranged between 15.0 and 64.1 (mg gallic acid g-1) and 2.3 and 11.4 (mg quercetin g-1), while the antioxidant activity (EC50) of the 50% ethanol extracts ranged between 28.0 and 123.2 (µg mL-1) in 1,1-Diphenyl-2-picrylhydrazyl radical scavenging assay. Among the identified compounds, chlorogenic acid and 1,5-dicaffeoylquinic acid were the main constituents in C. indicum leaves. The antioxidant activity demonstrated a positive correlation with 1,5-dicaffeoylquinic acid (R2 = 0.62) and 3,5-dicaffeoylquinic acid (R2 = 0.77). The content of chlorogenic acid and dicaffeoylquinic acid isomers varied significantly according to the effects of exchangeable magnesium, cation exchange capacity, annual temperature, and precipitation, based on analysis of variance. The habitat suitability map using the geographical information system and the MaxEnt model predicted very high and high regions, comprising 3.2% and 10.1% of the total area, respectively. These findings could be used in future cultivation to produce high-quality leaves of C. indicum.

In vitro and in planta investigation of succinate dehydrogenase inhibitor resistance conferred by target-site amino acid substitutions in Puccinia horiana, chrysanthemum white rust.

BACKGROUND: Resistance to succinate dehydrogenase inhibitor (SDHI) fungicides has been reported in some rust fungi within Pucciniales. However, measuring the resistance factors conferred by a specific substitution at the target site is difficult for most species because of the difficulty in performing in vitro experiments and the complexity of the binuclear state in these obligate parasites. We focused on Puccinia horiana because it easily forms homozygous basidiospores that are sensitive to SDHIs during in vitro germination, whereas the uredospores of other rust fungi are less sensitive. RESULTS: We identified two substitutions, SdhC-I88F and SdhD-C125Y, that drive SDHI resistance in Pu. horiana. Using basidiospore germination inhibition tests, we measured the resistance factors for six SDHI fungicides in Pu. horiana isolates harboring SdhC-I88F substitutions, wherein orthologous substitutions were most frequently observed in SDHI-resistant Pucciniales, such as soybean rust (Phakopsora pachyrhizi). The resistance factors were high for penthiopyrad and benzovindiflupyr (>150), moderate for oxycarboxin and inpyrfluxam (10-30), and low for mepronil and fluxapyroxad (3-10). The most potent SDHI against SdhC-I88F-harboring isolates was inpyrfluxam, with a half-maximal effective concentration (EC50) of 0.0082 mg L-1 owing to its high intrinsic activity. SdhD-C125Y played a minor, but significant role in increasing the resistance factors (one- to tenfold increases), depending on the individual SDHIs. CONCLUSION: This study is the first to use basidiospore germination inhibitory tests to quantify the resistance factors for SDHI-resistant Pucciniales. Owing to its homozygous binucleate nature and the high availability of basidiospores, Pu. horiana is useful for investigating SDHI resistance in Pucciniales. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genomic insights into the evolution of flavonoid biosynthesis and O-methyltransferase and glucosyltransferase in Chrysanthemum indicum.

Flavonoids are a class of secondary metabolites widely distributed in plants. Regiospecific modification by methylation and glycosylation determines flavonoid diversity. A rare flavone glycoside, diosmin (luteolin-4'-methoxyl-7-O-glucosyl-rhamnoside), occurs in Chrysanthemum indicum. How Chrysanthemum plants evolve new biosynthetic capacities remains elusive. Here, we assemble a 3.11-Gb high-quality C. indicum genome with a contig N50 value of 4.39 Mb and annotate 50,606 protein-coding genes. One (CiCOMT10) of the tandemly repeated O-methyltransferase genes undergoes neofunctionalization, preferentially transferring the methyl group to the 4'-hydroxyl group of luteolin with ortho-substituents to form diosmetin. In addition, CiUGT11 (UGT88B3) specifically glucosylates 7-OH group of diosmetin. Next, we construct a one-pot cascade biocatalyst system by combining CiCOMT10, CiUGT11, and our previously identified rhamnosyltransferase, effectively producing diosmin with over 80% conversion from luteolin. This study clarifies the role of transferases in flavonoid diversity and provides important gene elements essential for producing rare flavone.

The histone deacetylase SRT2 enhances the tolerance of chrysanthemum to low temperatures through the ROS scavenging system.

Low temperatures can severely affect plant growth and reduce their ornamental value. A family of plant histone deacetylases allows plants to cope with both biotic and abiotic stresses. In this study, we screened and cloned the cDNA of DgSRT2 obtained from transcriptome sequencing of chrysanthemum leaves under low-temperature stress. Sequence analysis showed that DgSRT2 belongs to the sirtuin family of histone deacetylases. We obtained the stable transgenic chrysanthemum lines OE-2 and OE-12. DgSRT2 showed tissue specificity in wild-type chrysanthemum and was most highly expressed in leaves. Under low-temperature stress, the OE lines showed higher survival rates, proline content, solute content, and antioxidant enzyme activities, and lower relative electrolyte leakage, malondialdehyde, hydrogen peroxide, and superoxide ion accumulation than the wild-type lines. This work suggests that DgSRT2 can serve as an essential gene for enhancing cold resistance in plants. In addition, a series of cold-responsive genes in the OE line were compared with WT. The results showed that DgSRT2 exerted a positive regulatory effect by up-regulating the transcript levels of cold-responsive genes. The above genes help to increase antioxidant activity, maintain membrane stability and improve osmoregulation, thereby enhancing survival under cold stress. It can be concluded from the above work that DgSRT2 enhances chrysanthemum tolerance to low temperatures by scavenging the ROS system.

The BTB/TAZ domain-containing protein CmBT1-mediated CmANR1 ubiquitination negatively regulates root development in chrysanthemum.

Roots are fundamental for plants to adapt to variable environmental conditions. The development of a robust root system is orchestrated by numerous genetic determinants and, among them, the MADS-box gene ANR1 has garnered substantial attention. Prior research has demonstrated that, in chrysanthemum, CmANR1 positively regulates root system development. Nevertheless, the upstream regulators involved in the CmANR1-mediated regulation of root development remain unidentified. In this study, we successfully identified bric-a-brac, tramtrack and broad (BTB) and transcription adapter putative zinc finger (TAZ) domain protein CmBT1 as the interacting partner of CmANR1 through a yeast-two-hybrid (Y2H) screening library. Furthermore, we validated this physical interaction through bimolecular fluorescence complementation and pull-down assays. Functional assays revealed that CmBT1 exerted a negative influence on root development in chrysanthemum. In both in vitro and in vivo assays, it was evident that CmBT1 mediated the ubiquitination of CmANR1 through the ubiquitin/26S proteasome pathway. This ubiquitination subsequently led to the degradation of the CmANR1 protein and a reduction in the transcription of CmANR1-targeted gene CmPIN2, which was crucial for root development in chrysanthemum. Genetic analysis suggested that CmBT1 modulated root development, at least in part, by regulating the level of CmANR1 protein. Collectively, these findings shed new light on the regulatory role of CmBT1 in degrading CmANR1 through ubiquitination, thereby repressing the expression of its targeted gene and inhibiting root development in chrysanthemum.

Overexpression of the Chrysanthemum lavandulifolium ROS1 gene promotes flowering in Arabidopsis thaliana by reducing the methylation level of CONSTANS.

DNA demethylation is involved in the regulation of flowering in plants, yet the underlying molecular mechanisms remain largely unexplored. The RELEASE OF SILENCING 1 (ROS1) gene, encoding a DNA demethyltransferase, plays key roles in many developmental processes. In this study, the ROS1 gene was isolated from Chrysanthemum lavandulifolium, where it was strongly expressed in the leaves, buds and flowers. Overexpression of the ClROS1 gene caused an early flowering phenotype in Arabidopsis thaliana. RNA-seq analysis of the transgenic plants revealed that differentially expressed genes (DEGs) were significantly enriched in the circadian rhythm pathway and that the positive regulator of flowering, CONSTANS (CO), was up-regulated. Additionally, whole-genome bisulphite sequencing (WGBS), PCR following methylation-dependent digestion with the enzyme McrBC, and bisulfite sequencing PCR (BSP) confirmed that the methylation level of the AtCO promoter was reduced, specifically in CG context. Overall, our results demonstrated that ClROS1 accelerates flowering by reducing the methylation level of the AtCO promoter. These findings clarify the epigenetic mechanism by which ClROS1-mediated DNA demethylation regulates flowering.

Nitrogen and spent coffee ground for enhancing nutritional, morphological, flowering and antioxidant properties of Chrysanthemum (Chrysanthemum morfolium Ramat).

Chrysanthemum is one of the most attractive flowering plants widely grown commercially worldwide. Having a good source of organic fertilizers plays an important role in meeting the increasing demand for these plants, which requires high-quality flowers and a high survival time for the longest period. The effect of nitrogen (N) coupled with spent coffee ground (SCG) at various levels (0.0, 2.5, 5.0, 7.5, 10.0°% w/w) was evaluated on growth performance and chemical components of the Chrysanthemum over two years in a pot scale. Overall, total dry matter (TDM) was significantly enhanced with N+ by 125 and 97°% over N- in the first and second years, respectively. SCG also enhanced TDM up to the highest level of application in the range of 27-98°% and 18-81°% over SCG (0.0°%) in the same years, respectively. The interaction effect between N and SCG was perfect on TDM, flower number, and flower dry weight. Similarly, total antioxidant activities when N and SCG were coupled together gave respective increments ranging from 11.8 to 45.9 U/g DW and from 2.1 to 15.9 U/g DW compared to N alone (5.8 and 0.9 U/g DW) in both leaves and flowers, respectively. Extracts of plant treated with N and 10°% SCG exhibited a higher content of rosmarinic, caffeic, chlorogenic, vanillic acids, and rutin in the leaves. SCG as a natural organic source is easy to obtain and is a practical and cost-effective solution to plant nutrition, which can be valuable for ornamental plants, especially when combined with nitrogen.

Chrysanthemum (Chrysanthemum morifolium) CmHRE2-like negatively regulates the resistance of chrysanthemum to the aphid (Macrosiphoniella sanborni).

BACKGROUND: The growth and ornamental value of chrysanthemums are frequently hindered by aphid attacks. The ethylene-responsive factor (ERF) gene family is pivotal in responding to biotic stress, including insect stress. However, to date, little is known regarding the involvement of ERF transcription factors (TFs) in the response of chrysanthemum to aphids. RESULTS: In the present study, CmHRE2-like from chrysanthemum (Chrysanthemum morifolium), a transcription activator that localizes mainly to the nucleus, was cloned. Expression is induced by aphid infestation. Overexpression of CmHRE2-like in chrysanthemum mediated its susceptibility to aphids, whereas CmHRE2-like-SRDX dominant repressor transgenic plants enhanced the resistance of chrysanthemum to aphids, suggesting that CmHRE2-like contributes to the susceptibility of chrysanthemum to aphids. The flavonoids in CmHRE2-like-overexpression plants were decreased by 29% and 28% in two different lines, whereas they were increased by 42% and 29% in CmHRE2-like-SRDX dominant repressor transgenic plants. The expression of Chrysanthemum-chalcone-synthase gene(CmCHS), chalcone isomerase gene (CmCHI), and flavonoid 3'-hydroxylase gene(CmF3'H) was downregulated in CmHRE2-like overexpression plants and upregulated in CmHRE2-like-SRDX dominant repressor transgenic plants, suggesting that CmHRE2-like regulates the resistance of chrysanthemum to aphids partially through the regulation of flavonoid biosynthesis. CONCLUSION: CmHRE2-like was a key gene regulating the vulnerability of chrysanthemum to aphids. This study offers fresh perspectives on the molecular mechanisms of chrysanthemum-aphid interactions and may bear practical significance for developing new strategies to manage aphid infestation in chrysanthemums.