AGAMOUS-LIKE 24 senses continuous inductive photoperiod in the inflorescence meristem to promote anthesis in chrysanthemum.

During the floral transition, many plant species including chrysanthemum (Chrysanthemum morifolium) require continuous photoperiodic stimulation for successful anthesis. Insufficient photoperiodic stimulation results in flower bud arrest or even failure. The molecular mechanisms underlying how continuous photoperiodic stimulation promotes anthesis are not well understood. Here, we reveal that in wild chrysanthemum (Chrysanthemum indicum), an obligate short-day (SD) plant, floral evocation is not limited to SD conditions. However, SD signals generated locally in the inflorescence meristem (IM) play a vital role in ensuring anthesis after floral commitment. Genetic analyses indicate that the florigen FLOWERING LOCUS T-LIKE3 (CiFTL3) plays an important role in floral evocation, but a lesser role in anthesis. Importantly, our data demonstrate that AGAMOUS-LIKE 24 (CiAGL24) is a critical component of SD signal perception in the IM to promote successful anthesis, and that floral evocation and anthesis are two separate developmental events in chrysanthemum. We further reveal that the central circadian clock component PSEUDO-RESPONSE REGULATOR 7 (CiPRR7) in the IM activates CiAGL24 expression in response to SD conditions. Moreover, our findings elucidate a negative feedback loop in which CiAGL24 and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (CiSOC1) modulate LEAFY (CiLFY) expression. Together, our results demonstrate that the CiPRR7-CiAGL24 module is vital for sustained SD signal perception in the IM to ensure successful anthesis in chrysanthemum.

The haplotype-resolved genome of diploid Chrysanthemum indicum unveils new acacetin synthases genes and their evolutionary history.

Acacetin, a flavonoid compound, possesses a wide range of pharmacological effects, including antimicrobial, immune regulation, and anticancer effects. Some key steps in its biosynthetic pathway were largely unknown in flowering plants. Here, we present the first haplotype-resolved genome of Chrysanthemum indicum, whose dried flowers contain abundant flavonoids and have been utilized as traditional Chinese medicine. Various phylogenetic analyses revealed almost equal proportion of three tree topologies among three Chrysanthemum species (C. indicum, C. nankingense, and C. lavandulifolium), indicating that frequent gene flow among Chrysanthemum species or incomplete lineage sorting due to rapid speciation might contribute to conflict topologies. The expanded gene families in C. indicum were associated with oxidative functions. Through comprehensive candidate gene screening, we identified five flavonoid O-methyltransferase (FOMT) candidates, which were highly expressed in flowers and whose expressional levels were significantly correlated with the content of acacetin. Further experiments validated two FOMTs (CI02A009970 and CI03A006662) were capable of catalyzing the conversion of apigenin into acacetin, and these two genes are possibly responsible acacetin accumulation in disc florets and young leaves, respectively. Furthermore, combined analyses of ancestral chromosome reconstruction and phylogenetic trees revealed the distinct evolutionary fates of the two validated FOMT genes. Our study provides new insights into the biosynthetic pathway of flavonoid compounds in the Asteraceae family and offers a model for tracing the origin and evolutionary routes of single genes. These findings will facilitate in vitro biosynthetic production of flavonoid compounds through cellular and metabolic engineering and expedite molecular breeding of C. indicum cultivars.

Transcription factor DgMYB recruits H3K4me3 methylase to DgPEROXIDASE to enhance chrysanthemum cold tolerance.

Cold affects the growth and development of plants. MYB transcription factors and histone H3K4me3 transferase ARABIDOPSIS TRITHORAXs (ATXs) play important regulatory functions in the process of plant resistance to low-temperature stress. In this study, DgMYB expression was responsive to low temperature, and overexpression of DgMYB led to increased tolerance, whereas the dgmyb mutant resulted in decreased tolerance of Chrysanthemum morifolium (Dendranthema grandiflorum var. Jinba) to cold stresses. Interestingly, we found that only peroxidase (POD) activity differed substantially between wild type (WT), overexpression lines, and the mutant line. A DgATX H3K4me3 methylase that interacts with DgMYB was isolated by further experiments. DgATX expression was also responsive to low temperature. Overexpression of DgATX led to increased tolerance, whereas the dgatx mutant resulted in decreased tolerance of chrysanthemum to cold stresses. Moreover, the dgmyb, dgatx, and dgmyb dgatx double mutants all led to reduced H3K4me3 levels at DgPOD, thus reducing DgPOD expression. Together, our results show that DgMYB interacts with DgATX, allowing DgATX to specifically target DgPOD, altering H3K4me3 levels, increasing DgPOD expression, and thereby reducing the accumulation of reactive oxygen species (ROS) in chrysanthemum.

Transcription factor CmHSFA4-CmMYBS3 complex enhances salt tolerance in chrysanthemum by repressing CmMYB121 expression.

Excessive soil salinity not only hampers plant growth and development but can also lead to plant death. Previously, we found that heat-shock factor A4 (CmHSFA4) enhances the tolerance of chrysanthemum (Chrysanthemum morifolium) to salt. However, the underlying molecular mechanism remains unclear. In this study, we identified a candidate MYB transcription factor, CmMYB121, which responded to salt stress. We observed that the CmMYB121 transcription is suppressed by CmHSFA4. Moreover, overexpression of CmMYB121 exacerbated chrysanthemum sensitivity to salt stress. CmHSFA4 directly bound to the promoter of CmMYB121 at the heat-shock element. Protein-protein interaction assays identified an interaction between CmHSFA4 and CmMYBS3, a transcriptional repressor, and recruited the corepressor TOPLESS (CmTPL) to inhibit CmMYB121 transcription by impairing the H3 and H4 histone acetylation levels of CmMYB121. Our study demonstrated that a CmHSFA4-CmMYBS3-CmTPL complex modulates CmMYB121 expression, consequently regulating the tolerance of chrysanthemum to salt. The findings shed light on the responses of plants to salt stress.

Integrated metabolomic and transcriptomic analysis reveals biosynthesis mechanism of flavone and caffeoylquinic acid in chrysanthemum.

BACKGROUND: Chrysanthemum morifolium 'HangBaiJu', a popular medicinal and edible plant, exerts its biological activities primarily through the presence of flavones and caffeoylquinic acids (CQAs). However, the regulatory mechanism of flavone and CQA biosynthesis in the chrysanthemum capitulum remains unclear. RESULTS: In this study, the content of flavones and CQAs during the development of chrysanthemum capitulum was determined by HPLC, revealing an accumulation pattern with higher levels at S1 and S2 and a gradual decrease at S3 to S5. Transcriptomic analysis revealed that CmPAL1/2, CmCHS1/2, CmFNS, CmHQT, and CmHCT were key structural genes in flavones and CQAs biosynthesis. Furthermore, weighted gene co-expression correlation network analysis (WGCNA), k-means clustering, correlation analysis and protein interaction prediction were carried out in this study to identify transcription factors (TFs) associated with flavone and CQA biosynthesis, including MYB, bHLH, AP2/ERF, and MADS-box families. The TFs CmERF/PTI6 and CmCMD77 were proposed to act as upstream regulators of CmMYB3 and CmbHLH143, while CmMYB3 and CmbHLH143 might form a complex to directly regulate the structural genes CmPAL1/2, CmCHS1/2, CmFNS, CmHQT, and CmHCT, thereby controlling flavone and CQA biosynthesis. CONCLUSIONS: Overall, these findings provide initial insights into the TF regulatory network underlying flavones and CQAs accumulation in the chrysanthemum capitulum, which laid a theoretical foundation for the quality improvement of C. morifolium 'HangBaiJu' and the high-quality development of the industry.

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.

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.

CmMYC2-CmMYBML1 module orchestrates the resistance to herbivory by synchronously regulating the trichome development and constitutive terpene biosynthesis in Chrysanthemum.

Trichomes are specialized epidermal outgrowths covering the aerial parts of most terrestrial plants. There is a large species variability in occurrence of different types of trichomes such that the molecular regulatory mechanism underlying the formation and the biological function of trichomes in most plant species remain unexplored. Here, we used Chrysanthemum morifolium as a model plant to explore the regulatory network in trichome formation and terpenoid synthesis and unravel the physical and chemical roles of trichomes in constitutive defense against herbivore feeding. By analyzing the trichome-related genes from transcriptome database of the trichomes-removed leaves and intact leaves, we identified CmMYC2 to positively regulate both development of T-shaped and glandular trichomes as well as the content of terpenoids stored in glandular trichomes. Furthermore, we found that the role of CmMYC2 in trichome formation and terpene synthesis was mediated by interaction with CmMYBML1. Our results reveal a sophisticated molecular mechanism wherein the CmMYC2-CmMYBML1 feedback inhibition loop regulates the formation of trichomes (non-glandular and glandular) and terpene biosynthesis, collectively contributing to the enhanced resistance to Spodoptera litura larvae feeding. Our findings provide new insights into the novel regulatory network by which the plant synchronously regulates trichome density for the physical and chemical defense against herbivory.

Lysine malonylation of DgnsLIPID TRANSFER PROTEIN1 at the K81 site improves cold resistance in chrysanthemum.

Lysine malonylation (Kmal) is a recently discovered posttranslational modification, and its role in the response to abiotic stress has not been reported in plants. In this study, we isolated a nonspecific lipid transfer protein, DgnsLTP1, from chrysanthemum (Dendranthema grandiflorum var. Jinba). Overexpression and CRISPR-Cas9-mediated gene editing of DgnsLTP1 demonstrated that the protein endows chrysanthemum with cold tolerance. Yeast 2-hybrid, bimolecular fluorescence complementation, luciferase complementation imaging, and coimmunoprecipitation experimental results showed that DgnsLTP1 interacts with a plasma membrane intrinsic protein (PIP) DgPIP. Overexpressing DgPIP boosted the expression of DgGPX (glutathione peroxidase), increased the activity of GPX, and decreased the accumulation of reactive oxygen species (ROS), thereby enhancing the low-temperature stress tolerance of chrysanthemum, while the CRISPR-Cas9-mediated mutant dgpip inhibited this process. Transgenic analyses in chrysanthemum showed that DgnsLTP1 improves the cold resistance of chrysanthemum in a DgPIP-dependent manner. Moreover, Kmal of DgnsLTP1 at the K81 site prevented the degradation of DgPIP in Nicotiana benthamiana and chrysanthemum, further promoted DgGPX expression, enhanced GPX activity, and scavenged excess ROS produced by cold stress, thereby further enhancing the cold resistance of chrysanthemum.

PHOTOLYASE/BLUE LIGHT RECEPTOR2 regulates chrysanthemum flowering by compensating for gibberellin perception.

The gibberellins (GAs) receptor GA INSENSITIVE DWARF1 (GID1) plays a central role in GA signal perception and transduction. The typical photoperiodic plant chrysanthemum (Chrysanthemum morifolium) only flowers when grown in short-day photoperiods. In addition, chrysanthemum flowering is also controlled by the aging pathway, but whether and how GAs participate in photoperiod- and age-dependent regulation of flowering remain unknown. Here, we demonstrate that photoperiod affects CmGID1B expression in response to GAs and developmental age. Moreover, we identified PHOTOLYASE/BLUE LIGHT RECEPTOR2, an atypical photocleavage synthase, as a CRYPTOCHROME-INTERACTING bHLH1 interactor with which it forms a complex in response to short days to activate CmGID1B transcription. Knocking down CmGID1B raised endogenous bioactive GA contents and GA signal perception, in turn modulating the expression of the aging-related genes MicroRNA156 and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3. We propose that exposure to short days accelerates the juvenile-to-adult transition by increasing endogenous GA contents and response to GAs, leading to entry into floral transformation.

Flowering repressor CmSVP recruits the TOPLESS corepressor to control flowering in chrysanthemum.

Plant flowering time is induced by environmental and endogenous signals perceived by the plant. The MCM1-AGAMOUSDEFICIENS-Serum Response Factor-box (MADS-box) protein SHORT VEGETATIVE PHASE (SVP) is a pivotal repressor that negatively regulates the floral transition during the vegetative phase; however, the transcriptional regulatory mechanism remains poorly understood. Here, we report that CmSVP, a chrysanthemum (Chrysanthemum morifolium Ramat.) homolog of SVP, can repress the expression of a key flowering gene, a chrysanthemum FLOWERING LOCUS T-like gene (CmFTL3), by binding its promoter CArG element to delay flowering in the ambient temperature pathway in chrysanthemum. Protein-protein interaction assays identified an interaction between CmSVP and CmTPL1-2, a chrysanthemum homologue of TOPLESS (TPL) that plays critical roles as transcriptional corepressor in many aspects of plant life. Genetic analyses revealed the CmSVP-CmTPL1-2 transcriptional complex is a prerequisite for CmSVP to act as a floral repressor. Furthermore, overexpression of CmSVP rescued the phenotype of the svp-31 mutant in Arabidopsis (Arabidopsis thaliana), overexpression of AtSVP or CmSVP in the Arabidopsis dominant-negative mutation tpl-1 led to ineffective late flowering, and AtSVP interacted with AtTPL, confirming the conserved function of SVP in chrysanthemum and Arabidopsis. We have validated a conserved machinery wherein SVP partially relies on TPL to inhibit flowering via a thermosensory pathway.

Two B-box proteins orchestrate vegetative and reproductive growth in summer chrysanthemum.

Floral transition, the switch from vegetative to reproductive growth, is extremely important for the growth and development of flowering plants. In the summer chrysanthemum, CmBBX8, a member of the subgroup II B-box (BBX) family, positively regulates the transition by physically interacting with CmERF3 to inhibit CmFTL1 expression. In this study, we show that CmBBX5, a B-box subgroup I member comprising two B-boxes and a CCT domain, interacts with CmBBX8. This interaction suppresses the recruitment of CmBBX8 to the CmFTL1 locus without affecting its transcriptional activation activity. CmBBX5 overexpression led to delayed flowering under both LD (long-day) and SD (short-day) conditions, while lines expressing the chimeric repressor gene-silencing (CmBBX5-SRDX) exhibited the opposite phenotype. Subsequent genetic evidence indicated that in regulating flowering, CmBBX5 is partially dependent on CmBBX8. Moreover, during the vegetative growth period, levels of CmBBX5 expression were found to exceed those of CmBBX8. Collectively, our findings indicate that both CmERF3 and CmBBX5 interact with CmBBX8 to dampen the regulation of CmFTL1 via distinct mechanisms, which contribute to preventing the premature flowering of summer chrysanthemum.

A group VIIIa ethylene-responsive factor, CmERF4, negatively regulates waterlogging tolerance in chrysanthemum.

Ethylene-responsive factors (ERF) play an important role in plant responses to waterlogging stress. However, the function and mechanism of action of ERFVIII in response to waterlogging stress remain poorly understood. In this study, we found that expression of the ERF VIIIa gene CmERF4 in chrysanthemum was induced by waterlogging stress. CmERF4 localized to the nucleus when expressed in tobacco leaves. Yeast two-hybrid and luciferase assays showed that CmERF4 is a transcriptional inhibitor. CmERF4 overexpression in chrysanthemum reduced plant waterlogging tolerance, whereas overexpression of the chimeric activator CmERF4-VP64 reversed its transcriptional activity, promoting higher waterlogging tolerance than that observed in wild-type plants, indicating that CmERF4 negatively regulates waterlogging tolerance. Transcriptome profiling showed that energy metabolism and reactive oxygen species (ROS) pathway-associated genes were differentially expressed between CmERF4-VP64 and wild-type plants. RT-qPCR analysis of selected energy metabolism and reactive oxygen species-related genes showed that the gene expression patterns were consistent with the expression levels obtained from RNA-seq analysis. Overall, we identified new functions of CmERF4 in negatively regulating chrysanthemum waterlogging tolerance by modulating energy metabolism and ROS pathway genes.

A pattern for the early, middle, and late phase of tea chrysanthemum response to Fusarium oxysporum.

Chrysanthemum morifolium is cultivated worldwide and has high ornamental, tea, and medicinal value. With the increasing area of chrysanthemum cultivation and years of continuous cropping, Fusarium wilt disease frequently occurs in various production areas, seriously affecting the quality and yield and causing huge economic losses. However, the molecular response mechanism of Fusarium wilt infection remains unclear, which limits the molecular breeding process for disease resistance in chrysanthemums. In the present study, we analyzed the molecular response mechanisms of 'Huangju,' one of the tea chrysanthemum cultivars severely infested with Fusarium wilt in the field at the early, middle, and late phases of F. oxysporum infestation. 'Huangju' responded to the infestation mainly through galactose metabolism, plant-pathogen interaction, auxin, abscisic acid, and ethylene signalling in the early phase; galactose metabolism, plant-pathogen interaction, auxin, salicylic acid signal, and certain transcription factors (e.g., CmWRKY48) in the middle phase; and galactose metabolism in the late phase. Notably, the galactose metabolism was important in the early, middle, and late phases of 'Huangju' response to F. oxysporum. Meanwhile, the phytohormone auxin was involved in the early and middle responses. Furthermore, silencing of CmWRKY48 in 'Huangju' resulted in resistance to F. oxysporum. Our results revealed a new molecular pattern for chrysanthemum in response to Fusarium wilt in the early, middle, and late phases, providing a foundation for the molecular breeding of chrysanthemum for disease resistance.

Transcriptomic and metabolomic analyses reveal CmMYB308 as a key regulator in the pink flower color variation of 'Dante Purple' chrysanthemum.

KEY MESSAGE: CmMYB308 was identified as a key regulator in chrysanthemum flower color variation from purple to pink by conducting transcriptome and metabolome analysis. CmMYB308 can inhibit anthocyanin biosynthesis by suppressing the expression of CmPAL, CmC4H, and Cm4CL. Flower color variation is a widespread natural occurrence that plays a significant role in floral breeding. We discovered a variation in the flower of the chrysanthemum cultivar 'Dante Purple' (abbreviated as 'DP'), where the flower color shifted from purple to pink. We successfully propagated these pink flowers through tissue culture and designated them as DPM. By conducting transcriptome and metabolome analysis, we identified a reduction in the expression of critical genes involved in anthocyanin biosynthesis-CmPAL, CmC4H, and Cm4CL-in the DPM. This downregulation led to an accumulation of phenylalanine and cinnamic acid within the general phenylpropanoid pathway (GPP), which prevented their conversion into cyanidin and cyanidin 3-glucoside. As a result, the flowers turned pink. Additional transformation and biochemical experiments confirmed that the upregulation of CmMYB308 gene expression in the DPM directly suppressed CmPAL-1 and CmC4H genes, which indirectly affected Cm4CL-3 expression and ultimately inhibited anthocyanin biosynthesis in the DPM. This study offers a preliminary insight into the molecular mechanism underlying chrysanthemum flower color mutation, paving the way for genetic improvements in chrysanthemum flower color breeding.

DgHDA6 enhances the cold tolerance in chrysanthemum by improving ROS scavenging capacity.

Histone deacetylases have been demonstrated to play an important role in responding to low-temperature stress, but the related response mechanism in chrysanthemum remains unclear. In this study, we isolated a cold-induced gene, DgHDA6, from chrysanthemum (Chrysanthemum morifolium Ramat). DgHDA6 contains 474 amino acids and shares a typical deacetylation domain with RPD3/HDA1 family members. The overexpression of DgHDA6 enhanced cold resistance in chrysanthemums. After low-temperature stress, the overexpression lines showed a higher survival rate. The contents of proline, soluble proteins and sugars, and the activities of antioxidant enzymes were significantly increased while the contents of H2O2, O2- and MDA were lower. Moreover, cold-stress-responding genes such as DgCuZnSOD, DgCAT, DgP5CS, and DgFAD were upregulated after cold stress. These results suggest that the overexpression of DgHDA6 can improve cold tolerance in chrysanthemum by enhancing ROS scavenging capacity.

CmNAC25 targets CmMYB6 to positively regulate anthocyanin biosynthesis during the post-flowering stage in chrysanthemum.

BACKGROUND: Anthocyanin is a class of important secondary metabolites that determines colorful petals in chrysanthemum, a famous cut flower. 'Arctic Queen' is a white chrysanthemum cultivar that does not accumulate anthocyanin during the flowering stage. During the post-flowering stage, the petals of 'Arctic Queen' accumulate anthocyanin and turn red. However, the molecular mechanism underlying this flower color change remains unclear. RESULTS: In this study, by using transcriptome analysis, we identified CmNAC25 as a candidate gene promoting anthocyanin accumulation in the post-flowering stage of 'Arctic Queen'. CmNAC25 is directly bound to the promoter of CmMYB6, a core member of the MBW protein complex that promotes anthocyanin biosynthesis in chrysanthemum, to activate its expression. CmNAC25 also directly activates the promoter of CmDFR, which encodes the key enzyme in anthocyanin biosynthesis. CmNAC25 was highly expressed during the post-flowering stage, while the expression level of CmMYB#7, a known R3 MYB transcription factor interfering with the formation of the CmMYB6-CmbHLH2 complex, significantly decreased. Genetic transformation of both chrysanthemum and Nicotiana tabacum verified that CmNAC25 was a positive regulator of anthocyanin biosynthesis. Another two cultivars that turned red during the post-flowering stages also demonstrated a similar mechanism. CONCLUSIONS: Altogether, our data revealed that CmNAC25 positively regulates anthocyanin biosynthesis in chrysanthemum petals during the post-flowering stages by directly activating CmMYB6 and CmDFR. Our results thus revealed a crucial role of CmNAC25 in regulating flower color change during petal senescence and provided a target gene for molecular design breeding of flower color in chrysanthemum.

Time-Course Analysis and Transcriptomic Identification of a Group III ERF CmTINY2 Involved in Waterlogging Tolerance in Chrysanthemums × morifolium Ramat.

'Hangju' is a variety of Chrysanthemum × morifolium Ramat. with both edible and medicinal value, cultivated as a traditional Chinese medicine for four centuries. The cultivation of 'Hangju' is currently at risk due to waterlogging, yet there is a lack of comprehensive understanding regarding its response to waterlogging stress. This study compared the waterlogging-tolerant 'Hangju' variety Enhanced Waterlogging Tolerance (EWT) with the waterlogging-sensitive variety CK ('zaoxiaoyangju'). EWT exhibited a more developed aeration tissue structure and demonstrated rapid growth regarding the adventitious roots following waterlogging. The time-course transcriptome analysis indicated that EWT could swiftly adjust the expression of the genes involved in the energy metabolism signaling pathways to acclimate to the waterlogged environment. Through WGCNA analysis, we identified Integrase-Type DNA-Binding Protein (CmTINY2) as a key factor in regulating the waterlogging tolerance in EWT. CmTINY2, a transcription factor belonging to the ethylene-responsive factor (ERF) subfamily III, operated within the nucleus and activated downstream gene expression. Its role in enhancing the waterlogging tolerance might be linked to the control of the stomatal aperture via the Ethylene-Responsive Element (ERE) gene. In summary, our research elucidated that the waterlogging tolerance displayed by EWT is a result of a combination of the morphological structure and molecular regulatory mechanisms. Furthermore, the study of the functions of CmTINY2 from ERF subfamily III also broadened our knowledge of the role of the ERF genes in the waterlogging signaling pathways.

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.

Integrated Metabolomics and Transcriptomics Reveal the Key Role of Flavonoids in the Cold Tolerance of Chrysanthemum.

Chrysanthemum (Chrysanthemum morifolium, ground-cover Chrysanthemums), one of the important garden flowers, has a high ornamental and economic value. However, its ornamental value is significantly diminished by the low temperature experienced in northeastern China. Here, metabolomics and transcriptomics were performed on three Chrysanthemum cultivars before and after a low temperature to investigate the dynamic metabolite changes and the molecular regulatory mechanisms. The results showed that 1324 annotated metabolites were detected, among which 327 were identified as flavonoids derived from Chrysanthemum. The accumulation of metabolites and gene expression related to the flavonoid biosynthesis pathway significantly increased in the three cultivars under the low temperature, indicating flavonoid metabolism actively participates in the Chrysanthemum cold response. Specifically, the content of cyanidin and pelargonidin derivatives and the expression of anthocyanin biosynthesis genes significantly increases in XHBF, providing a reasonable explanation for the change in petal color from white to purple under the low temperature. Six candidate UDP-glycosyltransferase genes involved in the glycosylation of flavonoids were identified through correlation networks and phylogenetic analysis. CmNAC1, CmbZIP3, and other transcription factors potentially regulating flavonoid metabolism and responding to low temperatures were discovered by correlation analysis and weighted gene co-expression network analysis (WGCNA). In conclusion, this study elucidated the specific response of flavonoids to low temperatures in Chrysanthemums, providing valuable insights and metabolic data for investigating cold tolerance.