DcWRKY33 promotes petal senescence in carnation (Dianthus caryophyllus L.) by activating genes involved in the biosynthesis of ethylene and abscisic acid and accumulation of reactive oxygen species.

Carnation (Dianthus caryophyllus L.) is one of the most famous and ethylene-sensitive cut flowers worldwide, but how ethylene interacts with other plant hormones and factors to regulate petal senescence in carnation is largely unknown. Here we found that a gene encoding WRKY family transcription factor, DcWRKY33, was significantly upregulated upon ethylene treatment. Silencing and overexpression of DcWRKY33 could delay and accelerate the senescence of carnation petals, respectively. Abscisic acid (ABA) and H2 O2 treatments could also accelerate the senescence of carnation petals by inducing the expression of DcWRKY33. Further, DcWRKY33 can bind directly to the promoters of ethylene biosynthesis genes (DcACS1 and DcACO1), ABA biosynthesis genes (DcNCED2 and DcNCED5), and the reactive oxygen species (ROS) generation gene DcRBOHB to activate their expression. Lastly, relationships are existed between ethylene, ABA and ROS. This study elucidated that DcWRKY33 promotes petal senescence by activating genes involved in the biosynthesis of ethylene and ABA and accumulation of ROS in carnation, supporting the development of new strategies to prolong the vase life of cut carnation.

The mutual regulation between DcEBF1/2 and DcEIL3-1 is involved in ethylene induced petal senescence in carnation (Dianthus caryophyllus L.).

Carnation (Dianthus caryophyllus L.) is a respiratory climacteric flower, comprising one of the most important cut flowers that is extremely sensitive to plant hormone ethylene. Ethylene signaling core transcription factor DcEIL3-1 plays a key role in ethylene induced petal senescence in carnation. However, how the dose of DcEIL3-1 is regulated in the carnation petal senescence process is still not clear. Here, we screened out two EBF (EIN3 Binding F-box) genes, DcEBF1 and DcEBF2, which showed quick elevation by ethylene treatment according to the ethylene induced carnation petal senescence transcriptome. Silencing of DcEBF1 and DcEBF2 accelerated, whereas overexpression of DcEBF1 and DcEBF2 delayed, ethylene induced petal senescence in carnation by influencing DcEIL3-1 downstream target genes but not DcEIL3-1 itself. Furthermore, DcEBF1 and DcEBF2 interact with DcEIL3-1 to degrade DcEIL3-1 via an ubiquitination pathway in vitro and in vivo. Finally, DcEIL3-1 binds to the promoter regions of DcEBF1 and DcEBF2 to activate their expression. In conclusion, the present study reveals the mutual regulation between DcEBF1/2 and DcEIL3-1 during ethylene induced petal senescence in carnation, which not only expands our understanding about ethylene signal regulation network in the carnation petal senescence process, but also provides potential targets with respect to breeding a cultivar of long-lived cut carnation.

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

MAIN CONCLUSION: A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

DNA methylation remodeled amino acids biosynthesis regulates flower senescence in carnation (Dianthus caryophyllus).

Dynamic DNA methylation regulatory networks are involved in many biological processes. However, how DNA methylation patterns change during flower senescence and their relevance with gene expression and related molecular mechanism remain largely unknown. Here, we used whole genome bisulfite sequencing to reveal a significant increase of DNA methylation in the promoter region of genes during natural and ethylene-induced flower senescence in carnation (Dianthus caryophyllus L.), which was correlated with decreased expression of DNA demethylase gene DcROS1. Silencing of DcROS1 accelerated while overexpression of DcROS1 delayed carnation flower senescence. Moreover, among the hypermethylated differentially expressed genes during flower senescence, we identified two amino acid biosynthesis genes, DcCARA and DcDHAD, with increased DNA methylation and reduced expression in DcROS1 silenced petals, and decreased DNA methylation and increased expression in DcROS1 overexpression petals, accompanied by decreased or increased amino acids content. Silencing of DcCARA and DcDHAD accelerates carnation flower senescence. We further showed that adding corresponding amino acids could largely rescue the senescence phenotype of DcROS1, DcCARA and DcDHAD silenced plants. Our study not only demonstrates an essential role of DcROS1-mediated remodeling of DNA methylation in flower senescence but also unravels a novel epigenetic regulatory mechanism underlying DNA methylation and amino acid biosynthesis during flower senescence.

Histone H3K4 methyltransferase DcATX1 promotes ethylene induced petal senescence in carnation.

Petal senescence is controlled by a complex regulatory network. Epigenetic regulation like histone modification influences chromatin state and gene expression. However, the involvement of histone methylation in regulating petal senescence remains poorly understood. Here, we found that the trimethylation of histone H3 at Lysine 4 (H3K4me3) is increased during ethylene-induced petal senescence in carnation (Dianthus caryophyllus L.). H3K4me3 levels were positively associated with the expression of transcription factor DcWRKY75, ethylene biosynthetic genes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (DcACS1), and ACC oxidase (DcACO1), and senescence associated genes (SAGs) DcSAG12 and DcSAG29. Further, we identified that carnation ARABIDOPSIS HOMOLOG OF TRITHORAX1 (DcATX1) encodes a histone lysine methyltransferase which can methylate H3K4. Knockdown of DcATX1 delayed ethylene-induced petal senescence in carnation, which was associated with the down-regulated expression of DcWRKY75, DcACO1, and DcSAG12, whereas overexpression of DcATX1 exhibited the opposite effects. DcATX1 promoted the transcription of DcWRKY75, DcACO1, and DcSAG12 by elevating the H3K4me3 levels within their promoters. Overall, our results demonstrate that DcATX1 is a H3K4 methyltransferase that promotes the expression of DcWRKY75, DcACO1, DcSAG12 and potentially other downstream target genes by regulating H3K4me3 levels, thereby accelerating ethylene-induced petal senescence in carnation. This study further indicates that epigenetic regulation is important for plant senescence processes.

Physiology, gene expression, and epiphenotype of two Dianthus broteri polyploid cytotypes under temperature stress.

Increasing evidence supports a major role for abiotic stress response in the success of plant polyploids, which usually thrive in harsh environments. However, understanding the ecophysiology of polyploids is challenging due to interactions between genome doubling and natural selection. Here, we investigated physiological responses, gene expression, and the epiphenotype of two related Dianthus broteri cytotypes-with different genome duplications (4× and 12×) and evolutionary trajectories-to short extreme temperature events (42/28 °C and 9/5 °C). The 12× cytotype showed higher expression of stress-responsive genes (SWEET1, PP2C16, AI5L3, and ATHB7) and enhanced gas exchange compared with 4×. Under heat stress, both ploidies had greatly impaired physiological performance and altered gene expression, with reduced cytosine methylation. However, the 12× cytotype exhibited remarkable physiological tolerance (maintaining gas exchange and water status via greater photochemical integrity and probably enhanced water storage) while down-regulating PP2C16 expression. Conversely, 4× D. broteri was susceptible to thermal stress despite prioritizing water conservation, showing signs of non-stomatal photosynthetic limitations and irreversible photochemical damage. This cytotype also presented gene-specific expression patterns under heat, up-regulating ATHB7. These findings provide insights into divergent stress response strategies and physiological resistance resulting from polyploidy, highlighting its widespread influence on plant function.

Impact of Preparative Isolation of C-Glycosylflavones Derived from Dianthus superbus on In Vitro Glucose Metabolism.

Dianthus superbus L. has been extensively studied for its potential medicinal properties in traditional Chinese medicine and is often consumed as a tea by traditional folk. It has the potential to be exploited in the treatment of inflammation, immunological disorders, and diabetic nephropathy. Based on previous studies, this study continued the separation of another subfraction of Dianthus superbus and established reversed-phase/reversed-phase and reversed-phase/hydrophilic (RPLC) two-dimensional (2D) high-performance liquid chromatography (HPLC) modes, quickly separating two C-glycosylflavones, among which 2″-O-rhamnosyllutonarin was a new compound and isomer with 6‴-O-rhamnosyllutonarin. This is the first study to investigate the effects of 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin on cellular glucose metabolism in vitro. First, molecular docking was used to examine the effects of 2″-O-rhamnosyllutonarin and 6″-O-rhamnosyllutonarin on AKT and AMPK; these two compounds exhibited relatively high activity. Following this, based on the HepG2 cell model of insulin resistance, it was proved that both of the 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin demonstrated substantial efficacy in ameliorating insulin resistance and were found to be non-toxic. Simultaneously, it is expected that the methods developed in this study will provide a basis for future studies concerning the separation and pharmacological effects of C-glycosyl flavonoids.

Melatonin strongly enhances the Agrobacterium- mediated transformation of carnation in nitrogen-depleted media.

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for ß-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and ß-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

An insertion of transposon in DcNAP inverted its function in the ethylene pathway to delay petal senescence in carnation (Dianthus caryophyllus L.).

Petal senescence is the final stage of flower development. Transcriptional regulation plays key roles in this process. However, whether and how post-transcriptional regulation involved is still largely unknown. Here, we identified an ethylene-induced NAC family transcription factor DcNAP in carnation (Dianthus caryophyllus L.). One allele, DcNAP-dTdic1, has an insertion of a dTdic1 transposon in its second exon. The dTdic1 transposon disrupts the structure of DcNAP and causes alternative splicing, which transcribes multiple domain-deleted variants (DcNAP2 and others). Conversely, the wild type allele DcNAP transcribes DcNAP1 encoding an intact NAC domain. Silencing DcNAP1 delays and overexpressing DcNAP1 accelerates petal senescence in carnation, while silencing and overexpressing DcNAP2 have the opposite effects, respectively. Further, DcNAP2 could interact with DcNAP1 and interfere the binding and activation activity of DcNAP1 to the promoters of its downstream target ethylene biosynthesis genes DcACS1 and DcACO1. Lastly, ethylene signalling core transcriptional factor DcEIL3-1 can activate the expression of DcNAP1 and DcNAP2 in the same way by binding their promoters. In summary, we discovered a novel mechanism by which DcNAP regulates carnation petal senescence at the post-transcriptional level. It may also provide a useful strategy to manipulate the NAC domains of NAC transcription factors for crop genetic improvement.

Matrix-independent boron isotope analysis of silicate and carbonate reference materials by ultraviolet femtosecond laser ablation multi-collector inductively coupled plasma mass spectrometry with application to the cold-water coral Desmophyllum dianthus.

RATIONALE: Boron isotopes are a powerful tool for pH reconstruction in marine carbonates and as a tracer for fluid-mineral interaction in geochemistry. Microanalytical approaches based on laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) often suffer from effects induced by the sample matrix. In this study, we investigate matrix-independent analyses of B isotopic ratios and apply this technique to cold-water corals. METHODS: We employ a customized 193 nm femtosecond laser ablation system (Solstice, Spectra-Physics) coupled to a MC-ICP-MS system (Nu Plasma II, Nu Instruments) equipped with electron multipliers for in situ measurements of B isotopic ratios (11 B/10 B) at the micrometric scale. We analyzed various reference materials of silicate and carbonate matrices using non-matrix matched calibration without employing any correction. This approach was then applied to investigate defined increments in coral samples from a Chilean fjord. RESULTS: We obtained accurate B isotopic ratios with a reproducibility of ±0.9‰ (2 SD) for various reference materials including silicate glasses (GOR132-G, StHs6/80-G, ATHO-G and NIST SRM 612), clay (IAEA-B-8) and carbonate (JCp-1) using the silicate glass NIST SRM 610 as calibration standard, which shows that neither laser-induced nor ICP-related matrix effects are detectable. The application to cold-water corals (Desmophyllum dianthus) reveals minor intra-skeleton variations in δ11 B with average values between 23.01‰ and 25.86‰. CONCLUSIONS: Our instrumental set-up provides accurate and precise B isotopic ratios independently of the sample matrix at the micrometric scale. This approach opens a wide field of application in geochemistry, including pH reconstruction in biogenic carbonates and deciphering processes related to fluid-mineral interaction.