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.

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.

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.

Potential candidate genes and pathways related to cytoplasmic male sterility in Dianthus spiculifolius as revealed by transcriptome analysis.

KEY MESSAGE: We introduced the candidate gene DsHSP70 into Arabidopsis thaliana, resulting in male gametophyte sterility and abnormal degeneration of sepals and petals. Cytoplasmic male sterility (CMS) is a useful tool for hybrid production. However, the regulatory mechanism of CMS in Dianthus spiculifolius remains unclear. In this study, we investigated whether male-sterile line of D. spiculifolius has a malformed tapetum and fails to produce normal fertile pollen. RNA sequencing technology was used to compare the gene expression patterns of the D. spiculifolius male-sterile line and its male fertility maintainer line during anther development. A total of 12,365 differentially expressed genes (DEGs) were identified, among which 1765 were commonly expressed in the S1, S2 and S3 stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these DEGs were mainly involved in oxidation-reduction processes, signal transduction and programmed cell death. Additionally, weighted correlation network analysis (WGCNA) showed that three modules may be related to male sterility. A putative regulatory pathway for the male sterility traits was constructed based on the reproductive development network. After introducing the candidate DsHSP70 gene into Arabidopsis thaliana, we found that overexpressing plants showed anther abortion and shorter filaments, and accompanied by abnormal degeneration of sepals and petals. In summary, our results identified potential candidate genes and pathways related to CMS in D. spiculifolius, providing new insights for further research on the mechanism of male sterility.

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.

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.

Cadmium/lead tolerance of six Dianthus species and detoxification mechanism in Dianthus spiculifolius.

Toxic heavy metal contaminants seriously affect plant growth and human health. Reducing the accumulation of toxic metals by phytoremediation is an effective way to solve this environmental problem. Dianthus spiculifolius Schur is an ornamental plant with strong cold and drought tolerance. Because of its fast growth, well-developed root system, and large accumulation of biomass, D. spiculifolius has potential applications as a heavy metal hyperaccumulator. Therefore, the aim of this study was evaluate the ability of D. spiculifolius and other Dianthus species to remediate heavy metals, with an ultimate goal to identify available genetic resources for toxic metal removal. The cadmium (Cd) and lead (Pb) tolerance and accumulation of six Dianthus species were analyzed comparatively in physiological and biochemical experiments. Compared with the other Dianthus species, D. spiculifolius showed higher tolerance to, and greater accumulation of, Cd and Pb. Second-generation transcriptome analysis indicated that glutathione transferase activity was increased and the glutathione metabolism pathway was enriched with genes encoding antioxidant enzymes (DsGST, DsGST3, DsGSTU10, DsGGCT2-1, and DsIDH-2) that were up-regulated under Cd/Pb treatment by RT-qPCR in D. spiculifolius. When expressed in yeast, DsGST, DsGST3, DsGSTU10 and DsIDH-2 enhanced Cd or Pb tolerance. These results indicate that D. spiculifolius has potential applications as a new ornamental hyperaccumulator plant, and that antioxidant enzymes might be involved in regulating Cd/Pb accumulation and detoxification. The findings of this study reveal some novel genetic resources that can be used to breed new plant varieties that tolerate and accumulate heavy metals.

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.

Genome-wide characterization of DcHsp90 gene family in carnation (Dianthus caryophyllus L.) and functional analysis of DcHsp90-6 in heat tolerance.

Plant heat shock protein 90 (Hsp90) participates in various physiological processes including protein folding, degradation, and signal transduction. However, the DcHsp90 gene family in carnation (Dianthus caryophyllus L.) has not been systematically analyzed. We thoroughly examined and comprehensively analyzed the carnation DcHsp90 gene family in this study and discovered 9 DcHsp90 genes. Based on the phylogenetic examination, DcHsp90 proteins may be divided into two groups. DcHsp90 structural features were similar but varied between groups. Promoter analysis revealed the presence of many cis-acting elements, most of which were connected to growth and development, hormones, and stress. DcHsp90 genes may play distinct functions in heat stress response, according to gene expression analyses. The DcHsp90-6 was isolated, and its role in the reaction to heat stress was studied. Thermotolerance and superoxide dismutase activity in transgenic seedlings were enhanced by Arabidopsis overexpression of DcHsp90-6. After heat stress, transgenic plants' electrolyte leakage and malondialdehyde levels were much lower than wild-type plants. Furthermore, overexpression of DcHsp90-6 altered the expressions of stress-responsive genes such as AtHsp101, AtHsp90, AtGolS1, AtRS4/5, and AtHsfB1. This study provides comprehensive information on the DcHsp90 gene family and suggests that overexpressed DcHsp90-6 positively regulates thermotolerance highlighting the adaptation mechanism of carnation under heat stress.

Comprehensive Comparative Analysis and Development of Molecular Markers for Dianthus Species Based on Complete Chloroplast Genome Sequences.

Dianthus spp. is a genus with high economic and ornamental value in the Caryophyllaceae, which include the famous fresh-cut carnation and the traditional Chinese herbal medicine, D. superbus. Despite the Dianthus species being seen everywhere in our daily lives, its genome information and phylogenetic relationships remain elusive. Thus, we performed the assembly and annotation of chloroplast genomes for 12 individuals from seven Dianthus species. On this basis, we carried out the first comprehensive and systematic analysis of the chloroplast genome sequence characteristics and the phylogenetic evolution of Dianthus. The chloroplast genome of 12 Dianthus individuals ranged from 149,192 bp to 149,800 bp, containing 124 to 126 functional genes. Sequence repetition analysis showed the number of simple sequence repeats (SSRs) ranged from 75 to 80, tandem repeats ranged from 23 to 41, and pair-dispersed repeats ranged from 28 to 43. Next, we calculated the synonymous nucleotide substitution rates (Ks) of all 76 protein coding genes to obtain the evolution rate of these coding genes in Dianthus species; rpl22 showed the highest Ks (0.0471), which suggested that it evolved the swiftest. By reconstructing the phylogenetic relationships within Dianthus and other species of Caryophyllales, 16 Dianthus individuals (12 individuals reported in this study and four individuals downloaded from NCBI) were divided into two strongly supported sister clades (Clade A and Clade B). The Clade A contained five species, namely D. caryophyllus, D. barbatus, D. gratianopolitanus, and two cultivars ('HY' and 'WC'). The Clade B included four species, in which D. superbus was a sister branch with D. chinensis, D. longicalyx, and F1 '87M' (the hybrid offspring F1 from D. chinensis and 'HY'). Further, based on sequence divergence analysis and hypervariable region analysis, we selected several regions that had more divergent sequences, to develop DNA markers. Additionally, we found that one DNA marker can be used to differentiate Clade A and Clade B in Dianthus. Taken together, our results provide useful information for our understanding of Dianthus classification and chloroplast genome evolution.

The transcription factors DcHB30 and DcWRKY75 antagonistically regulate ethylene-induced petal senescence in carnation (Dianthus caryophyllus).

Although numerous transcription factors with antagonistic activities have been shown to contribute to growth and development, whether and how they regulate senescence in plants is largely unknown. In this study, we investigated the role of antagonistic transcription factors in petal senescence in carnation (Dianthus caryophyllus), one of the most common types of ethylene-sensitive cut flowers produced worldwide. We identified DcHB30 that encodes a ZF-HD transcription factor that is down-regulated in ethylene-treated petal transcriptomes. We found that silencing DcHB30 accelerated ethylene-induced petal senescence and that DcHB30 physically interacts with DcWRKY75, a positive regulator of ethylene-induced petal senescence. Phenotypic characterization and molecular evidence indicated that DcHB30 and DcWRKY75 competitively regulate the expression of their co-targeted genes DcACS1, DcACO1, DcSAG12, and DcSAG29 by reciprocally inhibiting the DNA-binding activity of each other on the gene promoters. This transcriptional regulation mechanism demonstrates that these transcription factors serve as positive and negative regulators in ethylene-induced petal senescence in carnation. Thus, our study provides insights into how antagonizing transcription factors regulate plant senescence.

Morphological and physiological responses of Dianthus spiculifolius high wax mutant to low-temperature stress.

Cuticular wax plays a role in plant responses to environmental stresses. To understand the contribution of cuticular wax to plant responses to low-temperature stress, the morphological and physiological responses of a Dianthus spiculifolius high-wax (HW) mutant and wild type (WT) were compared. Under low-temperature stress (0 and -10 °C), HW plants showed a lower mortality rate and electrolyte leakage (El) than that WT plants. In plants treated with low-temperature stress (0 and -10 °C), HW mutant leaves exhibited higher soluble sugar and free proline contents and lower malondialdehyde contents than those WT leaves. The photosynthetic capacity, net photosynthetic rate, stomatal conductance, and maximal photochemical efficiency of photosystem II in HW mutant leaves were the least inhibited by low temperature than those in WT leaves. The dewaxing experiments showed no significant difference in the phenotype and El between the dewaxed-treated HW mutant and WT leaves under low-temperatures stress, indicating that cuticular wax causes differences in resistance to low-temperatures between HW and WT. Principal component analysis and the membership function value of the physiological data showed that the average membership value of the HW mutant was greater than that in WT. In general, the results indicated that high cuticular wax contributes positively to the response to low-temperature stress by D. spiculifolius.

Integrated multi-omic data and analyses reveal the pathways underlying key ornamental traits in carnation flowers.

Carnation (Dianthus caryophyllus) is one of the most popular ornamental flowers in the world. Although numerous studies on carnations exist, the underlying mechanisms of flower color, fragrance, and the formation of double flowers remain unknown. Here, we employed an integrated multi-omics approach to elucidate the genetic and biochemical pathways underlying the most important ornamental features of carnation flowers. First, we assembled a high-quality chromosome-scale genome (636 Mb with contig N50 as 14.67 Mb) of D. caryophyllus, the 'Scarlet Queen'. Next, a series of metabolomic datasets was generated with a variety of instrumentation types from different parts of the flower at multiple stages of development to assess spatial and temporal differences in the accumulation of pigment and volatile compounds. Finally, transcriptomic data were generated to link genomic, biochemical, and morphological patterns to propose a set of pathways by which ornamental traits such as petal coloration, double flowers, and fragrance production are formed. Among them, the transcription factors bHLHs, MYBs, and a WRKY44 homolog are proposed to be important in controlling petal color patterning and genes such as coniferyl alcohol acetyltransferase and eugenol synthase are involved in the synthesis of eugenol. The integrated dataset of genomics, transcriptomics, and metabolomics presented herein provides an important foundation for understanding the underlying pathways of flower development and coloration, which in turn can be used for selective breeding and gene editing for the development of novel carnation cultivars.

DcWRKY75 promotes ethylene induced petal senescence in carnation (Dianthus caryophyllus L.).

Carnation (Dianthus caryophyllus L.) is one of the most important and typical ethylene sensitive cut flowers worldwide, although how ethylene influences the petal senescence process in carnation remains largely unknown. Here, we screened out one of the key transcription factors, DcWRKY75, using a constructed ethylene induced petal senescence transcriptome in carnation and found that it shows quick induction by ethylene treatment. Silencing of DcWRKY75 delays ethylene induced petal senescence in carnation. Molecular evidence confirms that DcWRKY75 can bind to the promoter regions of two main ethylene biosynthetic genes (DcACS1 and DcACO1) and a couple of senescence associated genes (DcSAG12 and DcSAG29) to activate their expression. Furthermore, we show that DcWRKY75 is a direct target gene of DcEIL3-1, which is a homolog of the ethylene signaling core transcription factor EIN3 in Arabidopsis. DcEIL3-1 can physically interact with DcWRKY75 and silencing of DcEIL3-1 also delays ethylene induced petal senescence in carnation and inhibits the ethylene induced expression of DcWRKY75 and its target genes. The present study demonstrates that the transcriptional regulation network is vitally important for ethylene induced petal senescence process in carnation and potentially in other ethylene sensitive cut flowers.

From generalist to specialists: Variation in the host range and performance of anther-smut pathogens on Dianthus.

Determining the processes that drive the evolution of pathogen host range can inform our understanding of disease dynamics and the potential for host shifts. In natural populations, patterns of host range could be driven by genetically based differences in pathogen infectivity or ecological differences in host availability. In northwestern Italy, four reproductively isolated lineages of the fungal plant-pathogen Microbotryum have been shown to co-occur on several species in the genus Dianthus. We carried out cross-inoculation experiments to determine whether patterns of realized host range in these four lineages were driven by differences in infectivity and to test whether there was evidence of a trade-off between host range and within-host reproduction. We found strong concordance between field patterns of host range and pathogen infectivity on different Dianthus species using experimental inoculation, indicating that infection ability is a major driving force of host range. However, we found no evidence of a trade-off between the ability to infect a wider range of host species and spore production on a shared host.

Phenotypic diploidization in plant functional traits uncovered by synthetic neopolyploids in Dianthus broteri.

Whole-genome duplication and post-polyploidization genome downsizing play key roles in the evolution of land plants; however, the impact of genomic diploidization on functional traits still remains poorly understood. Using Dianthus broteri as a model, we compared the ecophysiological behaviour of colchicine-induced neotetraploids (4xNeo) to diploids (2x) and naturally occurring tetraploids (4xNat). Leaf gas-exchange and chlorophyll fluorescence analyses were performed in order to asses to what extent post-polyploidization evolutionary processes have affected 4xNat. Genomic diploidization and phenotypic novelty were evident. Distinct patterns of variation revealed that post-polyploidization processes altered the phenotypic shifts directly mediated by genome doubling. The photosynthetic phenotype was affected in several ways but the main effect was phenotypic diploidization (i.e. 2x and 4xNat were closer to each other than to 4xNeo). Overall, our results show the potential benefits of considering experimentally synthetized versus naturally established polyploids when exploring the role of polyploidization in promoting functional divergence.

Comprehensive analysis of sucrolytic enzyme gene families in carnation (Dianthus caryophyllus L.).

Sucrose plays crucial roles in growth and responses of plants to the environment, including those in ornamental species. During post-harvest handling of cut flowers, sucrose degradation is an essential process of inter- and intra-cellular carbon partitioning affecting flower opening and senescence and, subsequently, flower quality. However, complete information about the molecular basis of sucrose degradation in ornamental flowers, which can be catalyzed by two kinds of sucrolytic enzymes, invertase (INV), and sucrose synthase (SUS), is not available from past reports. The present study shows that sucrose treatment of carnation (Dianthus caryophyllus L.) florets increased starch content in petals, accompanied by decreased vacuolar INV (VIN) activity and increased SUS activity. However, hypoxic treatment of carnation florets decreased sucrose content and cell-wall INV (CWIN) activity in petals. In silico analysis using the carnation genome database identified six CWIN, three VIN, eight cytoplasmic INV (CIN), and five SUS genes. Real-time RT-PCR analysis confirmed that these genes are differentially expressed in carnation petals in response to sucrose and hypoxic treatments, partially corresponding to the changes in enzyme activities. In contrast to DcSUS1 (Dca4507.1), a SUS gene already reported in carnation, which showed preferential expression under aerated conditions, the expression of DcSUS2 (Dca22218.1), an undescribed carnation SUS gene, was enhanced under hypoxia similarly to an alcohol dehydrogenase gene DcADH1 (Dca18671.1). These results suggest that sugar metabolism in carnation petals is regulated in response to environmental cues, accompanied by modulated activities and gene expression of a set of sucrolytic enzymes.

Esterified carotenoids are synthesized in petals of carnation (Dianthus caryophyllus) and accumulate in differentiated chromoplasts.

Although yellow and orange petal colors are derived from carotenoids in many plant species, this has not yet been demonstrated for the order Caryophyllales, which includes carnations. Here, we identified a carnation cultivar with pale yellow flowers that accumulated carotenoids in petals. Additionally, some xanthophyll compounds were esterified, as is the case for yellow flowers in other plant species. Ultrastructural analysis showed that chromoplasts with numerous plastoglobules, in which flower-specific carotenoids accumulate, were present in the pale yellow petals. RNA-seq and RT-qPCR analyses indicated that the expression levels of genes for carotenoid biosynthesis and esterification in pale yellow and pink petals (that accumulate small amounts of carotenoids) were similar or lower than in green petals (that accumulate substantial amounts of carotenoids) and white petals (that accumulate extremely low levels of carotenoids). Pale yellow and pink petals had a considerably lower level of expression of genes for carotenoid degradation than white petals, suggesting that reduced degradation activity caused accumulation of carotenoids. Our results indicate that some carnation cultivars can synthesize and accumulate esterified carotenoids. By manipulating the rate of biosynthesis and esterification of carotenoids in these cultivars, it should be feasible to produce novel carnation cultivars with vivid yellow flowers.

Flower color mutation caused by spontaneous cell layer displacement in carnation (Dianthus caryophyllus).

A change of layer arrangement of shoot apical meristem (SAM) organized by three cell layers (L1, L2 and L3) is thought to be one of the provocations of bud sport, which often induces changes in phenotypic colors in periclinal chimeras. This paper describes a cell layer rearrangement which is the cause of spontaneous flower color mutation by using two carnation (Dianthus caryophyllus L.) cultivars that are presumably periclinal chimeras, 'Feminine Minami' (deep pink flower) and its recessive sport 'Tommy Minami' (pinkish red flower). The genotype of the acyl-glucose-dependent anthocyanin 5-glucosyltransferase (AA5GT) which is responsible for the color change of red to pink, in each cell layer was deduced by genomic analysis using tissues originated from specific cell layer and investigation of partial petal color mutations. In the results, the genotype of the L1 of 'Feminine Minami' was heterozygous for functional AA5GT and non-functional AA5GT carrying retrotransposon Ty1dic1 (AA5GT-Ty1dic1), and its inner cell layer hid red flower genotype, whereas AA5GT-Ty1dic1 of the L1 of 'Tommy Minami' became homogenic in absence of the insertion of a new Ty1dic1. Our outcomes concluded that the L1 of 'Tommy Minami' harboring the recessive AA5GT alleles are attributed to the inner cell layer of 'Feminine Minami' possessing red flower genotype.

Mutations in orthologous PETALOSA TOE-type genes cause a dominant double-flower phenotype in phylogenetically distant eudicots.

The double-flower phenotype has been selected by humans for its attractiveness in various plant species and it is of great commercial value for the ornamental market. In this study we investigated the genetic determinant of the dominant double-flower trait in carnation, petunia, and Rosa rugosa, and identified mutant alleles of TARGET OF EAT (TOE)-type genes characterized by a disruption of the miR172 target sequence and of the C-terminal portion of the encoded protein. Despite the phylogenetic distance between these eudicots, which diverged in the early Cretaceous, the orthologous genes carrying these mutations all belong to a single TOE-type subgroup, which we name as PETALOSA (PET). Homology searches allowed us to identify PET sequences in various other species. To confirm the results from naturally occurring mutations, we used CrispR-Cas9 to induce lesions within the miR172 target site of Nicotiana tabacum PET genes, and this resulted in the development of supernumerary petaloid structures. This study describes pet alleles in economically important ornamental species and provides evidence about the possibility of identifying and engineering PET genes to obtain the desirable double-flower trait in different plants.