Genome-wide identification and characterization analysis of RWP-RK family genes reveal their role in flowering time of Chrysanthemum lavandulifolium.

BACKGROUND: RWP-RKs are plant specific transcription factors, which are widely distributed in plants in the form of polygenic families and play key role in nitrogen absorption and utilization, and are crucial to plant growth and development. However, the genome-wide identification and function of RWP-RK in Compositae plants are widely unknown. RESULTS: In this study, 101 RWP-RKs in Chrysanthemum lavandulifolium were identified and tandem repeat was an important way for the expansion of RWP-RKs in Compositae species. 101 RWP-RKs contain 38 NIN-like proteins (NLPs) and 31 RWP- RK domain proteins (RKDs), as well as 32 specific expansion members. qRT-PCR results showed that 7 ClNLPs in leaves were up-regulated at the floral transition stage, 10 ClNLPs were negatively regulated by low nitrate conditions, and 3 of them were up-regulated by optimal nitrate conditions. In addition, the flowering time of Chrysanthemum lavandulifolium was advanced under optimal nitrate conditions, the expression level of Cryptochromes (ClCRYs), phytochrome C (ClPHYC) and the floral integration genes GIGANTEA (ClGI), CONSTANS-LIKE (ClCOL1, ClCOL4, ClCOL5), FLOWERING LOCUS T (ClFT), FLOWERING LOCUS C (ClFLC), SUPPRESSOR OF OVER-EXPRESSION OF CONSTANS 1 (ClSOC1) also were up-regulated. The expression level of ClCRY1a, ClCRY1c, ClCRY2a and ClCRY2c in the vegetative growth stage induced by optimal nitrate reached the expression level induced by short-day in the reproductive growth stage, which supplemented the induction effect of short-day on the transcription level of floral-related genes in advance. CONCLUSIONS: It was speculated that ClNLPs may act on the photoperiodic pathway under optimal nitrate environment, and ultimately regulate the flowering time by up-regulating the transcription level of ClCRYs. These results provide new perspective for exploring the mechanism of nitrate/nitrogen affecting flowering in higher plants.

Transcriptomic analysis reveals the formation mechanism of anemone-type flower in chrysanthemum.

BACKGROUND: The ray and disc florets on the chrysanthemum capitulum are morphologically diverse and have remarkably abundant variant types, resulting in a rich variety of flower types. An anemone shape with pigmented and elongated disk florets is an important trait in flower shape breeding of chrysanthemums. The regulatory mechanism of their anemone-type disc floret formation was not clear, thus limiting the directional breeding of chrysanthemum flower types. In this study, we used morphological observation, transcriptomic analysis, and gene expression to investigate the morphogenetic processes and regulatory mechanisms of anemone-type chrysanthemum. RESULT: Scanning electron microscopy (SEM) observation showed that morphological differences between non-anemone-type disc florets and anemone-type disc florets occurred mainly during the petal elongation period. The anemone-type disc florets elongated rapidly in the later stages of development. Longitudinal paraffin section analysis revealed that the anemone-type disc florets were formed by a great number of cells in the middle layer of the petals with vigorous division. We investigated the differentially expressed genes (DEGs) using ray and disc florets of two chrysanthemum cultivars, 082 and 068, for RNA-Seq and their expression patterns of non-anemone-type and anemone-type disc florets. The result suggested that the CYCLOIDEA2 (CYC2s), MADS-box genes, and phytohormone signal-related genes appeared significantly different in both types of disc florets and might have important effects on the formation of anemone-type disc florets. In addition, it is noteworthy that the auxin and jasmonate signaling pathways might play a vital role in developing anemone-type disc florets. CONCLUSIONS: Based on our findings, we propose a regulatory network for forming non-anemone-type and anemone-type disc florets. The results of this study lead the way to further clarify the mechanism of the anemone-type chrysanthemum formation and lay the foundation for the directive breeding of chrysanthemum petal types.

Comprehensive transcriptomic analysis provides new insights into the mechanism of ray floret morphogenesis in chrysanthemum.

BACKGROUND: The ray floret shapes referred to as petal types on the chrysanthemum (Chrysanthemum × morifolium Ramat.) capitulum is extremely abundant, which is one of the most important ornamental traits of chrysanthemum. However, the regulatory mechanisms of different ray floret shapes are still unknown. C. vestitum is a major origin species of cultivated chrysanthemum and has flat, spoon, and tubular type of ray florets which are the three basic petal types of chrysanthemum. Therefore, it is an ideal model material for studying ray floret morphogenesis in chrysanthemum. Here, using morphological, gene expression and transcriptomic analyses of different ray floret types of C. vestitum, we explored the developmental processes and underlying regulatory networks of ray florets. RESULTS: The formation of the flat type was due to stagnation of its dorsal petal primordium, while the petal primordium of the tubular type had an intact ring shape. Morphological differences between the two ray floret types occurred during the initial stage with vigorous cell division. Analysis of genes related to flower development showed that CYCLOIDEA genes, including CYC2b, CYC2d, CYC2e, and CYC2f, were differentially expressed in different ray floret types, while the transcriptional levels of others, such as MADS-box genes, were not significantly different. Hormone-related genes, including SMALL AUXIN UPREGULATED RNA (SAUR), GRETCHEN HAGEN3 (GH3), GIBBERELLIN 2-BETA-DIOXYGENASE 1 (GA2OX1) and APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF), were identified from 1532 differentially expressed genes (DEGs) in pairwise comparisons among the flat, spoon, and tubular types, with significantly higher expression in the tubular type than that in the flat type and potential involvement in the morphogenesis of different ray floret types. CONCLUSIONS: Our findings, together with the gene interactional relationships reported for Arabidopsis thaliana, suggest that hormone-related genes are highly expressed in the tubular type, promoting petal cell division and leading to the formation of a complete ring of the petal primordium. These results provide novel insights into the morphological variation of ray floret of chrysanthemum.

Comparative transcriptomics identifies patterns of selection in roses.

BACKGROUND: Roses are important plants for human beings with pivotal economical and biological traits like continuous flowering, flower architecture, color and scent. Due to frequent hybridization and high genome heterozygosity, classification of roses and their relatives remains a big challenge. RESULTS: Here, to identify potential markers for phylogenetic reconstruction and to reveal the patterns of natural selection in roses, we generated sets of high quality and comprehensive reference transcriptomes for Rosa chinensis 'Old Blush' (OB) and R. wichuriana 'Basye's Thornless' (BT), two species exhibiting contrasted traits of high economical importance. The assembled reference transcriptomes showed transcripts N50 above 2000 bp. Two roses shared about 10,073 transcripts (N50 = 2282 bp), in which a set of 5959 transcripts was conserved within genera of Rosa. Further comparison with species in Rosaceae identified 4447 transcripts being common (Rosaceae-common) in Rosa, Malus, Prunus, Rubus, and Fragaria, while a pool of 164 transcripts being specific for roses (Rosa-specific). Among the Rosaceae-common transcripts, 409 transcripts showed a signature of positive selection and a clustered expression in different tissues. Interestingly, nine of these rapidly evolving genes were related to DNA damage repair and responses to environmental stimulus, a potential associated with genome confliction post hybridization. Coincident with this fast evolution pattern in rose genes, 24 F-box and four TMV resistant proteins were significantly enriched in the Rosa-specific genes. CONCLUSIONS: We expect that these Rosaceae-common and Rosa-specific transcripts should facilitate the phylogenetic analysis of Rosaceae plants as well as investigations of Rosa-specific biology. The data reported here could provide fundamental genomic tools and knowledge critical for understanding the biology and domestication of roses and for roses breeding.

Transcriptomic analyses reveal species-specific light-induced anthocyanin biosynthesis in chrysanthemum.

BACKGROUND: The flower colour of agricultural products is very important for their commercial value, which is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis. However, the deep molecular mechanism remains elusive, and many problems regarding the phenotypic change and the corresponding gene regulation are still unclear. In the present study, Chrysanthemum × morifolium 'Purple Reagan', a light-responding pigmentation cultivar, was selected to investigate the mechanism of light-induced anthocyanin biosynthesis using transcriptomic analyses. RESULTS: Only cyanidin derivatives were identified based on the analyses of the pigmentation in ray florets. Shading experiments revealed that the capitulum was the key organ and that its bud stage was the key phase responding to light. These results were used to design five libraries for transcriptomic analyses, including three capitulum developmental stages and two light conditions. RNA sequences were de novo assembled into 103,517 unigenes, of which 60,712 were annotated against four public protein databases. As many as 2,135 unigenes were differentially expressed between the light and dark libraries with 923 up-regulated and 1,212 down-regulated unigenes in response to shading. Next, interactive pathway analysis showed that the anthocyanin biosynthetic pathway was the only complete metabolic pathway both modulated in response to light and related to capitulum development. Following the shading treatment, nearly all structural genes involved in the anthocyanin biosynthetic pathway were down-regulated. Moreover, three CmMYB genes and one CmbHLH gene were identified as key transcription factors that might participate in the regulation of anthocyanin biosynthesis under light conditions based on clustering analysis and validation by RT-qPCR. Finally, a light-induced anthocyanin biosynthesis pathway in chrysanthemums was inferred. CONCLUSION: The pigmentation of the ray florets of chrysanthemum cultivar 'Purple Reagan' is dependent on light. During the light-induced pigmentation process, the expression of seven structural genes in the anthocyanin biosynthetic pathway (regulated by at least four transcription factors in response to light) are the main contributors to the pigmentation of chrysanthemums. This information will further our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in ornamental plants.

Identification and characterization of the CONSTANS-like gene family in the short-day plant Chrysanthemum lavandulifolium.

The CONSTANS (CO) and CONSTANS-like (COL) genes play key roles in the photoperiodic flowering pathways, and studying their functions can elucidate the molecular mechanisms underlying flowering control in photoperiod-regulated plants. We identified eleven COL genes (ClCOL1-ClCOL11) in Chrysanthemum lavandulifolium and divided them into three groups that are conserved among the flowering plants based on phylogenetic analysis. Most of the ClCOL genes are primarily expressed in the leaf and shoot apices, except for ClCOL6-ClCOL9, which belong to Group II. The expression levels of ClCOL4-ClCOL5 and ClCOL7-ClCOL8 are up-regulated under inductive short-day (SD) conditions, whereas ClCOL6 is down-regulated under inductive SD conditions. The ClCOL genes exhibit four different diurnal rhythm expressions (Type I-Type IV). The Type I genes (ClCOL4-ClCOL5) are highly transcribed under light. The Type II genes (ClCOL1-ClCOL2, ClCOL10) display increased expression in darkness and are rapidly suppressed under light. Transcripts of ClCOL6-ClCOL9 and ClCOL11, belonging to Type III, are abundant in the late light period or at the beginning of the dark period. ClCOL3 belongs to Type IV, with high expression in the early light period and dark period. The peak expression levels of ClCOL4-ClCOL6 are decreased and postponed in the non-inductive night break (NB) and under long-day (LD) conditions, indicating that those genes may play an essential role in the flowering regulation of C. lavandulifolium. The overexpression of ClCOL5 promotes the flowering of Arabidopsis grown under LD conditions, suggesting that ClCOL5 may function as a flowering enhancer in C. lavandulifolium. This study will be useful not only for the study of the C. lavandulifolium photoperiod-dependent flowering process but also for the genetic manipulation of flowering time-related genes to change the flowering time in the chrysanthemum.

cla-miR164-NO APICAL MERISTEM (ClNAM) regulates the inflorescence architecture development of Chrysanthemum lavandulifolium.

Chrysanthemum × morifolium has great ornamental and economic value on account of its exquisite capitulum. However, previous studies have mainly focused on the corolla morphology of the capitulum. Such an approach cannot explain the variable inflorescence architecture of the chrysanthemum. Previous research from our group has shown that NO APICAL MERISTEM (ClNAM) is likely to function as a hub gene in capitulum architecture in the early development stage. In the present study, ClNAM was used to investigate the function of these boundary genes in the capitulum architecture of Chrysanthemum lavandulifolium, a closely related species of C. × morifolium in the genus. Modification of ClNAM in C. lavandulifolium resulted in an advanced initiation of the floral primordium at the capitulum. As a result, the receptacle morphology was altered and the number of florets decreased. The ray floret corolla was shortened, but the disc floret was elongated. The number of capitula increased significantly, arranged in more densely compounded corymbose synflorescences. The yeast and luciferase reporter system revealed that ClAP1, ClRCD2, and ClLBD18 target and activate ClNAM. Subsequently, ClNAM targets and activates ClCUC2a/c, which regulates the initiation of floral and inflorescence in C. lavandulifolium. ClNAM was also targeted and cleaved by cla-miR164 in this process. In conclusion, this study established a boundary gene regulatory network with cla-miR164-ClNAM as the hub. This network not only influences the architecture of capitulum, but also affects compound corymbose synflorescences of the C. lavandulifolium. These results provide new insights into the mechanisms regulating inflorescence architecture in chrysanthemum.

Genome-Wide Analysis of MYB Gene Family in Chrysanthemum ×morifolium Provides Insights into Flower Color Regulation.

MYBs constitute the second largest transcription factor (TF) superfamily in flowering plants with substantial structural and functional diversity, which have been brought into focus because they affect flower colors by regulating anthocyanin biosynthesis. Up to now, the genomic data of several Chrysanthemum species have been released, which provides us with abundant genomic resources for revealing the evolution of the MYB gene family in Chrysanthemum species. In the present study, comparative analyses of the MYB gene family in six representative species, including C. lavandulifolium, C. seticuspe, C. ×morifolium, Helianthus annuus, Lactuca sativa, and Arabidopsis thaliana, were performed. A total of 1104 MYBs, which were classified into four subfamilies and 35 lineages, were identified in the three Chrysanthemum species (C. lavandulifolium, C. seticuspe, and C. ×morifolium). We found that whole-genome duplication and tandem duplication are the main duplication mechanisms that drove the occurrence of duplicates in CmMYBs (particularly in the R2R3-MYB subfamily) during the evolution of the cultivated chrysanthemums. Sequence structure and selective pressure analyses of the MYB gene family revealed that some of R2R3-MYBs were subjected to positive selection, which are mostly located on the distal telomere segments of the chromosomes and contain motifs 7 and 8. In addition, the gene expression analysis of CmMYBs in different organs and at various capitulum developmental stages of C. ×morifolium indicated that CmMYBS2, CmMYB96, and CmMYB109 might be the negative regulators for anthocyanin biosynthesis. Our results provide the phylogenetic context for research on the genetic and functional evolution of the MYB gene family in Chrysanthemum species and deepen our understanding of the regulatory mechanism of MYB TFs on the flower color of C. ×morifolium.

Isolation and functional analysis of a homolog of flavonoid 3',5'-hydroxylase gene from Pericallis × hybrida.

As the key enzyme in the biosynthesis of blue flower color pigments, flavonoid 3',5'-hydroxylase (F3'5'H) can catalyze the conversion of its major substrates, 2-S naringenin and dihydrokaempferol, into 3',4',5'-hydroxylated pentahydroxyflavanone and dihydromyricetin, respectively. Unlike other F3'5'Hs belonging to the CYP75A subfamily, Asteraceae-specific F3'5'Hs belong to the CYP75B subfamily. Furthermore, cineraria F3'5'H expressed in yeast exhibited not only F3'H (flavonoid 3'-hydroxylase) activity but also F3'5'H activity in vitro. In this study, Southern blotting showed that there was only one copy of a homolog of the F3'5'H gene PCFH in the Pericallis × hybrida genome. This gene could be detected by Northern blot in the primary developmental stages of ligulate florets of the purple- and blue-flowered cultivars, and its transcripts also accumulated in the leaves. Heterologous expression of PCFH could produce new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and lead to the blue- and red-shifting of flower color in T0 generation plants. These results indicate that cineraria F3'5'H exhibited both F3'5'H- and F3'H-activity in vivo. The types and contents of anthocyanins and flower color phenotypes of the T1 generation were similar to those of T0 generation plants. PCFH exhibited stable inheritance and normal functions between generations. This study supplies new evidence to understand Asteraceae-specific F3'5'Hs and provides important references for the further study of molecular breeding of blue-flowered chrysanthemums using the PCFH gene.

Genome-wide identification of the MIKCc-type MADS-box gene family in Chrysanthemum lavandulifolium reveals their roles in the capitulum development.

Chrysanthemum ×morifolium is well known throughout the world for its diverse and exquisite flower types. However, due to the complicated genetic background of C. ×morifolium, it is difficult to understand the molecular mechanism of its flower development. And it limits the molecular breeding of improving chrysanthemum flower types. C. ×morifolium has the typical radial capitulum, and many researches showed that the members of the MIKCc-type MADS box gene family play a key role in the formation and development of the capitulum. However, it has been difficult to isolate the important MIKCc and investigate their roles in this process due to the lack of genomic information in chrysanthemum. Here, we identified MIKCc-type MADS box genes at whole genome-wide level in C. lavandulifolium, a diploid species closely related to C. ×morifolium, and investigated their roles in capitulum development by gene expression pattern analysis and protein interaction analysis. A total of 40 ClMIKCc were identified and were phylogenetically grouped into 12 clades. Members of all clades showed different enriched expression patterns during capitulum formation. We speculate that the E-class genes in C. lavandulifolium underwent subfunctionalization because they have a significantly expanded, more diverse expression patterns, and specifically tissue expression than AtSEPs. Meanwhile, we detected the C-class expressed in disc floret corolla, which could be the clue to explore the morphological differences between disc and ray floret corolla. In addition, the potential roles of some MIKCcs in complex inflorescence formation were explored by comparing the number and phylogenetic relationship of MIKCc subfamily members in Asteraceae with different capitulum types. Members of the FLC branch in Asteraceae were found to be possibly related to the differentiation and development of the ray floret.

Genome-wide identification of the TCP gene family in Chrysanthemum lavandulifolium and its homologs expression patterns during flower development in different Chrysanthemum species.

TCP proteins, part of the transcription factors specific to plants, are recognized for their involvement in various aspects of plant growth and development. Nevertheless, a thorough investigation of TCPs in Chrysanthemum lavandulifolium, a prominent ancestral species of cultivated chrysanthemum and an excellent model material for investigating ray floret (RF) and disc floret (DF) development in Chrysanthemum, remains unexplored yet. Herein, a comprehensive study was performed to analyze the genome-wide distribution of TCPs in C. lavandulifolium. In total, 39 TCPs in C. lavandulifolium were identified, showing uneven distribution on 8 chromosomes. Phylogenetic and gene structural analyses revealed that ClTCPs were grouped into classes I and II. The class II genes were subdivided into two subclades, the CIN and CYC/TB1 subclades, with members of each clade having similar conserved motifs and gene structures. Four CIN subclade genes (ClTCP24, ClTCP25, ClTCP26, and ClTCP27) contained the potential miR319 target sites. Promoter analysis revealed that ClTCPs had numerous cis-regulatory elements associated with phytohormone responses, stress responses, and plant growth/development. The expression patterns of ClTCPs during capitulum development and in two different florets were determined using RNA-seq and qRT-PCR. The expression levels of TCPs varied in six development stages of capitula; 25 out of the 36 TCPs genes were specifically expressed in flowers. Additionally, we identified six key ClCYC2 genes, which belong to the class II TCP subclade, with markedly upregulated expression in RFs compared with DFs, and these genes exhibited similar expression patterns in the two florets of Chrysanthemum species. It is speculated that they may be responsible for RFs and DFs development. Subcellular localization and transactivation activity analyses of six candidate genes demonstrated that all of them were localized in the nucleus, while three exhibited self-activation activities. This research provided a better understanding of TCPs in C. lavandulifolium and laid a foundation for unraveling the mechanism by which important TCPs involved in the capitulum development.

Floral Development Stage-Specific Transcriptomic Analysis Reveals the Formation Mechanism of Different Shapes of Ray Florets in Chrysanthemum.

The formation mechanism of different ray floret shapes of chrysanthemum (Chrysanthemum × morifolium) remains elusive due to its complex genetic background. C. vestitum, with the basic ray floret shapes of the flat, spoon, and tubular types, is considered a model material for studying ray floret morphogenesis. In this study, the flat and tubular type lines of C. vestitum at specific stages were used to investigate the key genes that regulate morphological differences in ray florets. We found that the expression levels of genes related to auxin synthesis, transport, and response were generally higher in the tubular type than in the flat type. CvARF3 was highly expressed in the flat type, while CvARF5 and CvARF6 were highly expressed in the tubular type. Additionally, the transcription levels of Class B and E genes closely related to petal development, including CvPI, CvAP3, Cvdefh21, CvSEP3, and CvCDM77, were expressed at higher levels in the tubular type than the flat type. Based on the results, it is proposed that auxin plays a key role in the development of ray florets, and auxin-related genes, especially CvARFs, may be key genes to control the morphological difference of ray florets. Simultaneously, MADS-box genes are involved in the co-regulation of ray floret morphogenesis. The results provide novel insights into the molecular mechanism of different petal type formation and lay a theoretical foundation for the directional breeding of petal type in chrysanthemums.

The mechanisms of optimal nitrogen conditions to accelerate flowering of Chrysanthemum vestitum under short day based on transcriptome analysis.

Nitrogen (N) plays an important role in the development of plants, with N application having been shown to accelerate flowering of cultivated plants. However, the mechanism of optimal N conditions to accelerate flowering of short-day plants is still unclear. In this study, it was found that Chrysanthemum vestitum is a typical short-day plant like most chrysanthemum varieties, and its flowering must go through a short-day induction stage. Further observations on the growth of C. vestitum showed that the N range of external application for growth was limited to between 0.25 and 2.50 mM. The results showed that, under optimal N (ON, 1.25 mM) conditions, the plants increased rapidly and flowering time was advanced; under high N (HN, 2.50 mM) or limited N (LN, 0.25 mM) conditions, the growth of plants were inhibited and flowering time was delayed. On the basis of transcriptome data, analysis of differentially expressed genes (DEGs) revealed that the floral-related genes B-box19 (BBX19), Cryptochromes (CRYs), CONSTANS-like (COLs), nitrate transporter protein (NRT), and NIN-like protein (NLP) could respond to N availability. Most of the genes in the photoperiod pathway were upregulated by ON conditions, and their expression was inhibited under HN and LN conditions. Our findings indicated that N could affect flowering by regulating the transcription levels of genes that are involved mainly in the photoperiod pathway. These candidate genes provide important clues for the subsequent analysis of the mechanism of N-induced flowering of short-day plants, and provide a possibility to improve the flowering of chrysanthemum by molecular breeding.

Multi-Information Model for Large-Flowered Chrysanthemum Cultivar Recognition and Classification.

The traditional Chinese large-flowered chrysanthemum is one of the cultivar groups of chrysanthemum (Chrysanthemum × morifolium Ramat.) with great morphological variation based on many cultivars. Some experts have established several large-flowered chrysanthemum classification systems by using the method of comparative morphology. However, for many cultivars, accurate recognition and classification are still a problem. Combined with the comparative morphological traits of selected samples, we proposed a multi-information model based on deep learning to recognize and classify large-flowered chrysanthemum. In this study, we collected the images of 213 large-flowered chrysanthemum cultivars in two consecutive years, 2018 and 2019. Based on the 2018 dataset, we constructed a multi-information classification model using non-pre-trained ResNet18 as the backbone network. The model achieves 70.62% top-5 test accuracy for the 2019 dataset. We explored the ability of image features to represent the characteristics of large-flowered chrysanthemum. The affinity propagation (AP) clustering shows that the features are sufficient to discriminate flower colors. The principal component analysis (PCA) shows the petal type has a better interpretation than the flower type. The training sample processing, model training scheme, and learning rate adjustment method affected the convergence and generalization of the model. The non-pre-trained model overcomes the problem of focusing on texture by ignoring colors with the ImageNet pre-trained model. These results lay a foundation for the automated recognition and classification of large-flowered chrysanthemum cultivars based on image classification.

Functional analysis of the ScAG and ScAGL11 MADS-box transcription factors for anthocyanin biosynthesis and bicolour pattern formation in Senecio cruentus ray florets.

Cineraria (Senecio cruentus) is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, ScCHS2, ScF3H1, ScDFR3, ScANS, and ScbHLH17, were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, ScAG and ScAGL11, showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that ScAG inhibits anthocyanin accumulation in tobacco (Nicotiana tabacum). However, virus-induced gene silencing (VIGS) experiments showed that silencing of ScAG and ScAGL11 increases anthocyanin content in cineraria leaves. Similar results were observed when ScAG and ScAGL11 were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the ScAG/ScAGL11-silenced plants. The expression of ScCHS2, ScDFR3, and ScF3H1 increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, ScAG directly inhibited the transcription of ScF3H1 while ScAGL11 inhibited ScDFR3 expression by binding to their promoters separately. The findings reported herein indicate that ScAG and ScAGL11 negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.

The transcription factor complex CmAP3-CmPI-CmUIF1 modulates carotenoid metabolism by directly regulating carotenogenic gene CmCCD4a-2 in chrysanthemum.

Carotenoids are one of the most important pigments for the coloring in many plants, fruits and flowers. Recently, significant progress has been made in carotenoid metabolism. However, the specific understanding on transcriptional regulation controlling the expression of carotenoid metabolic genes remains extremely limited. Anemone-type chrysanthemum, as a special group of chrysanthemum cultivars, contain elongated disc florets in capitulum, which usually appear in different colors compared with the ray florets since accumulating distinct content of carotenoids. In this study, the carotenoid composition and content of the ray and disc florets of an anemone-type chrysanthemum cultivar 'Dong Li Fen Gui' were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and the key structural gene CmCCD4a-2, of which differential expression resulted in the distinct content of carotenoids accumulated in these two types of florets, was identified. Then the promoter sequence of CmCCD4a-2 was used as bait to screen a chrysanthemum flower cDNA library and two transcription factors, CmAP3 and CmUIF1 were identified. Y2H, BiFC and Y3H experiments demonstrated that these two TFs were connected by CmPI to form CmAP3-CmPI-CmUIF1 TF complex. This TF complex regulated carotenoid metabolism through activating the expression of CmCCD4a-2 directly. Furthermore, a large number of target genes regulated directly by the CmAP3-CmPI-CmUIF1 TF complex, including carotenoid biosynthetic genes, flavonoid biosynthetic genes and flower development-related genes, were identified by DNA-affinity purification sequencing (DAP-seq), which indicated that the CmAP3-CmPI-CmUIF1 TF complex might participate in multiple processes. These findings expand our knowledge for the transcriptional regulation of carotenoid metabolism in plants and will be helpful to manipulating carotenoid accumulation in chrysanthemum.

The chrysanthemum lavandulifolium genome and the molecular mechanism underlying diverse capitulum types.

Cultivated chrysanthemum (Chrysanthemum × morifolium Ramat.) is a beloved ornamental crop due to the diverse capitula types among varieties, but the molecular mechanism of capitulum development remains unclear. Here, we report a 2.60 Gb chromosome-scale reference genome of C. lavandulifolium, a wild Chrysanthemum species found in China, Korea and Japan. The evolutionary analysis of the genome revealed that only recent tandem duplications occurred in the C. lavandulifolium genome after the shared whole genome triplication (WGT) in Asteraceae. Based on the transcriptomic profiling of six important developmental stages of the radiate capitulum in C. lavandulifolium, we found genes in the MADS-box, TCP, NAC and LOB gene families that were involved in disc and ray floret primordia differentiation. Notably, NAM and LOB30 homologs were specifically expressed in the radiate capitulum, suggesting their pivotal roles in the genetic network of disc and ray floret primordia differentiation in chrysanthemum. The present study not only provides a high-quality reference genome of chrysanthemum but also provides insight into the molecular mechanism underlying the diverse capitulum types in chrysanthemum.

ScGST3 and multiple R2R3-MYB transcription factors function in anthocyanin accumulation in Senecio cruentus.

Anthocyanins are important flavonoid pigments involved in the colouring of flowers and fruits. They are synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported into the vacuole for storage. Previous reports have suggested that glutathione S-transferase (GST) is involved in anthocyanin transport. However, due to the limitation of plant materials, most GSTs only participate in the cyanidin or delphinidin transport pathway. Here, an anthocyanin-related GST, ScGST3, was identified from the transcriptome of cineraria. The expression pattern of ScGST3 was highly consistent with anthocyanin accumulation in ray florets. Molecular complementation of Arabidopsis tt19 indicated that the overexpression of ScGST3 restores the anthocyanin-deficient phenotype of the mutant. Virus-induced gene silencing (VIGS) of ScGST3 in carmine and blue cineraria leaves could inhibit anthocyanin accumulation, further confirming the function of ScGST3 in anthocyanin accumulation. In vitro assays showed that ScGST3 increases the water solubility of cyanidin-3-O-glucoside (C3G) and delphinidin-3-O-glucosid (D3G). In addition, we also identified two anthocyanin-related MYB transcription factors, ScMYB3 and ScMYB6. The expression pattern of these two genes was also highly consistent with anthocyanin accumulation. Faded abaxial leaf phenotypes were observed after the silencing of ScMYB3 and ScMYB6, and the expression levels of partial structural genes were repressed. Based on the results from dual-luciferase assays and yeast one-hybrid assays, ScMYB3 can activate the promoter of ScGST3. Collectively, the transcription of ScGST3 is regulated by ScMYB3, which plays an important role in the transport of C3G and D3G in cineraria.

Metric learning for image-based flower cultivars identification.

BACKGROUND: The study of plant phenotype by deep learning has received increased interest in recent years, which impressive progress has been made in the fields of plant breeding. Deep learning extremely relies on a large amount of training data to extract and recognize target features in the field of plant phenotype classification and recognition tasks. However, for some flower cultivars identification tasks with a huge number of cultivars, it is difficult for traditional deep learning methods to achieve better recognition results with limited sample data. Thus, a method based on metric learning for flower cultivars identification is proposed to solve this problem. RESULTS: We added center loss to the classification network to make inter-class samples disperse and intra-class samples compact, the script of ResNet18, ResNet50, and DenseNet121 were used for feature extraction. To evaluate the effectiveness of the proposed method, a public dataset Oxford 102 Flowers dataset and two novel datasets constructed by us are chosen. For the method of joint supervision of center loss and L2-softmax loss, the test accuracy rate is 91.88%, 97.34%, and 99.82% across three datasets, respectively. Feature distribution observed by T-distributed stochastic neighbor embedding (T-SNE) verifies the effectiveness of the method presented above. CONCLUSIONS: An efficient metric learning method has been described for flower cultivars identification task, which not only provides high recognition rates but also makes the feature extracted from the recognition network interpretable. This study demonstrated that the proposed method provides new ideas for the application of a small amount of data in the field of identification, and has important reference significance for the flower cultivars identification research.

Isolation and Functional Analysis of Genes Involved in Polyacylated Anthocyanin Biosynthesis in Blue Senecio cruentus.

Polyacylated anthocyanins with multiple glycosyl and aromatic acyl groups tend to make flowers display bright and stable blue colours. However, there are few studies on the isolation and functional characterization of genes involved in the polyacylated anthocyanin biosynthesis mechanism, which limits the molecular breeding of truly blue flowers. Senecio cruentus is an important potted ornamental plant, and its blue flowers contain 3',7-polyacylated delphinidin-type anthocyanins that are not reported in any other plants, suggesting that it harbours abundant gene resources for the molecular breeding of blue flowers. In this study, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of blue, carmine and white colours of cineraria cultivars "Venezia" (named VeB, VeC, and VeW, respectively), we found that 3',7-polyacylated anthocyanin, cinerarin, was the main pigment component that determined the blue colour of ray florets of cineraria. Based on the transcriptome sequencing and differential gene expression (DEG) analysis combined with RT- and qRT-PCR, we found two genes encoding uridine diphosphate glycosyltransferase, named ScUGT1 and ScUGT4; two genes encoding acyl-glucoside-dependent glucosyltransferases which belong to glycoside hydrolase family 1 (GH1), named ScAGGT11 and ScAGGT12; one gene encoding serine carboxypeptidase-like acyltransferase ScSCPL2; and two MYB transcriptional factor genes ScMYB2 and ScMYB4, that were specifically highly expressed in the ray florets of VeB, which indicated that these genes may be involved in cinerarin biosynthesis. The function of ScSCPL2 was analysed by virus-induced gene silencing (VIGS) in cineraria leaves combined with HPLC-MS/MS. ScSCPL2 mainly participated in the 3' and 7-position acylation of cinerarin. These results will provide new insight into the molecular basis of the polyacylated anthocyanin biosynthesis mechanism in higher plants and are of great significance for blue flower molecular breeding of ornamental plants.