CmNAC73 Mediates the Formation of Green Color in Chrysanthemum Flowers by Directly Activating the Expression of Chlorophyll Biosynthesis Genes HEMA1 and CRD1.
ABSTRACT: Chrysanthemum is one of the most beautiful and popular flowers in the world, and the flower color is an important ornamental trait of chrysanthemum. Compared with other flower colors, green flowers are relatively rare. The formation of green flower color is attributed to the accumulation of chlorophyll; however, the regulatory mechanism of chlorophyll metabolism in chrysanthemum with green flowers remains largely unknown. In this study, we performed Illumina RNA sequencing on three chrysanthemum materials, Chrysanthemum vestitum and Chrysanthemum morifolium cultivars 'Chunxiao' and 'Green anna', which produce white, light green and dark green flowers, respectively. Based on the results of comparative transcriptome analysis, a gene encoding a novel NAC family transcription factor, CmNAC73, was found to be highly correlated to chlorophyll accumulation in the outer whorl of ray florets in chrysanthemum. The results of transient overexpression in chrysanthemum leaves showed that CmNAC73 acts as a positive regulator of chlorophyll biosynthesis. Furthermore, transactivation and yeast one-hybrid assays indicated that CmNAC73 directly binds to the promoters of chlorophyll synthesis-related genes HEMA1 and CRD1. Thus, this study uncovers the transcriptional regulation of chlorophyll synthesis-related genes HEMA1 and CRD1 by CmNAC73 and provides new insights into the development of green flower color in chrysanthemum and chlorophyll metabolism in plants.
Project description:Green chrysanthemums are difficult to breed but have high commercial value. The molecular basis for the green petal color in chrysanthemum is not fully understood. This was investigated in the present study by RNA sequencing analysis of white and green ray florets collected at three stages of flower development from the F<sub>1</sub> progeny of the cross between <i>Chrysanthemum × morifolium</i> "Lüdingdang" with green-petaled flowers and <i>Chrysanthemum vistitum</i> with white-petaled flowers. The chlorophyll content was higher and chloroplast degradation was slower in green pools than in white pools at each developmental stage. Transcriptome analysis revealed that genes that were differentially expressed between the two pools were enriched in pathways related to chlorophyll metabolism and photosynthesis. We identified the transcription factor genes <i>CmCOLa</i>, <i>CmCOLb</i>, <i>CmERF</i>, and <i>CmbHLH</i> as regulators of the green flower color in chrysanthemum by differential expression analysis and weighted gene co-expression network analysis. These findings can guide future efforts to improve the color palette of chrysanthemum flowers through genetic engineering.
Project description:<i>Nicotiana alata</i> is an ornamental horticultural plant with a variety of flower colors and a long flowering period. The genes in four different colored <i>N. alata</i> (white, purple, red, and lemon green) were analyzed to explain the differences in flower color using transcriptomes. A total of 32 differential expression genes in the chlorophyll biosynthesis pathway and 41 in the anthocyanin biosynthesis pathway were identified. The enrichment analysis showed that the chlorophyll biosynthesis pathway and anthocyanin biosynthesis pathway play critical roles in the color differences of <i>N. alata</i>. The <i>HEMA</i> of the chlorophyll biosynthesis pathway was up-regulated in lemon green flowers. Compared with white flowers, in the red and purple flowers, <i>F3H</i>, <i>F3'5'H</i> and <i>DFR</i> were significantly up-regulated, while <i>FLS</i> was significantly down-regulated. Seventeen differential expression genes homologous to transcription factor coding genes were obtained, and the homologues of <i>HY5</i>, <i>MYB12, AN1</i> and <i>AN4</i> were also involved in flower color differences. The discovery of these candidate genes related to flower color differences is significant for further research on the flower colors formation mechanism and color improvements of <i>N. alata</i>.
Project description:Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)-glucose:anthocyanin 3',5'-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3'5'H. Newly synthesized 3',5'-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed "copigmentation," with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.
Project description:Green petals pose a challenge for pollinators to distinguish flowers from leaves, but they are valuable as a specialty flower trait. However, little is understood about the molecular mechanisms that underlie the development of green petals. Here, we report that CINCINNATA (CIN)-like TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) proteins play key roles in the control of petal color. The septuple tcp2/3/4/5/10/13/17 mutant produced flowers with green petals due to chlorophyll accumulation. Expression of TCP4 complemented the petal phenotype of tcp2/3/4/5/10/13/17. We found that chloroplasts were converted into leucoplasts in the distal parts of wild-type petals but not in the proximal parts during flower development, whereas plastid conversion was compromised in the distal parts of tcp2/3/4/5/10/13/17 petals. TCP4 and most CIN-like TCPs were predominantly expressed in distal petal regions, consistent with the green-white pattern in wild-type petals and the petal greening observed in the distal parts of tcp2/3/4/5/10/13/17 petals. RNA-sequencing data revealed that most chlorophyll biosynthesis genes were downregulated in the white distal parts of wild-type petals, but these genes had elevated expression in the distal green parts of tcp2/3/4/5/10/13/17 petals and the green proximal parts of wild-type petals. We revealed that TCP4 repressed chlorophyll biosynthesis by directly binding to the promoters of PROTOCHLOROPHYLLIDE REDUCTASE (PORB), DIVINYL REDUCTASE (DVR), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), which are known to promote petal greening. We found that the conversion of chloroplasts to leucoplasts and the green coloration in the proximal parts of petals appeared to be conserved among plant species. Our findings uncover a major molecular mechanism that underpins the formation of petal color patterns and provide a foundation for the breeding of plants with green flowers.
Project description:<h4>Background</h4>Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils.<h4>Results</h4>We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding.<h4>Conclusions</h4>The whole-transcriptome analysis of DEGs in mutant and normal C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.
Project description:Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium 'LPi' and C × morifolium 'LPu', that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3'H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3'H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3'H using RNAi and overexpressed the Senecio cruentus F3'5'H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3'H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3'5'H only exhibited F3'H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum.
Project description:Chinese narcissus (Narcissus tazetta var. chinensis) is one of the ten traditional flowers in China and a famous bulb flower in the world flower market. However, only white color tepals are formed in mature flowers of the cultivated varieties, which constrains their applicable occasions. Unfortunately, for lack of genome information of narcissus species, the explanation of tepal color formation of Chinese narcissus is still not clear. Concerning no genome information, the application of transcriptome profile to dissect biological phenomena in plants was reported to be effective. As known, pigments are metabolites of related metabolic pathways, which dominantly decide flower color. In this study, transcriptome profile and pigment metabolite analysis methods were used in the most widely cultivated Chinese narcissus "Jinzhanyintai" to discover the structure of pigment metabolic pathways and their contributions to white tepal color formation during flower development and pigmentation processes. By using comparative KEGG pathway enrichment analysis, three pathways related to flavonoid, carotenoid and chlorophyll pigment metabolism showed significant variations. The structure of flavonoids metabolic pathway was depicted, but, due to the lack of F3'5'H gene; the decreased expression of C4H, CHS and ANS genes; and the high expression of FLS gene, the effect of this pathway to synthesize functional anthocyanins in tepals was weak. Similarly, the expression of DXS, MCT and PSY genes in carotenoids synthesis sub-pathway was decreased, while CCD1/CCD4 genes in carotenoids degradation sub-pathway was increased; therefore, the effect of carotenoids metabolic pathway to synthesize adequate color pigments in tepals is restricted. Interestingly, genes in chlorophyll synthesis sub-pathway displayed uniform down-regulated expression, while genes in heme formation and chlorophyll breakdown sub-pathways displayed up-regulated expression, which also indicates negative regulation of chlorophyll formation. Further, content change trends of various color metabolites detected by HPLC in tepals are consistent with the additive gene expression patterns in each pathway. Therefore, all three pathways exhibit negative control of color pigments synthesis in tepals, finally resulting in the formation of white tepals. Interestingly, the content of chlorophyll was more than 10-fold higher than flavonoids and carotenoids metabolites, which indicates that chlorophyll metabolic pathway may play the major role in deciding tepal color formation of Chinese narcissus.
Project description: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.
Project description:Flower type is an important and extremely complicated trait of chrysanthemum. The corolla tube merged degree (CTMD) and the relative number of ray florets (RNRF) are the two key factors affecting chrysanthemum flower type. However, few reports have clarified the inheritance of these two complex traits, which limits directed breeding for flower-type improvement. In this study, 305 F<sub>1</sub> hybrids were obtained from two parents with obvious differences in CTMD and RNRF performance. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, we constructed a high-density genetic linkage map with an average map distance of 0.76 cM. Three major QTLs controlling CTMD and four major QTLs underlying RNRF were repeatedly detected in the 2 years. Moreover, the synteny between the genetic map and other Compositae species was investigated, and weak collinearity was observed. In QTL regions with a high degree of genomic collinearity, eight annotated genes were probed in the <i>Helianthus annuus</i> L. and <i>Lactuca sativa</i> L. var. ramosa Hort. genomes. Furthermore, 20 and 11 unigenes were identified via BLAST searches between the SNP markers of the QTL regions and the <i>C. vestitum</i> and <i>C. lavandulifolium</i> transcriptomes, respectively. These results lay a foundation for molecular marker-assisted breeding and candidate gene exploration in chrysanthemum without a reference assembly.
Project description:Flowering patterns are crucial to understand the dynamics of plant reproduction and resource availability for pollinators. Seasonal climate constrains flower and leaf phenology, where leaf and flower colors likely differ between seasons. Color is the main floral trait attracting pollinators; however, seasonal changes in the leaf-background coloration affect the perception of flower color contrasts by pollinators. For a seasonally dry woody cerrado community (Brazilian savanna) mainly pollinated by bees, we verified whether seasonality affects flower color diversity over time and if flower color contrasts of bee-pollinated species differ between seasons due to changes in the leaf-background coloration. For 140 species, we classified flower colors based on human-color vision, and for 99 species, we classified flower colors based on bee-color vision (spectral measurements). We described the community's flowering pattern according to the flower colors using a unique 11 years phenological database. For the 43 bee-pollinated species in which reflectance data were also available, we compared flower color diversity and contrasts against the background between seasons, considering the background coloration of each season. Flowering was markedly seasonal, peaking at the end of the dry season (September), when the highest diversity of flower colors was observed. Yellow flowers were observed all year round, whereas white flowers were seasonal, peaking during the dry season, and pink flowers predominated in the wet season, peaking in March. Bee-bluegreen flowers peaked between September and October. Flowers from the wet and dry seasons were similarly conspicuous against their corresponding background. Regardless of flowering season, the yellowish background of the dry season promoted higher flower color contrast for all flower species, whereas the greener background of the wet season promoted a higher green contrast. Temporal patterns of flower colors and color contrasts were related to the cerrado seasonality, but also to bee's activity, visual system, and behavior. Background coloration affected flower contrasts, favoring flower conspicuousness to bees according to the season. Thus, our results provide new insights regarding the temporal patterns of plant-pollinator interactions.