Carotenoid biosynthesis profiling unveils the variance of flower coloration in Tagetes erecta and enhances fruit pigmentation in tomato.

Carotenoids play a pivotal role in plant. Tagetes erecta, commonly called marigold, has increasing nutritional and economic value due to its high level of carotenoids in flower. However, the functional genes in the carotenoid biosynthesis of T. erecta have not been studied. In this work, three T. erecta varieties with flowers of yellow, yellow-orange and orange color, respectively, were examined for carotenoids composition and corresponding expression profiling of biosynthetic genes at four developmental stages. The results indicated that the varieties with higher lutein content, orange-flower 'Juwang' and yellow-orange 'Taishan', exhibited significant upregulation of genes in the upstream biosynthesis pathway, especially PDS (phytoene desaturase), PSY (phytoene synthase) and ZDS (zeta-carotene desaturase), whereas downstream carotenoid cleavage genes CCD (carotenoid cleavage dioxygenase) were markedly downregulated throughout flower development in the highest lutein containing variety 'Juwang'. Furthermore, marigold TePDS, TePSYS3 and TeZDS were isolated and transformed into tomato. Overexpression of TePDS or TeZDS resulted in the promotion of fruit ripening and accumulation of carotenoids in the transgenic lines. On the other hand, marigold TePSYS3 showed multiple effects, not only on fruit carotenogenesis but also on pigmentation patterns in vegetative tissues and plant growth. Taken together, the variations in expression profiles of the biosynthetic genes contribute to dynamic change in carotenoid levels and diversity of flower coloration in T. erecta. These functional genes of T. erecta were verified in tomato and provide targets for genetic improvement of fruit carotenoids accumulation.

Overexpression of chrysanthemyl diphosphate synthase (CDS) gene in Tagetes erecta leads to the overproduction of pyrethrin.

Pyrethrins are widely accepted as natural insecticides and offers several advantages of synthetic compounds, i.e., rapidity of action, bioactivity against a wide range of insects, comparatively lesser costs and the like. A significant source of pyrethrin is Chrysanthemum cinerariaefolium; cultivated in restricted areas, as a result; natural pyrethrins are not produced in a large amount that would meet the ongoing global market demand. However, increasing its content and harnessing the desired molecule did not attract much attention. To enhance the production of pyrethrins in Tagetes erecta, the Chrysanthemyl diphosphate synthase (CDS) gene was overexpressed under the promoter CaMV35S. Hypocotyls were used as explant for transformation, and direct regeneration was achieved on MS medium with 1.5 mg L-1 BAP and 5.0 mg L-1 GA3. Putative transgenics were screened on 10 mgL-1 hygromycin. After successful regeneration, screening and rooting process, the transgenic plants were raised inside the glass house and PCR amplification of CDS and HYG-II was used to confirm the transformation. Biochemical analysis using HPLC demonstrated the expression levels of the pyrethrin, which was approx. twenty-six fold higher than the non-transformed Tagetes plant.

Chryseobacterium tagetis sp. nov., a plant growth promoting bacterium with an antimicrobial activity isolated from the roots of medicinal plant (Tagetes patula).

A novel plant growth-promoting and indole acetic acid (IAA) producing strain designated RG1T was isolated from the roots of Tagetes patula (marigold) collected from Goyang, South Korea. The cells of strain RG1T is aerobic, yellow, Gram-stain-negative, pleomorphic and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain RG1T belongs to the genus Chryseobacterium and is closely related to Chryseobacterium gwangjuense THG-A18T (98.6%). The strain produced IAA (70.5 µg ml-1) in the presence of L-tryptophan and showed antimicrobial activity against Gram-negative bacterium Xanthomonas campestris pv. campestris KACC 10377T. The isolate had a significant positive effect on rice plant shoot and root growth. The novel strain RG1T had a draft genome size of 4,430,189 bp, with ten scaffolds and 3969 protein-coding genes. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain RG1T and other closely related members ranged from 21.5 to 36.6% and from 79.2 to 86.6%, respectively. The genomic DNA G + C content was 34.8 mol%. Furthermore, anti-SMASH analysis of the whole genome revealed six putative biosynthetic gene clusters responsible for various secondary metabolites. The major respiratory quinone was MK-6 and the major fatty acids were iso-C15:0, summed feature 3 (comprising C16: 1ω7c and/or C16: 1ω6c) and summed feature 9 (comprising iso-C17: 1 ω9c and/or 10-methyl C16:0). The major polar lipid is phosphatidylethanolamine. Based on the genotypic, chemotaxonomic and physiological data, strain RG1T represents a novel species, for which the name Chryseobacterium tagetis sp. nov. is proposed. The type strain is designated as RG1T ( = KCTC 82696T = NBRC 115057T).

Functional Conservation and Divergence of Five AP1/FUL-like Genes in Marigold (Tagetes erecta L.).

Members of AP1/FUL subfamily genes play an essential role in the regulation of floral meristem transition, floral organ identity, and fruit ripping. At present, there have been insufficient studies to explain the function of the AP1/FUL-like subfamily genes in Asteraceae. Here, we cloned two euAP1 clade genes TeAP1-1 and TeAP1-2, and three euFUL clade genes TeFUL1, TeFUL2, and TeFUL3 from marigold (Tagetes erecta L.). Expression profile analysis demonstrated that TeAP1-1 and TeAP1-2 were mainly expressed in receptacles, sepals, petals, and ovules. TeFUL1 and TeFUL3 were expressed in flower buds, stems, and leaves, as well as reproductive tissues, while TeFUL2 was mainly expressed in flower buds and vegetative tissues. Overexpression of TeAP1-2 or TeFUL2 in Arabidopsis resulted in early flowering, implying that these two genes might regulate the floral transition. Yeast two-hybrid analysis indicated that TeAP1/FUL proteins only interacted with TeSEP proteins to form heterodimers and that TeFUL2 could also form a homodimer. In general, TeAP1-1 and TeAP1-2 might play a conserved role in regulating sepal and petal identity, similar to the functions of MADS-box class A genes, while TeFUL genes might display divergent functions. This study provides a theoretical basis for the study of AP1/FUL-like genes in Asteraceae species.

Production of Haploids and Doubled Haploids in Marigold (Tagetes spp.) Using Anther Culture.

Homozygous parental lines are indispensable for commercial hybrid seed production in many ornamental and vegetable crops. The in vitro induction of haploids and doubled haploids (DHs) through gametic embryogenesis is an effective approach for single-step development of complete homozygous lines from heterozygous donor plants. Anther culture is one of the most popular and widely employed techniques for development of haploids. Here we describe the detailed protocol for rapid and successful induction of haploids in Tagetes spp. using in vitro androgenesis approach. In this protocol, we have provided the comprehensive details of various steps of anther culture in marigold right from the growing of donor plants, selection of buds, pretreatment, embryogenesis and regeneration to ploidy analysis, and chromosome doubling for development of DHs.

Functional conservation and divergence of SEPALLATA-like genes in the development of two-type florets in marigold.

Marigold (Tagetes erecta), as one member of Asteraceae family, bears a typical capitulum with two morphologically distinct florets. The SEPALLATA genes are involved in regulating the floral meristem determinacy, organ identity, fruit maturation, seed formation, and plant architecture. Here, five SEP-like genes were cloned and identified from marigold. Sequence alignment and phylogenetic analysis demonstrated that TeSEP3-1, TeSEP3-2, and TeSEP3-3 proteins were grouped into SEP3 clade, and TeSEP1 and TeSEP4 proteins were clustered into SEP1/2/4 clade. Quantitative real-time PCR analysis revealed that TeSEP1 and TeSEP3-3 were broadly expressed in floral organs, and that TeSEP3-2 and TeSEP4 were mainly expressed in pappus and corollas, while TeSEP3-1 was mainly expressed in two inner whorls. Ectopic expression of TeSEP1, TeSEP3-2, TeSEP3-3, and TeSEP4 in arabidopsis and tobacco resulted in early flowering. However, overexpression of TeSEP3-1 in arabidopsis and tobacco caused no visible phenotypic changes. Notably, overexpression of TeSEP4 in tobacco decreased the number of petals and stamens. Overexpression of TeSEP1 in tobacco led to longer sepals and simpler inflorescence architecture. The comprehensive pairwise interaction analysis suggested that TeSEP proteins had a broad interaction with class A, C, D, E proteins to form dimers. The yeast three-hybrid analysis suggested that in ternary complexes, class B proteins interacted with TeSEP3 by forming heterodimer TePI-TeAP3-2. The regulatory network analysis of MADS-box genes in marigold further indicated that TeSEP proteins played a "glue" role in regulating floral organ development, implying functional conservation and divergence of MADS box genes in regulating two-type floret developments. This study provides an insight into the formation mechanism of floral organs of two-type florets, thus broadening our knowledge of the genetic basis of flower evolution.

Carotenoid metabolite and transcriptome dynamics underlying flower color in marigold (Tagetes erecta L.).

Marigold (Tagetes erecta L.) is an important ornamental plant with a wide variety of flower colors. Despite its economic value, few biochemical and molecular studies have explored the generation of flower color in this species. To study the mechanism underlying marigold petal color, we performed a metabolite analysis and de novo cDNA sequencing on the inbred line 'V-01' and its petal color mutant 'V-01M' at four flower developmental stages. A total of 49,217 unigenes were identified from 24 cDNA libraries. Based on our metabolites and transcriptomic analyses, we present an overview of carotenoid biosynthesis, degradation, and accumulation in marigold flowers. The carotenoid content of the yellow mutant 'V-01M' was higher than that of the orange inbred line 'V-01', and the abundances of the yellow compounds lutein, neoxanthin, violaxanthin, zeaxanthin, and antheraxanthin were significantly higher in the mutant. During flower development, the carotenoid biosynthesis genes were upregulated in both 'V-01' and 'V-01M', with no significant differences between the two lines. By contrast, the carotenoid degradation genes were dramatically downregulated in the yellow mutant 'V-01M'. We therefore speculate that the carotenoid degradation genes are the key factors regulating the carotenoid content of marigold flowers. Our research provides a large amount of transcriptomic data and insights into the marigold color metabolome.

Identification, characterization and functional analysis of AGAMOUS subfamily genes associated with floral organs and seed development in Marigold (Tagetes erecta).

BACKGROUND: AGAMOUS (AG) subfamily genes regulate the floral organs initiation and development, fruit and seed development. At present, there has been insufficient study of the function of AG subfamily genes in Asteraceae. Marigold (Tagetes erecta) belongs to Asteraceae family whose unique inflorescence structure makes it an important research target for understanding floral organ development in plants. RESULTS: Four AG subfamily genes of marigold were isolated and phylogenetically grouped into class C (TeAG1 and TeAG2) and class D (TeAGL11-1 and TeAGL11-2) genes. Expression profile analysis demonstrated that these four genes were highly expressed in reproductive organs of marigold. Subcellular localization analysis suggested that all these four proteins were located in the nucleus. Protein-protein interactions analysis indicated that class C proteins had a wider interaction manner than class D proteins. Function analysis of ectopic expression in Arabidopsis thaliana revealed that TeAG1 displayed a C function specifying the stamen identity and carpel identity, and that TeAGL11-1 exhibited a D function regulating seed development and petal development. In addition, overexpression of both TeAG1 and TeAGL11-1 leaded to curling rosette leaf and early flowering in Arabidopsis thaliana. CONCLUSIONS: This study provides an insight into molecular mechanism of AG subfamily genes in Asteraceae species and technical support for improvement of several floral traits.

Functional Analysis of the Marigold (Tagetes erecta) Lycopene ε-cyclase (TeLCYe) Promoter in Transgenic Tobacco.

Lycopene ε-cyclases (LCYEs) are key enzymes in carotenoid biosynthesis converting red lycopene to downstream lutein. The flowers of marigold (Tagetes erecta) have been superior sources to supply lutein. However, the transcriptional regulatory mechanisms of LCYe in lutein synthesis are still unclear in marigold. In this work, the expression pattern of the TeLCYe gene in marigold indicated that TeLCYe mainly expressed in floral organs. To gain a better understanding of the expression and regulatory mechanism of TeLCYe gene, the TeLCYe promoter was isolated, sequenced, and analyzed through bioinformatics tools. The results suggested that the sequence of TeLCYe promoter contained various putative cis-acting elements responsive to exogenous and endogenous factors. The full-length TeLCYe promoter and three 5'-deletion fragments were fused to the GUS reporter gene and transferred into tobacco to test the promoter activities. A strong GUS activity was observed in stems of seedlings, leaves of seedlings, middle stems, top leaves, petals, stamens, and stigmas in transgenic tobacco containing full-length TeLCYe promoter LP0-2086. Deletion of - 910 to - 429 bp 5' to ATG significantly increased the GUS activity in chloroplast-rich tissues and floral organs, while deletion occurring from 1170 to 910 bp upstream ATG decreased the TeLCYe promoter strength in stems of seedlings, leaves of seedlings, top leaves and sepals. Functional characterization of the full-length TeLCYe promoter and its' deletion fragments in stable transgenic tobacco indicated that the LP0-2086 contains some specific cis-acting elements, which might result in the high-level expression of in floral organs, and LP2-910 might contain some specific cis-acting elements which improved GUS activities in vegetable tissues.

The transcriptome analyses of Tagetes erecta provides novel insights into secondary metabolite biosynthesis during flower development.

Genomic and transcriptomic sequences of Tagetes erecta are very limited in public databases, despite its nutritional and economical value. In this study, nine cDNA libraries were constructed from leaves, immature and mature flowers and sequenced using Illumina Hiseq 2000. Over 45.66 Gb nucleotides were generated and a total of 72,499 unigenes were assembled, 34,892 (48.13%) of which were annotated in NR, Swiss-prot, COG, GO, KOG, KEGG and Pfam. 11,721 differentially expressed genes were identified in leaves, immature flowers and mature flowers. The differentially expressed genes between immature flowers and mature flowers were mainly involved in photosynthesis and secondary metabolite biosynthesis. Additionally, the catalysis-related unigenes and their expression involved in carotenoids biosynthesis were determined. Using our newly identified reference genes as internal control, the expression profiles of carotenoids biosynthetic genes were verified by real-time qRT-PCR, and four of the unigenes were cloned for full length sequences confirmation. Furthermore, gene expansions occurred among the catalytic gene families in carotenoids biosynthesis pathway, which might explain the high pigment content in T. erecta.

Characterization and Functional Analysis of Five MADS-Box B Class Genes Related to Floral Organ Identification in Tagetes erecta.

According to the floral organ development ABC model, B class genes specify petal and stamen identification. In order to study the function of B class genes in flower development of Tagetes erecta, five MADS-box B class genes were identified and their expression and putative functions were studied. Sequence comparisons and phylogenetic analyses indicated that there were one PI-like gene-TePI, two euAP3-like genes-TeAP3-1 and TeAP3-2, and two TM6-like genes-TeTM6-1 and TeTM6-2 in T. erecta. Strong expression levels of these genes were detected in stamens of the disk florets, but little or no expression was detected in bracts, receptacles or vegetative organs. Yeast hybrid experiments of the B class proteins showed that TePI protein could form a homodimer and heterodimers with all the other four B class proteins TeAP3-1, TeAP3-2, TeTM6-1 and TeTM6-2. No homodimer or interaction was observed between the euAP3 and TM6 clade members. Over-expression of five B class genes of T. erecta in Nicotiana rotundifolia showed that only the transgenic plants of 35S::TePI showed altered floral morphology compared with the non-transgenic line. This study could contribute to the understanding of the function of B class genes in flower development of T. erecta, and provide a theoretical basis for further research to change floral organ structures and create new materials for plant breeding.

Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling.

Tagetes erecta is an important commercial plant of Asteraceae family. The male sterile (MS) and male fertile (MF) two-type lines of T. erecta have been utilized in F1 hybrid production for many years, but no report has been made to identify the genes that specify its male sterility that is caused by homeotic conversion of floral organs. In this study, transcriptome assembly and digital gene expression profiling were performed to generate expression profiles of MS and MF plants. A cDNA library was generated from an equal mixture of RNA isolated from MS and MF flower buds (1 mm and 4 mm in diameter). Totally, 87,473,431 clean tags were obtained and assembled into 128,937 transcripts among which 65,857 unigenes were identified with an average length of 1,188 bp. About 52% of unigenes (34,176) were annotated in Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO (KEGG Ortholog database) and/or GO. Taking the above transcriptome as reference, 125 differentially expressed genes were detected in both developmental stages of MS and MF flower buds. MADS-box genes were presumed to be highly related to male sterility in T. erecta based on histological and cytological observations. Twelve MADS-box genes showed significantly different expression levels in flower buds 4 mm in diameter, whereas only one gene expressed significantly different in flower buds 1 mm in diameter between MS and MF plants. This is the first transcriptome analysis in T. erecta and will provide a valuable resource for future genomic studies, especially in flower organ development and/or differentiation.

An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 µM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.

Thiophene occurrence in different Tagetes species: agricultural biomasses as sources of biocidal substances.

BACKGROUND: Tagetes species produce thiophenes, polyacetylenic compounds that possess strong biocidal activity, thus making Tagetes plants very useful for suppressing nematode populations in the soil and as sources of natural pesticides. In the present study, Tagetes species (T. erecta, T. filifolia, T. lucida, T. minuta, T. patula and T. tenuifolia) grown in northern Italy were evaluated for their morphophenological parameters and thiophene pattern in different plant parts (roots, shoots and flowers). RESULTS: Roots had the highest diversity and contents of thiophenes (from 64 to 100% of the total thiophene amount), with 5-(3-buten-1-ynyl)-2,2'-bithienyl (BBT) as the main component followed by 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), 2,2':5',2''-terthienyl (alpha-T) and 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH). Tagetes lucida and T. tenuifolia possessed the highest amounts of total thiophenes (6717.3 and 6452.5 mg kg(-1) dry weight respectively), while T. minuta had the highest total thiophene yield (518.8 mg m(-2)), with BBT accounting for 98% of the total. CONCLUSION: Considering both thiophene concentrations and biomass yields, T. minuta and T. lucida appeared to be the most promising Tagetes species, with high potential for use as biocidal crops for the implementation of pest control practices that are less harmful to human health and natural resources.

Optimization of DNA extraction from seeds and fresh leaf tissues of wild marigold (Tagetes minuta) for polymerase chain reaction analysis.

Tagetes, a genus of flowering marigolds in the family Asteraceae (Compositeae), is reported to be a medicinal plant with hypotensive, spasmolytic, anti-inflammatory, antimicrobial, and antifungal properties. Tagetes minuta characteristically contains high concentrations of essential oils, flavonoids, polyphenols, and polysaccharides that interfere with DNA, causing erroneous or no PCR products. We tested and modified various standard protocols in an effort to isolate high-quality DNA from different plant tissues of T. minuta. We used sun-dried, shade-dried and fresh-leaf tissues, as well as seeds for DNA analysis. The DNA obtained from seeds and fresh-leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. We were able to obtain good quality DNA from fresh leaf tissues without using liquid nitrogen. A relatively large amount of DNA was also extracted from the sun- and shade-dried tissues, but its quality was not as good as that from seeds. The DNA extracted from seeds and fresh leaves was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extracting high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.

Marigold regeneration and molecular analysis of carotenogenic genes.

Marigold (Tagetes erecta) is an Asteraceous plant of industrial, ornamental and medicinal importance. Tagetes erecta species, popularly known as marigold, is grown as ornamental plant and is adapted to several agro climates. Inflorescences have been utilized as pigment source for food coloring, mainly of poultry skin and eggs. Lutein is the main pigment in marigold flowers. Some carotenoids are well known as provitamin A compounds. There are many reports on carotenoids and their effect on the prevention of certain ocular diseases, ischemic heart disease, strokes, photoprotection, immune response, aging and cancer. Marigold flowers are certainly a good source of carotenoids; they show very different pigmentation levels. This chapter describes the establishment of techniques for plant regeneration, gene expression, pigment extraction and pigment deposition in specific structures of marigold.

Potential of the hybrid marigolds for arsenic phytoremediation and income generation of remediators in Ron Phibun District, Thailand.

Nugget marigold, a triploid hybrid between American (Tagetes erecta L.) and French (Tagetes patula) marigolds, is a marketed flowering plant with a good ability in arsenic phytoremediation. During field trial in an arsenic-polluted area in Thailand, arsenic was found mostly in leaves (46.2%) while flowers contained the lowest arsenic content (5.8%). Arsenic species in aqueous extracts of nugget marigolds were determined by HPLC-UV-HG-QF-AAS. Inorganic arsenics, arsenite and arsenate, were the main arsenic chemical species found in roots, stems, and leaves of marigolds with accumulated arsenic. Nugget marigolds from experimental plots not only accumulated high levels of arsenic but also grew well in arsenic-contaminated areas. Phosphate fertilizer enhanced arsenic uptake when the plants were in the flowering stage. Arsenic remediation using nugget marigolds could also provide economic benefits to the remediators through marketing flowers. Therefore, marigolds should be considered as a potential economic crop for phytoremediation.