Transgenic Chrysanthemum Research Articles

Aphid alarm pheromone mimicry in transgenic Chrysanthemum morifolium: insights into the potential of (E)-β-farnesene for aphid resistance.

(E)-β-Farnesene (EBF) serves as the primary component of the alarm pheromone used by most aphid pest species. Pyrethrum (Tanacetum cinerariifolium) exhibits tissue-specific regulation of EBF accumulation and release, effectively mimicking the aphid alarm signal, deterring aphid attacks while attracting aphid predators. However, cultivated chrysanthemum (Chrysanthemum morifolium), a popular and economically significant flower, is highly vulnerable to aphid infestations. In this study, we investigated the high expression of the pyrethrum EBF synthase (TcEbFS) gene promoter in the flower head and stem, particularly in the parenchyma cells. Subsequently, we introduced the TcEbFS gene, under the control of its native promoter, into cultivated chrysanthemum. This genetic modification led to increased EBF accumulation in the flower stem and young flower bud, which are the most susceptible tissues to aphid attacks. Analysis revealed that aphids feeding on transgenic chrysanthemum exhibited prolonged probing times and extended salivation durations during the phloem phase, indicating that EBF in the cortex cells hindered their host-location behavior. Interestingly, the heightened emission of EBF was only observed in transgenic chrysanthemum flowers after mechanical damage. Furthermore, we explored the potential of this transgenic chrysanthemum for aphid resistance by comparing the spatial distribution and storage of terpene volatiles in different organs and tissues of pyrethrum and chrysanthemum. This study provides valuable insights into future trials aiming for a more accurate replication of alarm pheromone release in plants. It highlights the complexities of utilizing EBF for aphid resistance in cultivated chrysanthemum and calls for further investigations to enhance our understanding of this defense mechanism.

Molecular Cloning of a cDNA for Chrysanthemum morifolium Ramat Squalene Synthase Gene and Impact of Its Silencing on Transgenic Chrysanthemum Plants

Molecular Cloning of a cDNA for Chrysanthemum morifolium Ramat Squalene Synthase Gene and Impact of Its Silencing on Transgenic Chrysanthemum Plants

GERDH: an interactive multi-omics database for cross-species data mining in horticultural crops.

Horticultural plants contribute immensely to the quality of humans' life. The rapid development of omics studies on horticultural plants has resulted in large volumes of valuable growth- and development-related data. Genes that are essential for growth and development are highly conserved in evolution. Cross-species data mining reduces the impact of species heterogeneity and has been extensively used for conserved gene identification. Owing to the lack of a comprehensive database for cross-species data mining using multi-omics data from all horticultural plant species, the current resources in this field are far from satisfactory. Here, we introduce GERDH (https://dphdatabase.com), a database platform for cross-species data mining among horticultural plants, based on 12,961 uniformly processed publicly available omics libraries from more than 150 horticultural plant accessions, including fruits, vegetables, and ornamental plants. Important and conserved genes that are essential for a specific biological process can be obtained by cross-species analysis module with interactive web-based data analysis and visualization. Moreover, GERDH is equipped with 7 online analysis tools, including gene expression, in-species analysis, epigenetic regulation, gene co-expression, enrichment/pathway and phylogenetic analysis. By interactive cross-species analysis, we identified key genes contributing to postharvest storage. By gene expression analysis, we explored new functions of CmEIN3 in flower development, which was validated by transgenic chrysanthemum analysis. We believe that GERDH will be a useful resource for key gene identification and will allow for omics big data to be more available and accessible to horticultural plant community members.

BBX7 interacts with BBX8 to accelerate flowering in chrysanthemum

The quantitative control of FLOWERING LOCUS T (FT) activation is important for the floral transition in flowering plants. However, the flowering regulation mechanisms in the day-neutral, summer-flowering chrysanthemum plant remain unclear. In this study, the chrysanthemum BBX7 homolog CmBBX7 was isolated and its flowering function was identified. The expression of CmBBX7 showed a diurnal rhythm and CmBBX7 exhibited higher expression levels than CmBBX8. Overexpression of CmBBX7 in transgenic chrysanthemum accelerated flowering, whereas lines transfected with a chimeric repressor (pSRDX-CmBBX7) exhibited delayed flowering. Yeast single hybridization, luciferase, electrophoretic mobility shift, and chromatin immunoprecipitation assays showed that CmBBX7 directly targets CmFTL1. In addition, we found that CmBBX7 and CmBBX8 interact to positively regulate the expression of CmFTL1 through binding to its promoter. Collectively, these results highlight CmBBX7 as a key cooperator in the BBX8–FT module to control chrysanthemum flowering.Graphical

Bacterial Communities Associated with the Rhizosphere of Transgenic Chrysanthemum

Bacterial Communities Associated with the Rhizosphere of Transgenic Chrysanthemum

Overexpression of CmWRKY8-1-VP64 Fusion Protein Reduces Resistance in Response to Fusarium oxysporum by Modulating the Salicylic Acid Signaling Pathway in Chrysanthemum morifolium.

Chrysanthemum Fusarium wilt, caused by the pathogenic fungus Fusarium oxysporum, severely reduces ornamental quality and yields. WRKY transcription factors are extensively involved in regulating disease resistance pathways in a variety of plants; however, it is unclear how members of this family regulate the defense against Fusarium wilt in chrysanthemums. In this study, we characterized the WRKY family gene CmWRKY8-1 from the chrysanthemum cultivar 'Jinba', which is localized to the nucleus and has no transcriptional activity. We obtained CmWRKY8-1 transgenic chrysanthemum lines overexpressing the CmWRKY8-1-VP64 fusion protein that showed less resistance to F. oxysporum. Compared to Wild Type (WT) lines, CmWRKY8-1 transgenic lines had lower endogenous salicylic acid (SA) content and expressed levels of SA-related genes. RNA-Seq analysis of the WT and CmWRKY8-1-VP64 transgenic lines revealed some differentially expressed genes (DEGs) involved in the SA signaling pathway, such as PAL, AIM1, NPR1, and EDS1. Based on Gene Ontology (GO) enrichment analysis, the SA-associated pathways were enriched. Our results showed that CmWRKY8-1-VP64 transgenic lines reduced the resistance to F. oxysporum by regulating the expression of genes related to the SA signaling pathway. This study demonstrated the role of CmWRKY8-1 in response to F. oxysporum, which provides a basis for revealing the molecular regulatory mechanism of the WRKY response to F. oxysporum infestation in chrysanthemum.

A new CIPK gene CmCIPK8 enhances salt tolerance in transgenic chrysanthemum

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a key protein family in plant to withstand stress and promote growth. In this study, we cloned a novel CIPK gene CmCIPK8 from chrysanthemum, which is localized to the cytoplasm. CmCIPK8 is a protein containing a highly conserved S_TKc domain and a NAF domain. The expression of CmCIPK8 was highly induced by salt stress. Under salt stress, the chlorophyll content and relative water content of transgenic chrysanthemum were markedly higher than WT. Meanwhile, the transgenic chrysanthemum also has a lower content of reactive oxygen species, stronger antioxidant enzymes activity, and lower peroxidation degree of membrane lipid, accompanied by more soluble sugar, soluble protein and proline content. In addition, transgenic chrysanthemum also has a lower relative electrical conductivity, higher K+/Na+ and Ca2+/Na+ ratios, stronger photosynthetic capacity and root activity. Further studies showed that CmCIPK8 might enhance the expression of ion transport-related genes. Taken together, these results suggest that CmCIPK8 may promote tolerance to salt stress.

Characterization of the MADS-Box Gene CmFL3 in chrysanthemum

Chrysanthemummorifolium is one of the four major cut flowers in the world, with high ornamental and economic value. Flowering time is an important ornamental characteristic of chrysanthemum that affects its value in the market. In Arabidopsis, the FRUITFULL (FUL) gene plays a key role in inducing flowering. Here, we isolated an FUL clade MADS-box gene, CmFL3, from chrysanthemum inflorescence buds. CmFL3 localized in the cellular membrane and nucleus, and showed no transcriptional activity in yeast. The qRT-PCR assay showed that CmFL3 was strongly expressed in the leaves, receptacles, and disc floret petals. Furthermore, CmFL3 was mainly detected in the inflorescence meristem and bract primordia using in situ hybridization. Similar to Arabidopsis, overexpression of CmFL3 in chrysanthemum induced early flowering. Particularly, the expression level of CmAFT was downregulated, whereas that of CmFTL3 was upregulated in the leaves of transgenic chrysanthemum lines. Meanwhile, the overexpression of CmFL3 in Arabidopsis also led to earlier flowering. Furthermore, the expression of AtFT, AtAP1, AtLFY, and AtFUL was significantly increased in CmFL3 transgenic Arabidopsis. The present study verified the function of CmFL3 in regulating flowering time and further revealed that it could affect the expression of other flowering-related genes—CmAFT and CmFTL3. Therefore, the CmFL3 gene may be an important candidate for genetic breeding aimed at regulating flowering.

Performance of Transgenic Chrysanthemum Harbouring Wasabi Defensin Gene for White Rust Disease Resistance

This study was intended to obtain white rust (Puccinia horiana) disease resistance Chrysanthemum transformed with wasabi defensin gene through mediation of Agrobacterium tumefaciens from three explant sources, i.e., leaf, lateral shoot bud, and internode. Observations were made on transformation efficiency, PCR analysis, in vitro and ex vitro disease resistance tests. Results showed that efficiency of transgenic callus and shoot regeneration was found both highest from lateral shoot buds (57.5% and 50.0%, respectively). PCR analysis showed that three putative transgenic plantlets from lateral shoot buds and one from leaf explant were putative transgenic carrying the wasabi, hpt, and nptII genes. Rooting test showed that the highest number of rooted plants was found in treatment of hygromycin (Hg) 25 mg L-1 (81%) and lowest was in treatment combination of kanamycin (Km) 50 mg L-1 + Hg 25 mg L-1 (25%). In vitro disease resistance test with sorus inoculation of P. horiana, directly on the leaves, resulted in 20 resistant plants out of 30 putative transgenic plants (66.67%). Ex vitro testing on adult plants of the same samples in a confined closed greenhouse (CGH) resulted in average of 80% transgenic Chrysanthemum plants were resistant, whereas in control plants caused white rust disease symptom.

CmNF-YB8 affects drought resistance in chrysanthemum by altering stomatal status and leaf cuticle thickness.

Drought is a major abiotic stress that limits plant growth and development. Adaptive mechanisms have evolved to mitigate drought stress, including the capacity to adjust water loss rate and to modify the morphology and structure of the epidermis. Here, we show that the expression of CmNF-YB8, encoding a nuclear factor Y (NF-Y) B-type subunit, is lower under drought conditions in chrysanthemum (Chrysanthemum morifolium). Transgenic chrysanthemum lines in which transcript levels of CmNF-YB8 were reduced by RNA interference (CmNF-YB8-RNAi) exhibited enhanced drought resistance relative to control lines, whereas lines overexpressing CmNF-YB8 (CmNF-YB8-OX) were less tolerant to drought. Compared to wild type (WT), CmNF-YB8-RNAi plants showed reduced stomatal opening and a thicker epidermal cuticle that correlated with their water loss rate. We also identified genes involved in stomatal adjustment (CBL-interacting protein kinase 6, CmCIPK6) and cuticle biosynthesis (CmSHN3) that are more highly expressed in CmNF-YB8-RNAi lines than in WT, CmCIPK6 being a direct downstream target of CmNF-YB8. Virus-induced gene silencing of CmCIPK6 or CmSHN3 in the CmNF-YB8-RNAi background abolished the effects of CmNF-YB8-RNAi on stomatal closure and cuticle deposition, respectively. CmNF-YB8 thus regulates CmCIPK6 and CmSHN3 expression to alter stomatal movement and cuticle thickness in the leaf epidermis, thereby affecting drought resistance.

Lysine decrotonylation of glutathione peroxidase at lysine 220 site increases glutathione peroxidase activity to resist cold stress in chrysanthemum

Lysine crotonylation is a protein post-translational modification that has been newly discovered in recent years. There are few studies on the lysine crotonylation of proteins in plants, and their functions in response to cold stress are still unclear. In this study, the chrysanthemum (Chrysanthemum morifolium Ramat.) glutathione peroxidase (GPX) gene was selected and named DgGPX1, and was found to be responsive to low temperature. Overexpression of DgGPX1 improved the cold resistance of transgenic chrysanthemum by increasing GPX activity to reduce the accumulation of reactive oxygen species (ROS) under low-temperature conditions. Furthermore, the level of DgGPX1 lysine crotonylation at lysine (K) 220 decreased under low temperature in chrysanthemum. Lysine decrotonylation of DgGPX1 at K220 further increased GPX activity to reduce ROS accumulation under cold stress, and thereby enhanced the cold resistance of chrysanthemum. The above results show that lysine decrotonylation of DgGPX1 at K220 increases GPX activity to resist cold stress in chrysanthemum.

Transgenic Chrysanthemum indicum overexpressing cin-miR396a exhibits altered plant development and reduced salt and drought tolerance

The conserved microRNA396 (miR396) is involved in growth, development, and abiotic stress responses in a variety of plants by regulating target genes. Here, we obtained transgenic Chrysanthemum indicum (C. indicum) overexpressing the cin-miR396a gene. The transgenic plants (TGs) had longer internodes and fewer epidermal hairs in contrast with the wild-type (WT) control. cin-miR396a overexpression in C. indicum reduced salt tolerance and drought tolerance. After salt and drought stress compared with WT plants, the transgenic C. indicum exhibited a relative decrease in leaf water content, and the leaf free proline content, also exhibited a relative increase, in the leaf conductivity and leaf Malondialdehyde content, while the total chlorophyll content did not differ significantly from WT, and the Na+/K+ ratio in the roots of transgenic C. indicum increased after salt stress. We also identified two target genes of cin-miR396a, CiGRF1 and CiGRF5, whose expression was induced by salt and drought treatments and suppressed in transgenic C. indicum. Taken together, our results reveal a unique role for the regulatory module of miR396a-GRFs in C. indicum development and response to abiotic stresses. cin-miR396a plays a negative regulatory role in C. indicum in response to salt and drought stresses.

The transcription factor CmLEC1 positively regulates the seed-setting rate in hybridization breeding of chrysanthemum

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1’s overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1–CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA’s expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

Heterologous Biosynthesis of Artemisinin in Chrysanthemum morifolium Ramat

Artemisinin-based drugs are the most effective medicine against multidrug-resistant Plasmodium spp., the parasite that causes malaria. To this day, wormwood A. annua L. is the sole commercial source of artemisinin, where it is produced in minor amounts. The artemisinin yield depends on numerous poorly regulated agricultural factors and the genetic variability of this non-domesticated plant. This has aroused significant interest in the development of heterologous expression platforms for artemisinin production. Previously, we obtained lines of Chrysanthemum morifolium Ramat. (C. morifolium Ramat.), cvs. White Snowdon and Egyptianka, transformed with artemisinin biosynthesis genes. Here, we report the results of an analysis of artemisinin production in transgenic chrysanthemums. Transcription of heterologous amorpha-4,11-diene monooxygenase and cytochrome P450 reductase genes in transgenic lines was confirmed using high-resolution melting analysis. Artemisinin accumulation was detected using GC-MS in White Snowdon plants, but not in Egyptianka ones, thereby demonstrating the possibility of transplanting active artemisinin biosynthetic pathway into chrysanthemum. Ways of increasing its content in producer plants are discussed.

DgC3H1, a CCCH zinc finger protein gene, confers cold tolerance in transgenic chrysanthemum

Zinc finger proteins play a significant role in regulating plant metabolism and stress responses. Nevertheless, the role of CCCH-type zinc finger proteins in chrysanthemums remains unclear. In this study, DgC3H1 was isolated and cloned from chrysanthemum. The DgC3H1 predicted protein contained 282 amino acids and three CCCH zinc finger motifs (C-X5-14-C-X4-5-C-X3-H). Through subcellular localization, it was found that DgC3H1 is located in the nucleus and is a CCCH zinc finger transcription factor. DgC3H1 overexpression improved the low temperature resistance of chrysanthemum. The expression of endogenous C3H1 was downregulated under antisense inhibition, and the sensitivity of chrysanthemum to low temperature stress was increased. Under cold stress, the contents of proline and soluble sugar, and the activities of peroxidase and superoxide dismutase were enhanced in the DgC3H1 overexpression lines,and reduced in the antisense suppression lines. In addition, the low temperature stress response genes DgCOR413, DgDREBa, DgCSD1, and DgCSD2 were upregulated in the overexpression lines, and downregulated in the antisense suppression lines when compared to the wild type. These results indicate that DgC3H1 can improve the resistance of chrysanthemum to cold stress and can be selected as an excellent candidate gene for breeding cold-tolerant plants.

Lysine crotonylation of DgTIL1 at K72 modulates cold tolerance by enhancing DgnsLTP stability in chrysanthemum.

SummaryLysine crotonylation of proteins is a recently identified post‐translational modification (PTM) in plants. However, the function of lysine‐crotonylated proteins in response to abiotic stress in plants has not been reported. In this study, we identified a temperature‐induced lipocalin‐1‐like gene (DgTIL1) from chrysanthemum and showed that it was notably induced in response to cold stress. Overexpression of DgTIL1 enhanced cold tolerance in transgenic chrysanthemum. Ubiquitin membrane yeast two‐hybrid (MYTH) system and bimolecular fluorescence complementation (BIFC) assays showed that DgTIL1 interacts with a nonspecific lipid transfer protein (DgnsLTP), which can promote peroxidase (POD) gene expression and POD activity to reduce the accumulation of reactive oxygen species (ROS) and improve resistance to cold stress in DgnsLTP transgenic chrysanthemum. In addition, we found that DgTIL1 was lysine crotonylated at K72 in response to low temperature in chrysanthemum. Moreover, lysine crotonylation of DgTIL1 prevented DgnsLTP protein degradation in tobacco and chrysanthemum. Inhibition of DgnsLTP degradation by lysine crotonylation of DgTIL1 further enhanced POD expression and POD activity, reduced the accumulation of ROS under cold stress in DgTIL1 transgenic chrysanthemum, thus promoting the cold resistance of chrysanthemum.

Chrysanthemum CmWRKY53 negatively regulates the resistance of chrysanthemum to the aphid Macrosiphoniella sanborni

Chrysanthemum is frequently attacked by aphids, which greatly hinders the growth and ornamental value of this plant species. WRKY transcription factors play an important role in the response to biotic stresses such as pathogen and insect stresses. Here, chrysanthemum CmWRKY53 was cloned, and its expression was induced by aphid infestation. To verify the role of CmWRKY53 in resistance to aphids, CmWRKY53 transgenic chrysanthemum was generated. CmWRKY53 was found to mediate the susceptibility of chrysanthemum to aphids. The expression levels of secondary metabolite biosynthesis genes, such as peroxidase- and polyphenol oxidase-encoding genes, decreased in CmWRKY53-overexpressing (CmWRKY53-Oe) plants but dramatically increased in chimeric dominant repressor (CmWRKY53-SRDX) plants, suggesting that CmWRKY53 contributes to the susceptibility of chrysanthemum to aphids, possibly due to its role in the regulation of secondary metabolites.

Distribution of cell layers in floral organs of chrysanthemum analyzed with periclinal chimeras carrying a transgene encoding fluorescent protein.

A fluorescent protein visualized distributions of cell layers in floral organs of chrysanthemum using transgenic periclinal chimeras carrying a gene encoding a fluorescent compound. Plant meristems have three cell layers: the outermost layer (L1), the second layer (L2), and the inner layer (L3). The layers are maintained during development but there is limited knowledge of the details of cell layer patterns within floral organs. In this study, we visualized the distributions of cell layers in floral organs of chrysanthemum using periclinal chimeras carrying a gene encoding a fluorescent compound in the L1 or the L2/L3 layers. The L1 layer contributed most of the epidermal cells of organs including the receptacle, petal, anther, filament, style, stigma, and ovule. The transmitting tissue in the pistil and most of the internal area of the ovule were also derived from the L1. In crossing experiments, no progeny of the L1-chimeric plants showed fluorescence, indicating that the germ cells of chrysanthemum are not derived from the L1 layer. Since anthocyanin pigment is present only in the L1-derived epidermal cells of petals, L1-specific gene integration could be used to alter flower color in commercial cultivars, with a reduced risk of transgene flow from the transgenic chrysanthemums to wild relatives.

Ectopic expression of AtSOC1 gene driven by the inducible promoter rd29A, causes early flowering in Chrysanthemum

Suppressor of Overexpression of Constans1 of Arabidopsis (AtSOC1) plays an important role in floral induction and development as one of the flowering pathway integrators. rd29A is a stress inducible promoter. To obtain a new chrysanthemum germplasm with an inducible flowering time, we succeeded in making transgenic chrysanthemum plants carrying AtSOC1 genes driven by the rd29A promoter via Agrobacterium-mediated transformation. Molecular characterization confirmed the stable integration of the introduced gene cassette in the transformants. The result showed that AtSOC1 expression was elevated to varying extents in different transgenic lines. One of the lines showed an obvious phenotype of early flowering, which was induced by drought stress. These results indicated that AtSOC1, driven by the rd29A promoter, can regulate the flowering time when drought stress treatments are applied to chrysanthemum.

Effects of the silencing of CmMET1 by RNA interference in chrysanthemum (Chrysanthemum morifolium)

DNA methylation is an epigenetic modification involving many biological processes. It is known that epigenetic mechanisms such as cytosine methylation play a pivotal role in regulating plant development. In this current study, a conservative domain-spliced hairpin RNA (ihpRNA) plant expression vector aimed at gene of DNA METHYLTRANSFERASE1 (CmMET1) has been constructed. Transgenic chrysanthemum materials (Zijingling) were obtained by Agrobacterium-mediated transformation with expression vectors. Transgenic plants were used as rootstock, grafted onto non-transgenic the scions [Guoqinghong (GQH) and Huanshuijinqiu (HSJQ)], which silencing CmMET1 gene, and exhibited the early flower phenotypes. A high-performance liquid chromatography analysis indicated that the decrease of CmMET1 expression decreased the methylation level of genomic DNA. Similarly, a quantitative real-time polymerase chain reaction analysis revealed that CmMET1 expression levels decreased in transgenic chrysanthemum plants and in the scions grafted onto transgenic plants. This decrease of CmMET1 expression upregulated the expression of the methyltransferase gene, METHYLTRANSFERASE2 (CmDRM2), but downregulated the expression of the demethylating enzyme gene, DEMETER (CmDME), while the CHROMOMETHYLASEA (CmCMT3) expression level remained low and could be almost undetectable. Among the CmFT-likes genes that affect flowering time, CmFTL1 expression was downregulated, as well as CmFTL2 and CmFTL3 expression levels which were upregulated. Our data indicated that silencing CmMET1 could decrease plant height, change the phenotype of chrysanthemum, and promote earlier flowering. The transgenic plants bloomed 8 days earlier. GQH and HSJQ scions grafted onto transgenic plants were 12 and 9 days earlier than the scions grafted onto non-transgenic plants, respectively. Overall, the results have some meanings for promoting the flowering of chrysanthemum scion varieties using genetic modified rootstock.