CmWRKY41 activates CmHMGR2 and CmFPPS2 to positively regulate sesquiterpenes synthesis in Chrysanthemum morifolium.

Chrysanthemum morifolium is one of the most significant multipurpose crops with ornamental, medicinal, and edible value. Terpenoids, an essentials component of volatile oils, are abundant in chrysanthemum. However, the transcriptional regulation of terpenoid biosynthesis in chrysanthemums remains unclear. In the present investigation, we identified CmWRKY41, whose expression pattern is similar to that of terpenoid content in chrysanthemum floral scent, as a candidate gene that may promote terpenoid biosynthesis in chrysanthemum. Two structural genes 3-hydroxy-3-methylglutaryl-CoA reductase 2 (CmHMGR2) and farnesyl pyrophosphate synthase 2 (CmFPPS2), play key role in terpene biosynthesis in chrysanthemum. CmWRKY41 can directly bind to the promoters of CmHMGR2 or CmFPPS2 through GTGACA or CTGACG elements and activate its expression to promote sesquiterpene biosynthesis. In summary, these results indicate that CmWRKY41 targets CmHMGR2 and CmFPPS2 to positively regulate sesquiterpene biosynthesis in chrysanthemums. This study preliminarily revealed the molecular mechanism of terpenoid biosynthesis in chrysanthemum while enriching the secondary metabolism regulatory network.

Labour-saving construction of a target protein interaction network by selective culture and high-throughput sequencing.

Yeast two-hybrid (Y2H) is a classic method of screening for protein-protein interactions. However, the operation process is labor intensive and time consuming, and there is a high possibility of false positives and false negatives. Combined with wet lab operation and bioinformatics analysis, we developed a novel method of Y2H library screening using Chrysanthemum morifolium CmMPK3 as an example. The protocol can not only greatly simplify the steps of traditional Y2H library screening but also identify as many interacting proteins as possible. Furthermore, this protocol is applicable to any species, even if no genomic information is available yet.

Evaluation of a Yeast Two-Hybrid Library by High-Throughput Sequencing.

Yeast two-hybridization (Y2H) is a classical method to study protein-protein interactions in organisms, and the top limitation of Y2H is the high ratio of false negatives and false positives. The most efficient way to reduce this error is to improve the quality of the library. The traditional library quality evaluation method can only inform us of the capacity of the library and a very limited number of library insert lengths. Therefore, we developed a new method to evaluate the library by using only a 10 ng library, amplifying the inserted fragments through 15 cycles of PCR, and then carrying out high-throughput sequencing. This method can eliminate the randomness and one-sidedness of the traditional method and can be used to obtain key indicators, such as the number of inserted genes and gene abundance, to effectively evaluate the quality of the library. In addition, the new library quality assessment method can also reveal the gene sequences of species. This method is expected to greatly accelerate PPI research on nonmodel species.

Comprehensive characterization of a floral mutant reveals the mechanism of hooked petal morphogenesis in Chrysanthemum morifolium.

The diversity of form of the chrysanthemum flower makes this species an ideal model for studying petal morphogenesis, but as yet, the molecular mechanisms underlying petal shape development remain largely unexplored. Here, a floral mutant, which arose as a bud sport in a plant of the variety 'Anastasia Dark Green', and formed straight, rather than hooked petals, was subjected to both comparative morphological analysis and transcriptome profiling. The hooked petals only became discernible during a late stage of flower development. At the late stage of 'Anastasia Dark Green', genes related to chloroplast, hormone metabolism, cell wall and microtubules were active, as were cell division-promoting factors. Auxin concentration was significantly reduced, and a positive regulator of cell expansion was down-regulated. Two types of critical candidates, boundary genes and adaxial-abaxial regulators, were identified from 7937 differentially expressed genes in pairwise comparisons, which were up-regulated at the late stage in 'Anastasia Dark Green' and another two hooked varieties. Ectopic expression of a candidate abaxial gene, CmYAB1, in chrysanthemum led to changes in petal curvature and inflorescence morphology. Our findings provide new insights into the regulatory networks underlying chrysanthemum petal morphogenesis.

Comprehensive analysis of mitogen-activated protein kinase cascades in chrysanthemum.

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades, an important type of pathway in eukaryotic signaling networks, play a key role in plant defense responses, growth and development. METHODS: Phylogenetic analysis and conserved motif analysis of the MKK and MPK families in Arabidopsis thaliana, Helianthus annuus and Chrysanthemum morifolium classified MKK genes and MPK genes. qRT-PCR was used for the expression patterns of CmMPK and CmMKK genes, and yeast two-hybrid assay was applied to clear the interaction between CmMPKs and CmMKKs. RESULTS: We characterized six MKK genes and 11 MPK genes in chrysanthemum based on transcriptomic sequences and classified these genes into four groups. qRT-PCR analysis demonstrated that CmMKKs and CmMPKs exhibited various expression patterns in different organs of chrysanthemum and in response to abiotic stresses and phytohormone treatments. Furthermore, a yeast two-hybrid assay was applied to analyze the interaction between CmMKKs and CmMPKs and reveal the MAPK cascades in chrysanthemum. DISCUSSION: Our data led us to propose that CmMKK4-CmMPK13 and CmMKK2-CmMPK4 may be involved in regulating salt resistance and in the relationship between CmMKK9 and CmMPK6 and temperature stress.

The heterologous expression of a chrysanthemum TCP-P transcription factor CmTCP14 suppresses organ size and delays senescence in Arabidopsis thaliana.

TCP transcription factors are important for plant growth and development, but their activity in chrysanthemum (Chrysanthemum morifolium) has not been thoroughly explored. Here, a chrysanthemum TCP-P sequence, which encodes a protein harboring the conserved basic helix-loop-helix (bHLH) motif, was shown to be related phylogenetically to the Arabidopsis thaliana gene AtTCP14. A yeast-one hybrid assay showed that the encoding protein had no transcriptional activation ability, and a localization experiment indicated that it was localized in the nucleus. Transcription profiling established that the gene was most active in the stem and leaf. Its heterologous expression in A. thaliana down-regulated certain cell cycle-related genes, reduced the size of various organs and increased the chlorophyll and carotenoid contents of the leaf which led to delayed senescence and a prolonged flowering period. Moreover, by screening the cDNA library of chrysanthemum, we found that the CmTCP14 can interact with CmFTL2 and some CmDELLAs.