Transcriptional Responses and GCMS Analysis for the Biosynthesis of Pyrethrins and Volatile Terpenes in Tanacetum coccineum.

Natural pyrethrins have been widely used as natural pesticides due to their low mammalian toxicity and environmental friendliness. Previous studies have mainly focused on Tanacetumcinerariifolium, which contains high levels of pyrethrins and volatile terpenes that play significant roles in plant defense and pollination. However, there is little information on T. coccineum due to its lower pyrethrin content and low commercial value. In this study, we measured the transcriptome and metabolites of the leaves (L), flower buds (S1), and fully blossomed flowers (S4) of T. coccineum. The results show that the expression of pyrethrins and precursor terpene backbone genes was low in the leaves, and then rapidly increased in the S1 stage before decreasing again in the S4 stage. The results also show that pyrethrins primarily accumulated at the S4 stage. However, the content of volatile terpenes was consistently low. This perhaps suggests that, despite T. coccineum and T. cinerariifolium having similar gene expression patterns and accumulation of pyrethrins, T. coccineum attracts pollinators via its large and colorful flowers rather than via inefficient and metabolically expensive volatile terpenes, as in T. cinerariifolium. This is the first instance of de novo transcriptome sequencing reported for T. coccineum. The present results could provide insights into pyrethrin biosynthetic pathways and will be helpful for further understanding how plants balance the cost-benefit relationship between plant defense and pollination.

Ribozyme-mediated CRISPR/Cas9 gene editing in pyrethrum (Tanacetum cinerariifolium) hairy roots using a RNA polymerase II-dependent promoter.

BACKGROUND: Traditional CRISPR/Cas9 systems that rely on U6 or U3 snRNA promoters (RNA polymerase III-dependent promoters) can only achieve constitutive gene editing in plants, hampering the functional analysis of specifically expressed genes. Ribozyme-mediated CRISPR/Cas9 systems increase the types of promoters which can be used to transcribe sgRNA. Therefore, such systems allow specific gene editing; for example, transcription of the artificial gene Ribozyme-sgRNA-Ribozyme (RGR) is initiated by an RNA polymerase II-dependent promoter. Genetic transformation is indispensable for editing plant genes. In certain plant species, including pyrethrum, genetic transformation remains challenging to do, limiting the functional verification of novel CRISPR/Cas9 systems. Thus, this study's aim was to develop a simple Agrobacterium rhizogenes-mediated hairy root transformation system to analyze the function of a ribozyme-mediated CRISPR/Cas9 system in pyrethrum. RESULTS: A hairy root transformation system for pyrethrum is described, with a mean transformation frequency of 7%. Transgenic hairy roots transformed with the pBI121 vector exhibited significantly increased beta-glucuronidase staining as a visual marker of transgene expression. Further, a ribozyme-based CRISPR/Cas9 vector was constructed to edit the TcEbFS gene, which catalyzes synthesis of the defense-related compound (E)-ß-farnesene in pyrethrum. The vector was transferred into the hairy roots of pyrethrum and two stably transformed hairy root transgenic lines obtained. Editing of the TcEbFS gene in the hairy roots was evaluated by gene sequencing, demonstrating that both hairy root transgenic lines had DNA base loss at the editing target site. Gas chromatography-mass spectrometry showed that the (E)-ß-farnesene content was significantly decreased in both hairy root transgenic lines compared with the empty vector control group. Altogether, these results show that RGR can be driven by the CaMV35S promoter to realize TcEbFS gene editing in pyrethrum hairy roots. CONCLUSION: An A. rhizogenes-mediated hairy root transformation and ribozyme-mediated CRISPR/Cas9 gene editing system in pyrethrum was established, thereby facilitating gene editing in specific organs or at a particular developmental stage in future pyrethrum research.

Analysis of aroma-active compounds in three sweet osmanthus (Osmanthus fragrans) cultivars by GC-olfactometry and GC-MS.

OBJECTIVE: Aroma is the core factor in aromatherapy. Sensory evaluation of aromas differed among three sweet osmanthus (Osmanthus fragrans) cultivar groups. The purpose of this study was to investigate the aroma-active compounds responsible for these differences. METHODS: Gas chromatography-olfactometry (GC-O) and GC-mass spectrometry (GC-MS) were used to analyze the aroma-active compounds and volatiles of creamy-white ('Houban Yingui', HBYG), yellow ('Liuye Jingui', LYJG), and orange ('Gecheng Dangui', GCDG) cultivars. RESULTS: Seventeen aroma-active compounds were detected among 54 volatiles. trans-ß-Ocimene, trans-ß-ionone, and linalool, which were major volatiles, were identified as aroma-active, while cis-3-hexenyl butanoate, γ-terpinene, and hexyl butanoate were also aroma-active compounds, although their contents were low. Analysis of the odors was based on the sum of the modified frequency (MF) values of aroma-active compounds in different odor groups. HBYG contained more herb odors, contributed by cis-ß-ocimene and trans-ß-ocimene, while LYJG had more woody/violet/fruity odors released by trans-ß-ionone, α-ionone, and hexyl butanoate. In GCDG, the more floral odors were the result of cis-linalool oxide, trans-linalool oxide, and linalool. CONCLUSIONS: Aroma-active compounds were not necessarily only the major volatiles: some volatiles with low content also contributed to aroma. The aroma differences among the three cultivars resulted from variation in the content of different odor groups and in the intensities of aroma-active compounds.

Chlorocholine chloride and paclobutrazol treatments promote carbohydrate accumulation in bulbs of Lilium Oriental hybrids 'Sorbonne'.

The present study was to test the hypothesis that the plant growth retardants chlorocholine chloride (CCC) and paclobutrazol (PBZ) could improve the carbohydrate accumulation in lily bulbs by enhancing photosynthetic capacity and changing endogenous hormones. Plants of Lilium Oriental hybrids 'Sorbonne' were treated with a foliar spray of CCC or PBZ (both at 300 mg/L) solution, at six weeks after planting (6 WAP). The morphological parameters, endogenous hormone contents (gibberellic acid (GA), abscisic acid (ABA), and indole-3-acetic acid (IAA)), and carbohydrate contents were measured from 6 to 18 WAP, at 2-week intervals. The results showed that CCC increased the biomass of leaves and stems which might produce more photoassimilates available for transportation and utilization. However, PBZ treatment suppressed vegetative growth and favored photoassimilate transportation into bulbs. A slight delay of bud and anthesis formation was observed in both treated plants. CCC and PBZ treatments substantially enhanced the sucrose contents in leaves probably due to the increase of chlorophyll contents. Treatment with CCC or PBZ decreased GA but increased IAA contents in lily bulbs which might stimulate starch accumulation and formation of new scales. Our experiment suggested that CCC or PBZ treatment is an effective method to promote carbohydrate accumulation in lily bulbs.