Expression of MEP Pathway Genes and Non-volatile Sequestration Are Associated with Circadian Rhythm of Dominant Terpenoids Emission in Osmanthus fragrans Lour. Flowers

Osmanthus fragrans Lour. is one of the top 10 traditional ornamental flowers in China famous for its unique fragrance. Preliminary study proved that the terpenoids including ionone, linalool, and ocimene and their derivatives are the dominant aroma-active compounds that contribute greatly to the scent bouquet. Pollination observation implies the emission of aromatic terpenoids may follow a circadian rhythm. In this study, we investigated the variation of volatile terpenoids and its potential regulators. The results showed that both volatile and non-volatile terpenoids presented circadian oscillation with high emission or accumulation during the day and low emission or accumulation during the night. The volatile terpenoids always increased to reach their maximum values at 12:00 h, while free and glycosylated compounds continued increasing throughout the day. The depletion of non-volatile pool might provide the substrates for volatile emission at 0:00–6:00, suggesting the sequestration of non-volatile compounds acted like a buffer regulating emission of terpenoids. Further detection of MEP pathway genes demonstrated that their expressions increased significantly in parallel with the evident increase of both volatile and non-volatile terpenoids during the day, indicating that the gene expressions were also closely associated with terpenoid formation. Thus, the expression of MEP pathway genes and internal sequestration both played crucial roles in modulating circadian rhythm of terpenoid emission in O. fragrans

Induction of Tetraploid Male Sterile Tagetes erecta by Colchicine Treatment and Its Application for Interspecific Hybridization

Tagetes erecta is an annual multifunctional plant which can be cultivated under a broad range of climatic conditions. Polyploidization and interspecific hybridization are applied to facilitate breeding cultivars of T. erecta with improved ornamental qualities. Colchicine treatment to the germinating seeds was proved to be a useful tool for chromosome doubling of the male sterile two-type line ‘M525AB’, with the resulting frequency of polyploid seedlings ranging from 88.89% (following 0.05% w/v colchicine applied for a 3–6 h exposure period) to a maximum of 100.00% (following 0.1% for 3–6 h, or 0.2% for 3 h). Morphological observation, stomatal size and density analysis, flow cytometric analysis and chromosome counting were conducted to identify the tetraploid plants. Distinctive morphological changes were observed in a notable proportion of polyploid plants. The colchicine-treated polyploid T. erecta plants showed dwarfed and more robust growth, thicker, larger and greener leaves, larger inflorescences and florets. The mutant plants identified through morphological observation all aligned as polyploid plants, thus morphological observation could be an effective method for the detection of polyploidy. The polyploid plants had significant larger stomata size over the abaxial leaf surface, whereas the density of stomata distribution was remarkably reduced. The survival rate of tetraploid cuttings (i.e. 38%) was greatly reduced compared to that of diploid plants. The fertility of tetraploid plants was also decreased, as shown by cross-pollination yields. Interspecific hybridizations between colchicine-induced tetraploid plants of a male sterile T. erecta line and the naturally tetraploid fully fertile Tagetes patula species resulted in hybrid progeny. Most of these hybrids displayed the dwarfed growth stature and compact, larger-flower morphology which is the typical ideotype of herbaceous flowers. Thus, polyploidization may be employed effectively as a means to facilitate interspecific hybridization, thereby contributing significantly to the improvement of quantitative traits of Tagetes spp

TcMYB8, a R3-MYB Transcription Factor, Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium

Pyrethrins are a mixture of terpenes, with insecticidal properties, that accumulate in the aboveground parts of the pyrethrum (Tanacetum cinerariifolium). Numerous studies have been published on the positive role of MYB transcription factors (TFs) in terpenoid biosynthesis; however, the role of MYB TFs in pyrethrin biosynthesis remains unknown. Here, we report the isolation and characterization of a T. cinerariifolium MYB gene encoding a R3-MYB protein, TcMYB8, containing a large number of hormone-responsive elements in its promoter. The expression of the TcMYB8 gene showed a downward trend during the development stage of flowers and leaves, and was induced by methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA). Transient overexpression of TcMYB8 enhanced the expression of key enzyme-encoding genes, TcCHS and TcGLIP, and increased the content of pyrethrins. By contrast, transient silencing of TcMYB8 decreased pyrethrin contents and downregulated TcCHS and TcGLIP expression. Further analysis indicated that TcMYB8 directly binds to cis-elements in proTcCHS and proTcGLIP to activate their expression, thus regulating pyrethrin biosynthesis. Together, these results highlight the potential application of TcMYB8 for improving the T. cinerariifolium germplasm, and provide insight into the pyrethrin biosynthesis regulation network

CPTA treatment reveals potential transcription factors associated with carotenoid metabolism in flowers of Osmanthus fragrans

Osmanthus fragrans is one of the top ten traditional flowers in China. It is divided into three different groups according to its color. α-Carotene and β-carotene are the main determinants to distinguish the color differences between three groups. However, the dominant genes and transcription factors involved in carotenoid metabolism remain unclear. CPTA treatment (0.7 mmol · L−1) remarkably promoted lycopene, α-carotene and β-carotene contents in flowers. Transcriptome sequencing analysis revealed that CPTA treatment could trigger chain reactions in carotenoid metabolism pathway genes. Four up-regulated and 10 down-regulated transcription factors which have close association with carotenoid variation were significantly induced by CPTA treatment. The up-regulated TFs such as MYB43, MYB123, HSF, were further subjected to transcript expression determination in different cultivars with drastic colors. Among them, transcript expression of four up-regulated TFs coincided with the carotenoid accumulation in different cultivars. We selected up-regulated OfMYB43 to verify its function, which is related to stress tolerance and transcriptional regulation. Transient overexpression of OfMYB43 in O. fragrans flowers showed that it could remarkably promote the expression of PDS, ZISO, LCYE and CCD4, leading to increased accumulation of β-branch carotenoids. OfMYB43 was a potential positive regulator of carotenoid biosynthesis in O. fragrans flowers. This study provides insight into the molecular mechanism of carotenoid metabolism in O. fragrans

Comparative transcriptome analysis of different heat stress responses between self-root grafting line and heterogeneous grafting line in rose

Rose is sensitive to high temperature which will make the rose go into a semi-dormancy state. Grafting is an excellent way to enhance rose heat tolerance. Here, heat-tolerant Rosa multiflora ‘Huanong Wuci 1′ (W) and heat-sensitive Rosa chinensis ‘Old Blush’ (X) were selected as experimental materials. The RNA-seq technique was used to investigate the transcriptomes of self-root grafting line (XX0), heterogeneous grafting line (XW0), self-root grafting line under 6 h heat stress (XX6), and heterogeneous grafting line under 6 h heat stress (XW6). Under high temperature stress, multiple signaling pathways were activated, moreover, a large number of transcription factors and functional genes were induced, especially the HSFs and HSPs with remarkably upregulated expression levels. The GO analysis showed that the differences in the expressions of the genes related to fatty acids and carbohydrates were observed between self-root grafting line and heterogeneous grafting line. In addition, 14 P450s were differentially expressed, and one lectin gene was up-regulated in XW0 vs XW6, but down-regulated in XX0 vs XX6. Considering physiological and biochemical traits such as relative electrolyte leakage, SOD activity, proline, and total soluble sugars, DEGs involved in these processes may be key factors to resist high temperature. The present study provides an insight into the complex mechanism underlying grafting in response to heat stress. Our results indicate that grafting is an effective way to improve rose heat resistance

Tissue specificity of (E) -β - farnesene and germacrene D accumulation in pyrethrum flowers

Plant defensive mimicry based on the aphid alarm pheromone (E)-β-farnesene (EβF) was previously shown to operate in Tanacetum cinerariifolium (Asteraceae) flowers. Germacrene D (GD), is another dominant volatile of T. cinerariifolium flowers and may modulate both defense and pollination. Here, we find that the increase in GD/EβF ratio at later developmental stages is correlated with the tissue distribution in the flower head: the total content of EβF and GD is similar, but GD accumulates comparatively more in the upper disk florets. Naphthol and N, N-dimethyl-p-phenylenediamine dihydrochloride (NADI)-stained purple ducts containing EβF and GD, were observed in the five petal lips of the corolla and two-lobed stigma of disk florets. By contrast in the peduncle, EβF accounts for nearly 80% of total terpenes, compared to 5% for GD. EβF is accumulated inside inner cortex cells and parenchyma cells of the pith in young peduncle. This is followed by the formation of terpene-filled axial secretory cavities parallel to the vascular bundles. In conclusion, the observed developmental and diurnal emissions of different EβF/GD ratios appear to be regulated by their tissue distributio