Tanacetum cinerariifolium Schultz Bip.
Creators: name: Li, Jinjin
name: Hu, Hao
name: Chen, Yu
name: Xie, Jing
name: Li, Jiawen
name: Zeng, Tuo
name: Wang, Manqun
name: Luo, Jing
name: Zheng, Riru
name: Jongsma, Maarten A.
name: Wang, Caiyun
Publisher: name: Zenodo
Year: 2021
Subjects: Biodiversity
Taxonomy
Plantae
Tracheophyta
Magnoliopsida
Asterales
Asteraceae
Tanacetum
Tanacetum cinerariifolium

description: 2.1. Morphological and anatomical analysis of T. cinerariifolium flowers
The T. cinerariifolium flower head is supported by a long peduncle rising from the base of the plant, and composed of an outer ring of white ray florets and a yellow flower heart densely populated by disk florets in the center of the receptacle (Fig. 1A). Both disk and ray florets have ribbed achenes, which are seated on the slightly convex receptacle (Fig. 1A). Each disk floret has a yellow tubular corolla opening at the top into five lobes (Fig. 1B and C). SEM observation of the longitudinal sections of involucre bracts located at the outer side of the receptacle showed that the joining site constituted mostly of parenchyma cells and vascular bundles (Fig. 1D and E). During flower development, the peduncle below the flower head coarsened with the disintegration of the parenchyma cells in the pith to support the heavier flower head (Fig. S1). First signs of disintegration of parenchyma cells in the pith were visible in the upper flower peduncle of S2 flowers (Fig. 1F and G).
2.2. Localization of terpenes in young flowers
In our previous work (Li et al., 2019), we reported that flowering T. cinerariifolium plants emitted a blend of five terpenes and one green leaf volatile in changing ratios as the flowers matured. The ratios in this blend appeared to control ladybird beetle attraction and aphid repellence. To further investigate the location and production of these terpenes, we performed hexane extraction and GC-MS analysis on selected early stage flower tissues (S0–S2) showing a significant transition in terpene emission composition and quantity. Focusing on terpenes with the terpene-specific mass 93 shows that fewer compounds were in peduncle extracts compared to the flower head (Table 1, Fig. 2A). The principal volatile terpene constituents of the flower head were a set of isomeric sesquiterpenes with very similar mass spectra (Table 1). (E)-β- farnesene (EβF) and germacrene D (GD) were the predominant molecules, accounting for 68% of total terpenes (34% EβF and 34% GD) in the S0 flower head and 87% of total terpenes (80% EβF and 7.6% GD) in the upper flower peduncle (Table 1). To our surprise, these two dominant terpene volatiles EβF and GD were not present in common storage site trichomes, so this raised the question how these terpenes were then distributed inside the flower tissues. In fact, we found that approximately 88% of the total amount of GD was concentrated inside the S2 flower head, while in contrast 71% of EβF was stored in the flower peduncle (4 cm upper and 4 cm lower peduncle together) with less trichomes (Fig. 2B). Table 1 shows how during flower development, the amount of EβF and GD in the flower head decreased relative to the strongly accumulating of pyrethrins, but maintained a constant level in the flower peduncle. Also in terms of specific weight in the flower head, EβF (257 ng per mg fresh tissue) and GD (251 ng per mg fresh tissue) similarly decreased by a factor two during S0 – S2 flower development (Fig. 2B).
2.3. Detailed EβF and GD emission and distribution patterns during flower development
The early stage flowers contained similar levels of EβF and GD in the flower head (Fig. 2B), but in the peduncle EβF levels were ~10-fold higher than GD. From our earlier work (Li et al., 2019), we were interested to understand how the change in EβF/GD emission ratio was achieved during flower development, with GD becoming the dominant volatile in the blend (Fig. 3A). In a first step, we analyzed the kinetics of EβF and GD emissions in the S0-2 flowering plants, and found that EβF emission levels peaked at 10 a.m., whereas GD emissions peaked 4 h earlier at 6 a.m. and were significantly higher than EβF at night (Fig. 3B). The differences in emissions we presumed to be partly due to differences in storage tissues and gene expression patterns.
To analyze the temporal distribution of these two sesquiterpenes in the flower and peduncle in detail, we further analyzed hexane extracts of flower heads and flower peduncles from a wider range of developmental stages (S1–S5). Whole plant EβF and GD emissions both increased during flower development (Fig. 3A), but the content per fresh weight of EβF and GD stored in the flower head decreased and remained constant in the flower peduncle (Fig. 3C–D). On a per flower basis, the content of EβF and GD in the flower head peaked at stage 2 (Table S1). The majority of this stored amount is never emitted as only a small fraction (around 1%) is measured to be released into the environment during a flower lifetime per day (Fig. 3A). 100–200 ng /h EβF or GD was released from S0-2 whole flowering plant (15–20 flowers) in a 24 h collection period, which means about 120–310 ng EβF or GD was released from a single flower per day. Yet, we found nearly 18 μg EβF and 28 μg GD to be stored in a single S2 flower head (Table S1) (Li et al., 2019).
a Percentages obtained by peak area (TIC) normalization (SH-Rsi-5sil-MS column), mean ± SE of three biological replications.
b Not detected. The bold values consist in the major constituents of the essential oils.
c Mixture of the six components of pyrethrins.
To reveal the spatial-distribution of stored EβF and GD in more detail, we next dissected the flower head into four parts (upper disk floret; lower disk floret (ovary); bract; receptacle; flower stages S1–S5), extracted the samples in hexane, and analyzed by GC-MS. EβF and GD concentrations stored in the disk flower (upper and lower parts) both decreased during flower development, but the amount of GD in the disk flower and bract was 2–3 times higher than EβF (Fig. 4). We interpret the decrease in GD and EβF to result from volatile release when the corolla of the disk floret opens although the increase in flower weight will also play a role in the decrease. The much higher GD content of the upper disk floret (GD, 1181 ng /mg fresh tissue; EβF, 478 ng /mg fresh tissue) most likely caused the higher ratio of GD/EβF during flower development (Fig. 4).
2.4. Terpene volatiles in the flower stigma and corolla
As shown above, with the opening of the disk flower, EβF and GD stored in the disk floret were released. The higher GD emission is correlated with a higher stored content in the upper disk floret. We performed a NADI staining which shows that in closed S1 and S2 disk flowers purple stained terpene oil (EβF and/or GD based on content analysis) was present in the young two-lobed stigma of the pistil in the form of two channels/cavities/secretory ducts (Fig. 5A–C and S 3). Following the flower bud development, more blue-violet stained oils (mainly pyrethrins, nearly 66%–80% of the stained terpenes) began to emerge in the ovary wall of S1 disk floret, and this proceeded further in the S3 flower (Table S2, Fig. 5A and D). In open S3 and S4 flowers, we found NADI-stained terpenes (mainly EβF and GD, 70–80% of the stained terpene oil droplets) were also pushed upward in the two lobes of the stigma and five lips of the corolla in the opened disk floret with the elongation of the style (Table S2, Fig. 5B–E and S 3). We dissected the stigma and corolla of the opened disk florets of S4 and S5 flowers and then did hexane extraction for GC-MS analysis. We found stigma extracts to contain ten times higher concentrations of EβF and GD (EβF, 169 ± 73 ng /mg fresh weight; GD, 257 ± 126 ng /mg) compared to the corolla (EβF, 17.8 ± 4.9 ng /mg; GD, 38.6 ± 10.9 ng /mg) (Fig. 6). We, therefore, propose a scenario that when the corolla opens up, the style and stigma elongates and terpenes are much promoted upwards the terminal ends of the two separating stigma lobes resulting in a release into the air, presumably with the purpose of promoting pollination.
2.5. EβF stored in the flower peduncle
EβF was much more dominant and nearly pure in the flower peduncle extract (Fig. 2A) and shown earlier to be mostly produced in cortex cells neighboring vascular bundles (Li et al., 2019). Here we looked into the question of the fate of the oil droplets produced early on during later development. Cross sections of young upper flower peduncles showed that parenchymatous cortex with chloroplasts consisting of 6–7 layers and that lamellar collenchyma is discontinuous under the epidermis (Fig. S1). The peduncle has 13–15 open collateral type vascular bundles surrounded by schlerenchyma fibres. There were secretory cavities next to the schlerenchyma fibres that were especially abundant in the upper peduncle (Fig. S2). These secretory cavities developed parallel to the vascular system and may consist of elongated cavities extending into the cortex cells (cortical canals).
In T. cinerariifolium, NADI staining of cross sections of younger flower peduncles (S0–S1 stages) showed up positive for purple-violet terpene oil droplets that largely accumulated inside cortex cells surrounding the vascular bundles (Fig. 7A) sometimes also filling parenchyma cells of the pith (Fig. 7B). When flower peduncles develop to the S2 stage, the pith in the upper peduncle disintegrates. NADI coupled with aniline blue staining revealed the presence of terpene oil droplets in the intercellular space between the two vascular bundles at that stage (Fig. 7C and D).
Moreover, we also observed very large mature secretory cavities close to the vascular bundles filled with purple-violet terpene oil (Fig. 8A and B). Strings of purple (beaded) staining were also found in the cortex cells of longitudinal sections of the flower peduncles (Fig. 8C and D). These purple-violet stained oil droplets consist mainly of EβF based on: (i) our previous gene expression study (Li et al., 2019), (ii) the chemical content of peduncles dominated by EβF (Fig. 2A), and (iii) the correspondence of the violet color with pure EβF stained by NADI reagent (Fig. 8A). It may be that these droplets (dominated by EβF) are on a path towards the very large secretory cavities around the vascular bundles to be stored and possibly transported from there.
description: Published as part of Li, Jinjin, Hu, Hao, Chen, Yu, Xie, Jing, Li, Jiawen, Zeng, Tuo, Wang, Manqun, Luo, Jing, Zheng, Riru, Jongsma, Maarten A. & Wang, Caiyun, 2021, Tissue specificity of (E) - β-farnesene and germacrene D accumulation in pyrethrum flowers, pp. 1-11 in Phytochemistry (112768) (112768) 187 on pages 2-5, DOI: 10.1016/j.phytochem.2021.112768, http://zenodo.org/record/8258947