Glandular secretory trichomes formation mechanism study on synthesis and storage of terpenoids in lavender

doi:10.21203/rs.3.rs-2695243/v1

Background: Lavender belongs to the family Lamiaceae and is an aromatic plant that is widely grown as an ornamental plant. The chemical components of lavender are characterized by the presence of monoterpenoids and sesquiterpenoidsand other irregular types. These main compounds are primarily synthesized and stored in an epidermal secretory structure, glandular secretory trichomes (GSTs).

Results: Volatile organic compounds (VOCs) are responsible for the aroma characteristics of plant oil that drive consumer preference. It is usually regarded as a characteristic character in the classification of aromatic plants. Interestingly, VOCs are synthesized and stored in GSTs. In this study, we measured the VOCs of four lavenders by HS-SPME-GC-MS. Among them, 66 volatile organic compounds were identified, flowers were the main site of accumulation of VOCs and its prominent components were linalyl acetate and linalool. In Lamiaceae, GSTs usually include peltate glandular trichomes (PGTs) and capitate glandular trichomes (CGTs), like Perilla frutescens, peppermint, Ocimum basilicum, thyme, oregano, etc. But there were no reports about GSTs in lavender until now. We also examined the developmental processes of PGTs, it included the head and the stalk region, the head cells have a secretory, VOCs are produced by these secretory cells. Based on a reference genome from ‘Jingxun 2’, several genes related to GSTs belonged to R2R3-MYB subfamily had been identified. These results will give a directive sense for GSTs engineering and molecular breeding of lavender for targeting VOCs.

Conclusions: In this study, we used HS-SPME-GC/MS to identify VOCs of these lavenders. We analyzed the formation of GSTs in lavender and compared quantity and diameter size of four lavenders. In the meanwhile, we found four candidate genes belonging to R2R3-MYB family.

Lavender

Volatile organic compounds (VOCs)

Glandular secretory trichomes

Formation

The genus Lavandula of the Lamiaceae family comprises about 39 species. Among plants of this genus, L. angustifolia, L. latifolia, and their natural sterile hybrid L. × intermedia, are now cultivated worldwide and are widely used in perfumes, cosmetics, pharmaceuticals, and more fields recently, in aromatherapy products [1]. Lavender belongs to the family Lamiaceae and is an aromatic plant that is widely grown as an ornamental plant. [2–4]. Lavender ‘Jingxun 1’ and ‘Jingxun 2’ are improved varieties selectively to breed by Institute of Botany, Chinese Academy of Sciences with flower yield, yield of essential oil, and other factors as evaluation indexes (Fig. 1a and b). The color of the sepal of ‘Jingxun1’ is lighter than that ‘Jingxun 2’ (Fig. 1c-f). ‘Jingxun 2’ is characterized by high levels of linalyl acetate and linalool and low amounts of camphor in its essential oil, making it the most valued lavender oil additive to many over-the-counter complementary medicines and cosmetic products in China [5].

Lavender is also an important spice appreciated for its aroma. Volatile compounds of lavender contribute significantly to the aroma [6]. Volatile organic compounds (VOCs) are responsible for the aroma characteristics of plant oil that drive consumer preference. VOCs play a number of biotic roles, such as acting as cues between plants and other organisms (herbivores, pathogens, pollinators, parasitoids, and plants) [7]. Given their roles in plant survival and their environmental adaptations, they are of major interest in chemical ecology. The main groups of volatiles present in aromatic plants are terpenes like monoterpenes and sesquiterpenes. Aldehydes, alcohols, ketones, esters, and hydrocarbons create other part of aroma. Therefore there is much interest in the volatile organic compounds of lavender.

Trichomes are specialized tissues from epidermal cells with diverse structures and functions. According to their cell number and secretory ability, trichomes can be categorised into unicellular and multicellular or secretory and non-secretory types [8, 9]. Though unicellular trichomes have no secretory function, they can protect against natural hazards, such as herbivores, ultraviolet (UV) irradiation, pathogen attacks, excessive transpiration, seed spread, and seed protection [10–14]. Arabidopsis thaliana is a kind of typical unicellular nonglandular trichomes model plant. Glandular secretory trichomes can synthesize, store, or secrete large amounts of bioactive metabolites, such as terpenoids, phenylpropanoids, flavonoids, alkaloids, and acyl sugars [15]. Five plant species like tomato (Solanum lycopersicum), cucumber (Cucumis sativus), sweet wormwood (Artemisia annua), tobacco (Nicotiana tabacum), and cotton (Gossypium hirsutum) are key working materials for GSTs these years [16]. In Lamiaceae, GSTs usually include two kinds of trichomes, namely, peltate glandular trichomes (PGTs) and capitate glandular trichomes (CGTs), like Perilla frutescens [17], peppermint [18], Ocimum basilicum [19], thyme [20], and oregano [21], ect.

In this study, we selected four different varieties of lavenders and measured the volatile components of their leaves and flowers by HS-SPME-GC-MS. Meanwhile, we observed their fresh leaves and flowers by fluorescence microscopy and scanning electron microscopy, and calculated glandular trichomes. Morphogenetic process of peltate glandular trichomes was elucidated here. Based on a reference genome from ‘Jingxun 2’ [5], several genes related to glandular trichomes belonged to R2R3-MYB subfamily had been identified. These results will give a directive sense for GSTs engineering and molecular breeding of lavender.

Identification of volatile organic compounds (VOCs) profiling

A total of 66 volatile organic compounds (VOCs) were identified in the four lavender varieties by HS-SPME-GC/MS. The VOCs were mainly divided into monoterpenoids and sesquiterpenoids (Fig. 2a). In monoterpenoids, monoterpenes (10), and monoterpene oxides (10) had the same proprotion (14.49%). Besides, monoterpene alcohols (8) accounted for 11.59%, the rest of compounds were monoterpene esters (2), aldehydes (1), and ketones (4). Sesquiterpenes (16) accounted for the vast majority of sesquiterpenoids, there were no esters, aldehydes, and ketones in this group (Fig. 2b). Principal component analysis (PCA) is a technique for showing the general metabolic differences between groups and variation within the group. Then we performed PCA to further classify these samples (Fig. 2c). The results showed that the first principal component (PC1) could explain 42.00% of the total variance and separated samples. The second principal component (PC2) could explain 23.00% of the total variance. The samples with three replicates were gathered into each group and significantly varied among different groups among different lavenders. The VOCs of flowers in four lavenders were relatively close, but the VOCs of leaves had large variation. Therefore, these results suggested that the compounds of different varieties of plants had variation, the compounds of different tissue parts of the same varieties also were disparate.

Hierarchal clustering of the VOCs profile was performed, and the results are shown in Fig. 2d. There were three main clusters in these samples. For leaves, ‘Jingxun 1’ and ‘Jingxun 2’ were clustered into a group, ‘Luoshen’ and ‘Taikonglan’ were clustered into another group. For flowers, these four lavenders were clustered into one group. The results were in accord with PCA. It indicated that VOCs of flowers and leaves in lavenders were very important for elucidating the mechanisms of aroma variation. Accordingly, the details of these differentially expressed VOCs were analyzed in this study.

The chemical compositions of four lavenders are shown in Table 1. The prominent components were linalyl acetate and linalool in flowers. They were characteristic constituents. Linalyl acetate in ‘Jingxun 2’, ‘Luoshen’ and ‘Taikonglan’ were 85.16 mg/g, 93.19 mg/g and 88.28 mg/g, but the content was only 4.01 mg/g in ‘Jingxun 1’. Interestingly, no matter what kind of components of leaves in ‘Jingxun 2’ and ‘Luoshen’, the contents were all below 1.00 mg/g. These indicated that flowers were the main site of accumulation of VOCs. Besides, lavandulol acetate and β-caryophyllene placed a great deal of weight (Fig. 2e).

Table 1 Terpenoid components of leaves and flowers in four lavenders.

No.	RT	RI	Terpenoid	‘Jingxun 1’ (mg/g)		‘Jingxun 2’ (mg/g)		‘Luoshen’ (mg/g)		‘Taikonglan’ (mg/g)
No.	RT	RI	Terpenoid	Flower	Leaf	Flower	Leaf	Flower	Leaf	Flower	Leaf
1	5.176	929	α-Thujene	-	0.12 ± 0.02	-	-	-	-	-	-
2	5.234	937	α-Pinene	-	-	-	0.02 ± 0.01	-	-	-	-
3	5.515	952	Camphene	-	0.12 ± 0.02	-	0.08 ± 0.04	-	0.08 ± 0.02	-	0.12 ± 0.02
4	6.111	980	1-Octen-3-ol	-	0.25 ± 0.04	2.25 ± 0.17	0.03 ± 0.01	1.31 ± 0.03	0.02 ± 0.00	0.96 ± 0.06	0.02 ± 0.00
5	6.465	991	β-Myrcene	-	-	1.25 ± 0.26	-	-	-	-	-
6	6.975	1011	3-Carene	-	0.09 ± 0.00	-	0.01 ± 0.01	-	-	-	-
7	6.975	1011	Hexyl acetate	-	-	2.95 ± 0.46	-	-	-	-	-
8	7.286	1023	β-Cymene	-	1.92 ± 0.93	-	0.06 ± 0.03	-	0.08 ± 0.00	-	0.10 ± 0.00
9	7.324	1025	p-Cymene	-	-	1.50 ± 0.00	0.16 ± 0.08	-	0.21 ± 0.01	-	0.29 ± 0.01
10	7.423	1030	Limonene	0.10 ± 0.04	1.75 ± 0.80	3.68 ± 0.19	0.16 ± 0.00	2.23 ± 0.54	-	2.16 ± 0.16	0.07 ± 0.03
11	7.503	1032	Eucalyptol	0.19 ± 0.07	0.87 ± 0.13	6.45 ± 0.27	0.30 ± 0.06	8.89 ± 0.69	1.77 ± 0.31	7.78 ± 0.52	2.12 ± 0.19
12	7.632	1037	β-cis-Ocimene	1.47 ± 0.22	-	34.56 ± 4.50	-	11.66 ± 0.52	-	9.15 ± 1.06	-
13	7.875	1049	β-trans-Ocimene	-	-	-	-	1.70 ± 0.03	-	1.57 ± 0.06	-
14	8.388	1070	trans-Sabinene hydrate	-	0.08 ± 0.04	-	-	-	-	-	-
15	8.419	1067	cis-Sabinene hydrate	-	-	-	0.01 ± 0.01	-	-	-	-
16	8.559	1074	Linalool oxide	-	-	-	-	-	0.02 ± 0.00	-	-
17	8.856	1093	Rosefuran	-	0.09 ± 0.00	-	0.01 ± 0.00	-	-	-	-
18	8.977	1086	trans-Linalool oxide	-	-	-	-	-	0.02 ± 0.00	-	-
19	9.305	1099	Linalool	2.43 ± 0.21	-	49.03 ± 6.03	-	51.97 ± 1.94	-	47.11 ± 2.57	-

Continued Table 1

No.	RT	RI	Terpenoid	‘Jingxun 1’ (mg/g)		‘Jingxun 2’ (mg/g)		‘Luoshen’ (mg/g)		‘Taikonglan’ (mg/g)
No.	RT	RI	Terpenoid	Flower	Leaf	Flower	Leaf	Flower	Leaf	Flower	Leaf
20	9.571	1111	Amyl vinyl carbinol acetate	0.23 ± 0.03	0.48 ± 0.05	9.29 ± 1.87	0.05 ± 0.02	7.02 ± 0.58	0.23 ± 0.06	5.92 ± 0.06	0.16 ± 0.01
21	9.875	1122	cis-2-Menthenol	-	0.08 ± 0.07	-	0.01 ± 0.00	-	-	-	-
22	10.231	1134	Limonene oxide, cis-	-	0.18 ± 0.09	-	-	-	-	-	-
23	10.251	1144	trans-Verbenol	-	-	-	0.01 ± 0.01	-	-	-	-
24	10.303	1138	Limonene oxide, trans-	-	0.10 ± 0.01	-	0.01 ± 0.01	-	-	-	-
25	10.509	1145	Camphor	-	0.69 ± 0.02	1.49 ± 0.51	0.08 ± 0.04	1.62 ± 0.24	0.80 ± 0.10	1.55 ± 0.09	0.74 ± 0.06
26	11.075	1167	endo-Borneol	0.18 ± 0.06	1.78 ± 0.30	7.84 ± 0.89	0.20 ± 0.09	4.30 ± 0.40	0.90 ± 0.10	3.78 ± 0.28	1.03 ± 0.05
27	11.208	1174	(3E,5Z)-1,3,5-Undecatriene	0.04 ± 0.00	-	1.54 ± 0.10	-	-	-	-	-
28	11.398	1177	Terpinen-4-ol	0.54 ± 0.06	-	1.85 ± 0.36	0.01 ± 0.00	11.29 ± 0.63	-	9.52 ± 0.84	-
29	11.485	1183	p-Cymen-8-ol	-	0.21 ± 0.00	-	0.03 ± 0.01	-	0.05 ± 0.00	-	0.06 ± 0.01
30	11.581	1184	Cryptone	0.08 ± 0.03	1.02 ± 0.01	2.78 ± 0.20	0.11 ± 0.05	1.33 ± 0.10	0.06 ± 0.01	1.36 ± 0.14	0.11 ± 0.03
31	11.756	1192	Hexyl butyrate	0.04 ± 0.00	-	4.26 ± 0.46	-	2.25 ± 0.01	-	2.24 ± 0.11	-
32	11.76	1189	α-Terpineol	-	0.07 ± 0.00	-	0.01 ± 0.01	-	-	-	-
33	12.212	1208	trans-Piperitol	-	-	-	0.01 ± 0.00	-	-	-	-
34	12.269	1205	Verbenone	-	-	-	0.01 ± 0.00	-	-	-	-
35	12.634	1243	Eucarvone	-	-	-	0.01 ± 0.00	-	-	-	-
36	12.805	1232	Isobornyl formate	-	0.30 ± 0.01	-	0.04 ± 0.02	-	0.17 ± 0.03	-	0.16 ± 0.03
37	13.089	1239	Cuminal	-	0.57 ± 0.09	1.13 ± 0.10	-	-	0.06 ± 0.00	0.97 ± 0.12	0.11 ± 0.02
38	13.192	1242	Carvone	-	0.28 ± 0.02	-	0.03 ± 0.01	-	-	-	0.04 ± 0.01

Continued Table 1

No.	RT	RI	Terpenoid	‘Jingxun 1’ (mg/g)		‘Jingxun 2’ (mg/g)		‘Luoshen’ (mg/g)		‘Taikonglan’ (mg/g)
No.	RT	RI	Terpenoid	Flower	Leaf	Flower	Leaf	Flower	Leaf	Flower	Leaf
39	13.481	1253	Piperitone	-	0.19 ± 0.00	-	0.01 ± 0.00	-	0.02 ± 0.00	-	0.03 ± 0.01
40	13.546	1257	Linalyl acetate	4.01 ± 0.55	-	85.16 ± 5.28	-	93.19 ± 5.16	-	88.28 ± 0.07	-
41	14.344	1285	Bornyl acetate	-	0.07 ± 0.00	1.81 ± 0.10	0.05 ± 0.03	1.14 ± 0.13	0.09 ± 0.01	1.12 ± 0.04	0.12 ± 0.02
42	14.42	-	Lavandulol acetate	0.95 ± 0.02	0.94 ± 0.05	49.81 ± 3.85	0.36 ± 0.28	23.28 ± 1.83	-	25.28 ± 2.65	0.07 ± 0.03
43	16.104	1351	α-Cubebene	-	-	-	0.01 ± 0.00	-	-	-	-
44	16.35	1364	Nerol acetate	-	0.13 ± 0.03	1.19 ± 0.11	0.01 ± 0.01	-	-	-	-
45	16.738	1376	Copaene	-	0.26 ± 0.07	-	-	-	-	-	-
46	16.841	1382	Geranyl acetate	0.06 ± 0.01	0.16 ± 0.01	1.57 ± 0.20	0.16 ± 0.03	1.20 ± 0.05	-	1.25 ± 0.15	-
47	16.894	1384	Hexyl hexoate	0.04 ± 0.00	-	0.90 ± 0.06	-	-	-	-	-
48	17.708	1411	α-Cedrene	-	-	-	0.02 ± 0.01	-	-	-	-
49	17.711	1415	cis-α-Bergamotene	-	0.18 ± 0.02	-	-	-	-	-	-
50	17.844	1420	Santalene	-	4.93 ± 0.11	-	0.36 ± 0.05	-	-	-	0.04 ± 0.01
51	17.871	1425	β-Caryophyllene	0.91 ± 0.05	-	29.44 ± 3.61	-	19.24 ± 0.60	-	17.56 ± 2.48	-
52	18.217	1435	α-Bergamotene	0.04 ± 0.00	-	1.93 ± 0.15	-	-	-	-	-
53	18.22	1441	Coumarin	-	1.61 ± 0.42	-	0.36 ± 0.07	-	0.43 ± 0.08	-	0.22 ± 0.10
54	18.471	1449	α-Himachalene	-	0.26 ± 0.07	-	0.02 ± 0.01	-	-	-	-
55	18.528	1448	Epi-β-Santalene	-	0.21 ± 0.01	-	0.02 ± 0.01	-	-	-	-
56	18.703	1457	(E)-β-Famesene	0.95 ± 0.06	-	18.60 ± 2.59	-	6.67 ± 0.66	-	6.49 ± 0.73	-
57	18.856	1462	β-Santalene	-	-	-	0.01 ± 0.00	-	-	-	-

Continued Table 1

No.	RT	RI	Terpenoid	‘Jingxun 1’ (mg/g)		‘Jingxun 2’ (mg/g)		‘Luoshen’ (mg/g)		‘Taikonglan’ (mg/g)
No.	RT	RI	Terpenoid	Flower	Leaf	Flower	Leaf	Flower	Leaf	Flower	Leaf
58	19.395	1481	Germacrene D	0.23 ± 0.02	-	11.12 ± 1.70	-	4.22 ± 0.29	-	4.14 ± 0.60	-
59	19.961	1508	α-Farnesene	-	-	-	-	-	-	-	-
60	20.177	1513	γ-Cadinene	-	0.51 ± 0.02	1.33 ± 0.05	0.28 ± 0.07	3.94 ± 0.92	0.06 ± 0.01	4.02 ± 0.10	0.12 ± 0.05
61	20.269	1508	α-Chamigrene	-	-	-	0.01 ± 0.00	-	-	-	-
62	20.383	1523	Calamenene	-	-	-	0.01 ± 0.00	-	-	-	-
63	20.618	1681	Sandal	-	0.85 ± 0.04	-	-	-	-	-	-
64	21.835	1581	Caryophyllene oxide	0.16 ± 0.02	0.31 ± 0.09	3.60 ± 0.14	0.38 ± 0.01	1.20 ± 0.06	0.06 ± 0.02	1.37 ± 0.29	0.13 ± 0.03
65	23.115	1646	Cadinol	-	0.12 ± 0.01	-	-	2.13 ± 0.59	-	2.07 ± 0.28	0.05 ± 0.02
66	24.895	1732	Herniarin	-	0.25 ± 0.00	-	-	-	0.06 ± 0.02	-	0.04 ± 0.02

Morphology and development of the glandular trichomes on L. angustifolia

The surface of the leaves is covered with glandular trichomes (GTs) of three types, peltate, capitate, and non-glandular trichomes (Fig 3a-h). They are located on the sepals (Supplementary Fig. S1), adaxial and abaxial side of leaves. But there is only capitate glandular trichomes on the petals, there were no peltate glandular trichomes on the petals (Fig. 3c). Peltate and capitate glandular trichomes all contained three parts including basal cells, stalk cells, and head cells, like GTs in other Lamiaceae plants [22, 23]. The gland is responsible for the secretion of specialized metabolites, the stalk is the structure bearing the gland, and the base connects the stalk to surrounding epidermal cells.

These three types of GTs not only situated on leaves and flowers, but also distributed on stems slightly, which were captured using a stereomicroscope (Supplementary Fig. S2). PGTs consisted of multicellular heads, one stalk cell, and one basal cell, which consist of eight cell disk-shaped head like orange segments and assumed a globular dome shape at maturity (Supplementary Fig. S3). CGTs were smaller than PGTs, they included a basal cell, a stalk cell, and a head of one to 2-4 cells, with the length of the stalk more than half the height of the head (Fig. 3d, e). PGTs and CGTs both have a storage cavity where secreted metabolites often accumulate in them. The cavity can be subcuticular: in that case, molecules secreted at the top of gland cells accumulate under the cuticle, which is gradually pushed away from the cell wall, as seen in A. annua glandular trichomes [24]. Non-glandular trichomes were dendritic: they had 4-6 arms branching off from the basal region (Fig. 3g, h). These trichomes were ubiquitous and their length was variable, they located on leaves, sepals, and stems densely.

We examined the developmental processes of PGTs. Trichome initiation occured when anepidermal cell acquired a trichome identity according to signals received from surrounding cells. This cell then underwent tightly controlled cell divisions from a periclinal bipartition to two cells (Fig. 4a, b); this was followed by cell divisions to form the head region and the stalk (Fig. 4c-e). On the longitudinal section of the peltate trichome, it was seen that the head cells had a dense cytoplasm (Fig. 4f-h) called secretory. The secretory cells were arranged radially. The main feature of the neck cell was the development of a fully cutinized lateral wall, resembling a Casparian strip (Fig. 4g-h). The large lipid inclusions were both in the secretory cells and in the neck cell. At the last stage, cuticle had formed (Fig. 4i). In this trichome type, essential oil were produced by these secretory cells, consisting of eight arranged on a plane with the storage space located above them, designated as subcuticular.

Comparison of quantity and diameter size of peltate glandular trichomes of four lavender varieties

We use fluorescence microscope to observe the glandular trichomes on leaves and flowers of five lavender varieties. The number of peltate glandular trichomes was significantly different in different organs and varieties (Fig. 5). The numbers of PGTs on abaxial and adaxial surface on leaves of ‘Jingxun 1’and ‘Jingxun 2’ were higher than ‘Luoshen’ and ‘Taikonglan’, especially on the abaxial surface of the leaves (Fig. 5a). The total number of PGTs on leaves of ‘Jingxun 1’ was maximum, the number of PGTs on leaves of ‘Taikonglan’ was minimum, meanwhile its number of PGTs on abaxial or adaxial surface was least. No matter what variety, the number of PGTs on abaxial surface of leaves was often more than on adaxial surface (Fig. 5b). These results indicated that trichome density is varieties and plant organ-specific. The diameter of PGTs on sepal was larger than that on leave, but there were no obvious differences between four lavender varieties (Fig. 5c). The average diameters of PGTs on sepal varied from 94.56 to 101.17 μm, the smallest diameter is about 54.41 μm (on the adaxial surface), the largest diameter is about 109.32 μm (on the sepal) (Supplementary Table S1). These suggested that lavender's essential oil came mainly from flowers.

Identification and characterization of R2R3-MYB-coding genes involved in the plant tissue development

We screened the ‘Jingxun 2’ genome database [5] and identified 51 R2R3-MYB proteins by performing a BLASTP analysis with A. thaliana, A. annua, Petunia hybrida, Antirrhinum majus, G. hirsutum, Medicago truncatula, Salvia miltiorrhiza, Solanum lycopersicum, Camellia sinensis, and Lactuca sativa R2R3-MYB proteins. Combined with transcriptome database [25], we found several genes, La23G02448, La22G02482, La19G00023, and La04G00020, which had high expression in flower (Fig 6a). Phylogenetic analysis (Fig. 6b) of our identified candidate MYBs showed that these genes clustered with the AaMYB1 and AtMYB61 that have a known function in terpenoid metabolism and trichome development in A. annua and A. thaliana [26]. Further analysis indicated that these genes also clustered with MYB factors from other species. Therefore, these genes were selected as a candidate gene for further investigation.

The amino acid alignment was performed against all of the proteins clustering with these four genes. High sequence conservation was noted at the N-terminus within the two MYB repeat domains known to be important for DNA binding (Fig. 6c). The presence of signature domain ‘GIDP_XTHKP_XSEV’or‘DVF_XKDLQRMA’ demonstrated that they belonged to the subgroup 13 of R2R3-MYB factors [27]. Though previous studies reported that AtMYB61 played a pleiotropic role, influencing lignin deposition [28], mucilage production [29], and stomatal aperture [30]. AtMYB1 and AtMYB61 induced trichome initiation and branching in A. thaliana [26]. Here, we speculated these four candidate genes may relate to regulation of cell fate as the same as subgroup 9.

Volatile organic compounds (VOCs) constituted a large and diverse category of plant secondary metabolites, the main function of these compounds is to protect plants from herbivores and pathogenic microorganism [31–33]. Terpenoids have different biological activities [34]. Multiple studies have indicated that in general, monoterpenoids are more abundant than sesquiterpenoids in the germplasm of Chrysanthemum indicum, regardless of the geographical origin [35–39]. In this study, monoterpenoids were also most abundant compounds in lavender. Particularly, in our four lavenders, flowers were the main site of accumulation of VOCs. Plants synthesize an arsenal of terpenoid compounds that serve as a defense against pathogens or herbivores; in particular, flowering plants exhibit an unusually high number of terpenoids [40]. It is similar with our study. The previous study showed that inflorescences with sequential and successive blossoms leads to long flowering-life and lasting attraction to bees. Numerous and diverse volatiles emitted by flowers act as long-distance signals and important cues for attracting pollinators to spread pollen [41, 42]. These evidence all indicated that the flowers of lavender are major organs that release volatile chemicals.

It has been reported that the primordial cells of GSTs originate from the epidermis. After the meristematic cells are produced, they protrude to the outside and divide into basal cells and apical cells first, and then apical cells divide into stalk cells and head cells [43, 44]. Different stem cell shapes lead to GSTs transferring into different types. Tomato, cucumber, sweet wormwood, tobacco, and cotton have led to significant advances in our understanding of GSTs morphology and developmental biology [16]. In Artemisia annua, the GSTs were composed of two basal cells, two stem cells, four near-head cells, and two head cells. These ten cells were arranged in a double row [45]. In Artemisia argyi, morphogenetic process of GSTs and trichomes had been elucidated, it had two types of GSTs, GST-Ⅰ was composed of 10 biserial cells, GST-Ⅱ was composed of 5 uniserial cells [46]. Though the types of GSTs in these two species were different, they had the same pattern on the development of GSTs. Besides, in Lamiaceae, the pattern of peltate glandular trichomes initiation in peppermint (Mentha × piperita) leaves had been defined. In accordance with our data, PGTs consisted of multicellular heads, one stalk cell, and one basal cell, which consist of eight cell disk-shaped head like orange segments and assumed a globular dome shape at maturity which was the same as peppermint [47]. In number species of Lamiaceae, peltate and small capitate trichomes are developed on the leaves and bracts, while the large capitate trichomes present on the calyx [48]. Trichomes of several types can also exist on the same tissue. In this study, we found lavender had three types trichomes and they distributed on leaves and flowers including bract and calyx (Fig. 2). These results were same as the thyme whose GSTs were loacted mainly in leaves and flowers [20].

Glandular trichome initiation is a complex process (Fig. 7A). A differentiating protodermal cell integrates both environmental and endogenous signals. Molecular data pointing to genes playing a specific role in glandular trichome development already exist, especially concerning some transcription factors, cell cycle regulators, as well as receptors involved in phytohormone-induced signaling cascades (Fig. 7B). Some genes involved in the development of GSTs had been identified: in sweet wormwood, TAR2 had GST morphogenesis function, AaMYB1 and AaMIXTA1, AaHD1 can directly regulate the formation of GSTs, besides AaMIXTA1 can interact with AaHD8 forming a regulatory complex that promotes AaHD1 expression and positively regulate the initiation of GSTs [49–51]; in tomato, SlMYC1 can regulate type Ⅵ formation, SlCycB2 and Hair can respectively interact with Woolly as an important regulator of GSTs [52–54]; in tabacco, NbGIS regulates the trichome initiation and acts upstream of NbMYB123-like [55]. On the contrary, NbCyCB2 can inhibit trichome initiation by binding to the LZ domain of NbWo [56]. In this study, we did not focus on negative regulation factors to GSTs.

In this study, we explained the GSTs formation mechanism in lavender, its PGTs included the head and the stalk region, the head cells have a secretory, VOCs are produced by these secretory cells. We also compared quantity and diameter size of peltate glandular trichomes of four varieties, and found the numbers of PGTs on abaxial and adaxial surface on leaves of ‘Jingxun 1’ and ‘Jingxun 2’ were higher than ‘Luoshen’ and ‘Taikonglan’, especially on the abaxial surface of the leaves. In addition, we identified 66 volatile organic compounds in these four lavenders, the results indicated flowers were the main site of accumulation of VOCs and its prominent components were linalyl acetate and linalool. In the end, we selected genes belonging to R2R3-MYB family, such as La23G02448, La22G02482, La19G00023, and La04G00020, may regulate glandular trichomes development in lavender. This study provided new insights into the morphology of GSTs. In the future, further research is required to reveal a novel molecular mechanism of the regulatory networks involved in the formation of GSTs in lavender.

Materials and methods

Plant materials

The plant used in this study were ‘Jingxun 1’, ‘Jingxun 2’, ‘Luoshen’ and ‘Taikonglan’. ‘Jingxun 1’, ‘Jingxun 2’, and ‘Luoshen’ were three varieties which were bred by our own research team. ‘Taikonglan’ was a business variety maintained by our own research team. They were cultivated in the Institute of Botany, Chinese Academy of Sciences (IB-CAS), Beijing, China.

HS-SPME procedure

The HS-SPME parameters are described in detail as follows: about 0.10 g of flower samples and 0.15 g of leave samples were weighed and immediately placed into a 20 ml head-space vial (Agilent, Palo Alto, CA, USA) containing 20 μl of internal standard solution (7.5 mg/ml, 3-Octanol, Cas#589-98-O, Aladdin, Shanghai, China). The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent, Palo Alto, CA, USA). Subsequently, each vial was immediately incubated at 65 ℃ for 30 min, then a 100 μm coating fiber divinylbenzene/carboxen/polydimethylsilioxan (Supelco, Inc., Bellefonte, PA, USA) was exposed to the headspace to absorb the volatiles for 30 min at 65 ℃, split ratio was 70:1. All the volatile components on the coating fiber were then analyzed using gas chromatography.

GC-MS analysis

A Model 7890B GC instrument (Agilent , Palo Alto, CA, USA) and a 7000D mass spectrometer (Agilent, Palo Alto, CA, USA) were used to perform GC-MS analysis. GC-MS analysis was performed using the following conditions: after microextraction, desorption of the volatile organic compounds from the coating fiber was performed in the injection port of the GC apparatus at 250 ℃ for 4 min in splitless mode. A DB-5MS (5% phenyl-polymethylsiloxane, 30 m × 0.25 mm × 1.0 μm) capillary column was used for identification and quantification. Carrier gas was 99.999% helium at a column flow of 1.0 ml/ min. The front injector temperature was kept at 250 ℃ and the detector was kept at 280 ℃. The column temperature program was set to 60 ℃ hold on 1 min and then raised to 240 ℃ at a rate of 5 ℃/ min. The transfer line temperature, ion source temperature and quadrupole mass detector temperature were set to 280 ℃. Mass spectra was recorded in electron impact (EI) onization mode at 70 eV and was scanned in the range m/z 30–500 amu at 1 s intervals [57].

Compound identification and quantification

Volatile compounds were identified by comparing the mass spectra of the published standard substance retaining index as well as information from the National Institute of Standards and Technology (NIST) [58]. The original data acquired by GC–MS was first deconvoluted using Masshunter Agilent Analysis (B.08.00, Agilent, Palo Alto, CA, USA) to obtain the retention time, peak area and mass-to-charge ratio of characteristic peaks. Internal standards were used to normalize data processing. Each chromatographic peak area represented the relative content of the corresponding substance. Finally, the integral data of all chromatographic peak areas were exported for statistical analysis.

Stereoscope and fluorescence microscope

The fresh flowers and leaves were collected to observe trichomes using a stereomicoscope (Leica DVM6) and fluorescence microscope (Leica DM6 B). Because of plant materials were three-dimensional, we used the layer scan mode to take several photos and finally synthesize one image.

Scanning electron microscopy

Fresh tissues were collected and cut into small pieces of approximately 5 mm² , then quickly put into FAA solution (50% ethanol: glacial acetic acid: methyl aldehyde = 90:5:5, v/v/v) for 24 h at 4℃. The samples were dehydrated by 70%, 80%, 90%, 95%, and 100% ethanol for 15 min. Following by carbon dioxide critical point drying. Dried samples were glued to a SEM stub and gold-coated before imaging in the main chamber of the microscope, at high-vacuum and room-temperature conditions.

Light microscopy

For light study, the different stage of leaves had been fixed in a solution of 1.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M phosphate buffer with 2% sucrose for 6 h at 4 ℃, poste-fixed with osmium tetroxide, washed in phosphate buffer, dehydrated through an acetone series, and, finally, embedded in Spurr’s resin. Thin sections were prepared with a LEICA UC6I microtome [59].

Peltate glandular trichomes counting

The mature and expanded leaves were collected from pants grown under the same conditions. The number of PGTs were counted from micrographs using IMAGEJ software (http://rsb. info.nih.gov/ij) reported previously [60]. For each independent plant, we selected three different leaves at the same position for counting.

Statistical analysis

All samples were prepared and analyzed in triplicate, and data were expressed as the mean ± SD. Statistical analyses were performed by analysis of variance (ANOVA), and Duncan’s test was used to determine the significance of differences between groups.

Acknowledgements

We thank Lu Wang from Plant Science Facility of the Institute of Botany, Chinese Academy of Sciences for her excellent technical assistance in the GC-MS. We thank Xiuping Xu and Ronghua Liang from Plant Science Facility of the Institute of Botany, Chinese Academy of Science for their excellent technical assistance on scanning electron microscopy and fluorescence microscope.

Authors’ contributions

Y.N.Z. performed the experiments, analyzed the data, and wrote the manuscript; D.W. helped to make semi-thin slices of PGTs; H.L. and H.T.B assisted in sample collection. M.Y.S. and L.S. designed the experiments and revised the manuscript. All authors read and approved the manuscript.

Funding

This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA23080603).

Availability of data and materials

The raw genome and transcriptome sequencing data used in this paper have been deposited in the National Center for Biotechnology Information (NCBI) database under project number PRJNA642976. And the data and material in the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Plant data were collected with permission from the related institution, and complied with national or international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

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No competing interests reported.

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Glandular secretory trichomes formation mechanism study on synthesis and storage of terpenoids in lavender

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