Trichomes
are developed from epidermal cells, which consist of single or multiple cells
(Schuurink and Tissier 2019). It is a special adaptive structure
evolved to cope with biological and abiotic environmental stresses during the
long-term evolution of plants. It is widely distributed on the surface of the
aboveground parts of terrestrial plants (including gymnosperms, angiosperms and
bryophytes) and is one of the important characteristics in taxonomic study. They range in
size from a several microns to few centimetres, and can have different shapes
(Payne 1978). It can be divided into glandular trichomes (GTs) and
non-glandular trichomes (NGTs) according to their structure and function.
GTs are usually
multicellular, composed of differentiated basal, stalk and apical cells, which
produce a large number of different kinds of metabolites. It is a "cellular chemical
factory" for the synthesis of natural products such as terpenoids,
flavonoids, polysaccharides and alkaloids (Tissier
2012). Special chemical substances in plant GTs often have unique chemical
structures and important biological functions, which play a key role in plant
defense. These natural products have diverse structures, complex biosynthesis
and extensive biological activities, which are important sources of the
discovery of natural drugs. Therefore, more and more attention has been paid to
natural products chemistry and other related disciplines.
NGTs are non-secretory protuberance,
which are widely found in the epidermis of plant organs on the ground, such as
leaves, stems, flowers and fruits. It is a special structure formed by
long-term evolution to cope with harsh environment. In complex and changeable
environment, NGTs on plant surface plays a more
important role than GTs. The morphology and structure of NGTs vary greatly with plant species,
so the characteristics of NGTs are regarded as important
microscopic identification characteristics in plant medicinal materials.
According to current reports, NGTs have the functions of drought
resistance, water retention, ultraviolet radiation resistance, insect
resistance and pathogen resistance and photosynthesis regulation (Naydenova and
Georgiev 2013; Lusa et al. 2015; Verma 2017). In some Labiaceae and
Verbenaceae plants, NGTs are also involved in the
synthesis, storage and release of bioactive substances (Tozin et al. 2016; Schuurink and Tissier
2019).
The family
Asteraceae is rich in aromatic species used as herbs, folk medicines,
fragrances, etc. Many plants in this family have important economic value
because they can synthesize secondary metabolites with medicinal value. These
medicinal plants, such as Arnica montana L., Arctium lappa L., Chrysanthemum
lavandulifolium (Fisch.ex Trautv.) Ling et Shih, Centaurea cyanus L., Tagetes erecta L., and Achillea wilsoniana Heimerl ex Hand.-Mazz., are characterized by their leaves and flowers
containing flavonoids, saponins, sesquiterpene lactones and coumarins. In
addition, GTs exists on the vegetative organs surface of all these plants (Muravnik
et al. 2019). The genus Artemisia L. belongs to the Asteraceae
family, which including more than 500 species (Song et al. 2019). Although Artemisia
is the largest genus in the family, the characteristics of trichomes have only
been described in a few species. A detailed description of the morphology,
anatomy, ultrastructure, and histochemistry, has only been reported for the GTs
of Artemisia annua (Duke and Paul 1993; Olofsson et al. 2012).
Artemisia argyi Levl. et Vant. is called “Aicao” in Chinese and
“Gaiyou” in Japanese. It has been widely used in traditional Chinese medicine
for thousands of years. A. argyi is widely distributed in Asia,
Europe and North America (Bora and Sharma 2011). Moxibustion is a kind of
thermal therapy, which is still widely used in China, Korea, Japan and other
countries (Han et al. 2017; Liu et al. 2017; Zhu 2018). Moxa floss is a special moxibustion
material because of its excellent combustion quality and is made from the dried
and processed leaves of A. argyi
(Zhang et al. 2019). A. argyi exhibits extensive
pharmacological properties and is
traditionally used to treat dysmenorrhea, abdominal pain, and inflammation
(Chinese Pharmacopoeia Commission 2015). A number of chemical
constituents have been isolated and identified from A. argyi,
including essential
oils, flavonoids, terpenes, organic acids, and polysaccharides (Yoshikawa et al. 1996; Abad et al. 2012; Han et al.
2017; Zhang et al. 2018). These
chemical components have extensive pharmacological properties, which include anti-inflammatory, anti-tumour, antioxidant,
anticoagulant, anti-osteoporotic effect, and neuroprotection effects (Seo et al. 2003; Zeng et al. 2014; Kim et al.
2015a–b; Yun et al. 2016; Lv et al. 2018; Zhang et al. 2018). However, studies of the morphological feature and
chemical component of GTs for A.
argyi are not available. Therefore, in this study, we analysed the
morphology, distribution, density and secretion of trichomes in the different
of vegetative organs of A. argyi.
Materials
and Methods
Species
characteristics
A. argyi is an
erect, perennial and herbaceous plant. The taproot is obvious, slightly thick
and long, with diameters up to 1.5 cm. The stems are solitary, with a few short
branches and obvious longitudinal ribs; they are 80–150 cm long and densely
covered with tomentum. The surface of the leaves can be visually confirmed to
have short grey and white tomentum, and they also present white glandular spots
and small concave points. The back of the leaves is densely covered with grey and
white tomentum, and the basal leaves have long stipes. The leaves at the base
of the stems are suborbicular or broadly ovate, pinnate and deeply lobed, with
petioles that are approximately 0.5–0.8 cm long; the leaves at the middle of
the stem are ovate or subrhomboid, 5–8 cm in length, and 4–7 cm wide; the upper
leaves are pinnate, semi-lobed, lobed or not divided, but they are elliptic or
lanceolate. The capitula are elliptic, with diameters up to 2.5–3.5 mm, and
they are sessile or subsessile; the corolla is narrowly tubular, slender in
style, and achenes oblong or oblong (Fig. 1). The flowering-fruiting season for
A. argyi is from July to October.
Plant
material
In September to October 2019,
samples of aerial parts from A. argyi
were collected from Nanyang County in Henan Province (33°03′6.56″
N, 112°49′36.91″ E), and identified by Professor Xianzhang Huang
from Nanyang Institute of Technology. The voucher specimens of A. argyi (NY2019093002) are deposited in
the Nanyang Institute of Technology.
Scanning
electron microscopy (SEM)
The samples (1×1 cm) from leaves, stems and petioles were fixed in 4%
glutaraldehyde solution for 12 h at 4℃. These samples were washed three
times with phosphate buffer and post-fixed in 1% osmic acid for 2 h at 4℃. They have been dehydrated at room
temperature for 10 min each time with 30, 50, and 70% ethanol respectively,
washed two times with isoamyl acetate for 15 min, and critical point–dried with
Hitachi CPD-II (Hitachi, Japan). The samples were pasted on the
objective table and sprayed with a layer of gold. After that, they were
observed with a Hitachi Regulus 8220 (Hitachi, Japan) under
different magnification to describe of the morphological
feature, density and distribution of GTs and NGTs.
To observe the distribution and density of GTs or NGTs on the
leaves, stem and petiole of A. argyi, GTs on adaxial surface of leaves from three
different stages of development (n=30) were analyzed in an area of 1 mm2
at suitable magnification.
Extraction
of the secretion of GTs
The improved method from reported
literature (Severson et al. 1984; Asai and Fujimoto 2010;
Zhou et al. 2018)
was used to extract volatile exudates from glandular trichomes of A. argyi. Fresh leaves of A. argyi (3.5
g) for each group were collected. Three groups of experimental materials were
analyzed under the same treatments. Every group samples were dipped 4 times
into methylene chloride, and submerged in the solvent for 2 s each time.
After that, solvent (containing 20 g anhydrous sodium sulfate) was poured into brown
reagent bottle, and stored in the dark for 24 h. The washings were filtered
through vacuum filtration and concentrated to 10 mL at 38℃ using a rotary
evaporator until preparation for analysis.
Gas
chromatography/mass spectrometry analysis of the secretion of GTs
The secretion of GTs in A. argyi was analyzed using Shimadzu
GC-2010 and Shimadzu QP2010 plus MS (Shimadzu Corp., Kyoto, Japan). A capillary
column (30 m × 0.25 mm, 0.25 µm film thickness) (Restek, Bellefonte, PA) was
used. The volume of sample injecting the machine was 1 µL and the
parameter of split ratio was 20:1. The flow rate of helium was 1.3 mL/min. The
temperature was increased from 50℃ to 90℃ at 10℃/min, and
continued for about five minutes; increased to 160℃ at 10℃/min and
continued for about ten minutes;
%20359-365_files/image002.jpg)
Fig. 1: Images of A.
argyi from aerial parts (A–C).
%20359-365_files/image004.png)
Fig. 2:
Distribution of the different types of trichomes on the vegetative organ
surfaces of A. argyi (SEM). (A–-C) Part of the abaxial
surface of the young, intermediate and mature leaves. (D) Stem surface. (E–F) Upper and
lower petiole surfaces. Bars: 500 μm
%20359-365_files/image006.png)
Fig. 3: Light
microscopy images of the cross sections of vegetative organs of A. argyi: (A) stem; (B) petiole; and (C–D) leaf blades. GT:
glandular trichome; NGT: non-glandular trichome. Bars: A, 500 μm; B, 100
μm; C, D, E, 50 μm; F, 20 μm; and G, H, 10 μm
increased to 250℃ at 10℃/min and continued for about ten minutes.
The temperature of injection was 230℃. The range of scan mass was 40–1000
m/z. The MS was operated in the electron impact mode (70 eV). Compounds were
identified by the NIST05 mass spectral library. The relative percentage was
determined based on peak area normalization.
Results
Morphology
and distribution of trichomes in A. argyi
Different morphotypes of
trichomes covering the surfaces of the vegetative organs of A. argyi. GTs were observed on the leaf
(Fig. 2A–C; Fig. 3C–D), stem (Fig. 2D; Fig. 3A) and petiole surfaces (Fig.
2E–F; Fig. 3B). NGTs were also observed on all analysed vegetative organs (Fig.
2A–D).
Based on
the morphological survey, the trichomes from
A. argyi included a total of three types of morphologically distinct
trichomes. Among them, there were two types of GTs (I and II) and one type of
NGTs. Table 1:
Comparative GT density (mm2) on the adaxial surface of leaves of A. argyi at different ontogenetic stages
|
Fully
expanded leaves |
|
|
|
Young * |
Intermediate
* |
Adult * |
|
Adaxial
surface |
Adaxial
surface |
Adaxial
surface |
|
36.16 ±
6.37 |
28.23 ±
3.79 |
17.43 ±
4.24 |
* Data obtained for n= 30 measurements for each
developmental stage. Mean ± SD.
%20359-365_files/image008.png)
Fig. 4: SEM images
of different types of trichomes at different developmental stages in A. argyi. A. NGTs at different
ontogenetic stages. B–C.
Different types of GTs with plump gland surfaces. D. NGTs at the early
ontogenetic stage. E–F.
Different types of GTs with wrinkled gland surfaces. GT: glandular trichome;
NGT: non-glandular trichomes.
%20359-365_files/image010.png)
Fig. 5: The adaxial
surface of leaves from A. argyi at different developmental
stages (SEM). A–B: completely unfolded young leaves. C–D: intermediate
developmental stage. E–F mature leaves
The results showed that
types I and II represented two predominant types of GTs (Fig. 4B, C). Only a
few type I GTs were observed, as most of them were type II GTs. The morphology
of type I GTs was circular, slightly sunken in the middle and made up of four
cells (Fig. 3F, H). The length of type I GTs was approximately 40 nm
(Fig. 4B). The morphology of type II GTs was non-circular, including two
layers, which were made up of eight cells (Fig. 3E, G). Additionally, the
bottom was slightly wider than the top. The length of type II GTs was
approximately 50 nm (Fig. 4C). Immature
GTs were usually distributed in the dent of the leaf epidermis, but mature GTs
are often higher than the leaf surface. NGTs
are T-shaped and branched (Fig. 4A, D). Trichomes of different stages of
development are distributed in the abaxial surface of young leaves (Fig. 4A).
A type of T-shaped NGTs was observed at the early stage of development (Fig.
4D). Morphological differences between the two types of GTs were obvious during
the vigorous growth and senescence periods (Fig. 4B, C, E, F).
Density
of glandular trichomes in A. argyi
SEM observations revealed
that GTs and NGTs were distributed on the abaxial and adaxial surfaces of
leaves. However, the density of the trichomes exhibited an obvious
difference between the abaxial and adaxial surfaces of the leaves. The NGTs
density was lower on the adaxial surface than on the abaxial surface, and the
NGTs were dense at intermediate stages of development. The density of NGTs on
the abaxial surface of the leaves was so high that the GTs could not be
counted. The number of trichomes per unit area of mature leaves was less than
young leaves. Different morphotypes of trichomes in early ontogenetic stages
were not observed in mature leaves. On the adaxial surface, the GTs were dense,
and their number gradually decreased as the leaves reached maturity in the
early developmental stage (Table 1 and Fig. 5).
Constituents
of the secretion of GTs from A. argyi
Compositions of the secretion of GTs
from A. argyi and their relative
percentages (%) are shown in Table 2. Twelve compounds were identified from the secretion of GTs
from A. argyi, including one alkene,
one monoterpene, three phenols, four esters, one alcohol, one ketone and one
heterocyclic compound. The peak area of these compounds accounted for 41.33% of the total
peak area of GC-MS (Fig. 6).
(1S,3S,5S)-1-Isopropyl-4-methylenebicyclo[3.1.0]hexan-3-yl acetate and
eucalyptol were the major components, and other notable components were
1-naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-,
[1R-(1.alpha.,4a.beta.,8a.alpha.)]-, 5,
8-dimethyl-1,4,6,7-tetrahydronaphthalene-1,4-dicarboxylic acid, 1,4-dimethyl
ester, etc.
Discussion
It is the
first report that two different morphotypes of GTs and one type of NGTs that
were observed on differential vegetative tissues of A. argyi in this study. According to reports, A. annua was also covered with peltate GTs and T-shaped NGTs. GT is composed
of 10 cells (Duke and Paul 1993; Duke et
al. 1994; Ferreira and Janick 1995). To date, only a few detailed
morphological studies have been carried out on GTs and NGTs structures in the
genus Artemisia (Kelsey and
Shafizadeh 1980; Ascensão and Pais 1982; 1987). However, most plants from the
family Asteraceae can produce special aromatic secondary metabolites. In
future, more and more in-depth studies on the morphological characteristics and
histochemistry of GTs and NGTs for different morphological types of plants in
the family Asteraceae will help us to understand the potential role of these
structures in these plant species.
The previous studies have
shown that large amounts of T-shaped NGTs and few GTs from A. argyi exist in moxa floss (Wu
et al. 2018). The GTs on the surface of leaves and stems could secrete more
kinds of volatile oils; therefore, we hypothesized that GTs and NGTs morphology
and density are closely associated with the quality of moxa floss. Most of the
secondary metabolites in the trichomes of plants are related to key pathway
genes, which are specifically or abundantly expressed in GTs specific cells.
For example, A. annua can synthesize
artemisinin, which is largely used as an anti-malarial agent (Graham et al. 2010). A few of significant
genes, such as Aldh1, CYP71AV1 and Dbr2, which are responsible for the biosynthesis of artemisinin,
are preferentially expressed in trichomes (Teoh et al. 2006; 2009; Zhang et
al. 2008). To date, no key genes for trichome development in A. argyi have been reported. In summary,
this study has important reference value for further research on the molecular
regulation of GT and NGT development for improving the yield and quality of
moxa floss, plant classification and agricultural applications in the future.
Table 2: Components
of the secretion of GTs from A. argyi
|
Compounds |
Retention
time (min) |
Relative
percentage (%) |
|
|
1 |
(1S)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene |
4.75 |
0.51 |
|
2 |
Eucalyptol |
5.90 |
8.87 |
|
3 |
Bicyclo[3.1.0]hexan-3-ol,
4-methylene-1-(1-methylethyl)-, (1.alpha.,3.alpha.,5.alpha.)- |
9.51 |
2.46 |
|
4 |
(1S,3S,5S)-1-Isopropyl-4-methylenebicyclo[3.1.0]hexan-3-yl
acetate |
13.36 |
10.93 |
|
5 |
Sabinol,
3-methylbut-2-enoate |
17.82 |
0.87 |
|
6 |
1-Naphthalenol,
decahydro-1,4a-dimethyl-7-(1-methylethylidene)-,
[1R-(1.alpha.,4a.beta.,8a.alpha.)]- |
20.23 |
3.72 |
|
7 |
5,8-Dimethyl-1,4,6,7-tetrahydronaphthalene-1,4-dicarboxylic
acid, 1,4-dimethyl ester |
34.78 |
3.08 |
|
8 |
Phenol,
2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl- |
35.46 |
1.28 |
|
9 |
2H-Pyran-2-carboxaldehyde,
3,4-dihydro-2,5-dimethyl- |
35.82 |
3.08 |
|
10 |
9,11-Dehydroprogesterone |
36.01 |
2.81 |
|
11 |
2-Butenoic
acid, 2-methyl-, dodecahydro-8-hydroxy-8a-methyl-3,5-bis(methylene)-2-oxonaphtho[2,3-b]furan-4-yl
ester, [3ar-[3a.al |
37.45 |
2.46 |
|
12 |
Cyclohexanecarboxylic
acid, 2-tridecyl ester |
39.23 |
1.26 |
%20359-365_files/image012.jpg)
Fig. 6: Total ion chromatogram of GTs volatile exudates
from three groups of A. argyi by
GC-MS
During the present investigation,
the volatile components present in the GTs of A. argyi were determined and identified by GC-MS. The secretion of GTs was
extracted by methylene chloride. According to a previous report, methylene
chloride can quickly extract GTs exudates from plant leaf surfaces (Wagner et al. 2004). Moreover, the components
cannot penetrate the epidermis and be extracted in internal leaves by using
this extraction method. Therefore, compared with other methods, it is an
efficient and convenient method for the study of GT secondary metabolism.
Twelve compounds were identified from the secretion of GTs of A. argyi. Among these compounds, the
contents of eucalyptol and (1S,3S,5S)-1-isopropyl-4-
methylenebicyclo[3.1.0]hexan-3-yl acetate were higher than the other components
in A. argyi. To a certain extent,
eucalyptol can reflect the quality of medicinal plants and has been used as a
quality control marker of A. argyi in
the Pharmacopoeia of P.R. China (Committee for the Pharmacopoeia of PR China
2015). Previous reports indicated that there were some differences in the
contents of total flavonoids, total phenolic acids and bioactive compounds of A. argyi in different harvest periods
(Xue et al. 2019). Only a few compounds of the volatile exudates were isolated from A. argyi in this study. This result may be closely associated with the harvest
period. In addition, the other chemical components isolated from A. argyi exhibit a wide range of
biological activities (Song et al. 2019), which deserves to be
deeply investigated. A
number of main components of the volatile
exudates were analysed from the whole GTs of A. argyi in this experiment. For the next step, differential
analysis of the secretory cells in different parts of the GTs of A. argyi is expected.
GTs are sites of biosynthesis
and storage of large quantities of specialized metabolites (Schuurink and
Tissier 2019) and widely exist in plants of the Labiaceae, Compositae,
Solanaceae families and plants, such as A. annua and Mentha
haplocalyx. GTs are also called “natural plant factories”. GTs can usually
adjust their density to adapt to changes in the environment or when under
stress (Huchelmann et al. 2017). For example, the
density of GTs from Schizonepeta
tenuifolia, Madia sativa
and Solanum lycopersicum exhibited
obvious responses to environmental stress, which increased with the aggravation
of stress (Gonzáles et al. 2008;
Galdon-Armero et al. 2018; Li et al. 2019). A GTs counting method and secretion analysis
method were successfully established in this study. Therefore, this study can
provide a basis for future studies on the effects of environmental stress on
the GT density and inclusion composition of A.
argyi.
In addition, there are
developmental issues beyond the scope of this study that could be used in
future research. For example, NGTs are so long and dense on the leaf surfaces
of A. argyi that the density of NGTs
could not be evaluated in this experiment. Establishing an evaluation method
for determining the density of NGTs has important practical significance for
the quality evaluation of A. argyi
and moxa floss in the future.
Conclusion
Three
different morphotypes of trichomes covering the surfaces of the vegetative
organs of A. argyi. A number of volatile secondary
metabolites were analyzed and identified from the whole GTs of A. argyi in this study. The detailed
studies on the morphological characteristics and histochemistry of GTs and NGTs
from A. argyi will
contribute us to understand the potential role of trichome structures for the
plants in the family Asteraceae.
Acknowledgements
We thank Jiang Chang for assistance in obtaining the SEM micrographs and
Yuhang Jiang for GC-MS analysis from College of Horticulture and College of
Agriculture, Fujian Agriculture and Forestry University. This study was supported
by the National Key Research and Development Program of China (2017YFC1700704),
the National Natural Science Foundation of China (81803661)
and Science and Technology Open Cooperation Project of Henan Province of China
(172106000053).
Author Contributions
XZH, DHL and ZYZ planned the experiments, ZHC, CL, ZL and LG interpreted
the results, ZHC and LG made the write up and MJL statistically analyzed the
data and made illustrations.
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