Cichoric acid is the main phenolic compound in the root and rhizome of the medicinal part, Echinacea purpurea that is known for possessing immune enhancing characteristics. In this study, we analysis the the synthesis and storage sites of phenolic compound in E. purpurea. We used fluorescent microscopy, transmission electron microscopy, cytochemical and immunocytochemical localization to observe the distribution of phenolic compounds. Our results show that the phenolic compounds were mostly distributed in the cortex parenchyma cells, vascular parenchyma cells and pith parenchyma cells in the root and rhizome, and mainly present in the vacuoles, large intercellular spaces and their surrounding cell walls. No phenolic compounds were observed in the cytoplasm and the organelles. We concluded that the phenolic compounds were synthetized in the cortex parenchyma cells, vascular parenchyma cells and pith parenchyma cells in the root and rhizome, and stored in the vacuoles of parenchyma cells. The above results provided significantly cytological information for further approaching the metabolic regulation and transfer pathways of phenolic compounds in biochemistry and molecular biology.
Echinacea purpurea (L.) Moench. (Compositae) is a perennial herb native to North America. It is widely used in western countries and is a widely recognized immune enhancing herb, with anti-inflammatory, antivirus, and antitumor activity [1-4]. Its main active constituents are caffeic acid derivatives, alkylamide and polysaccharides [5,6]. Cichoric acid is the most abundant phenolic compound in the root and rhizome of E. purpurea, and is the important characteristic and immune active constituent [7,8]. And it was reported to have HIV-1 and HIV-1 integrase inhibiting effect [
Seeds of E. Purpurea were bought from Easyliving Wildflowers seed company. The seeds were grown in Ningxi teaching and scientific research base of South China Agricultural University. The roots (with rhizome) of E. Purpurea grown for two years were harvested in July, 2010. The base locates in Zengcheng city, Guangdong province, China, at altitude of 26 meters, of 23˚14'35'' north latitude and 113˚38'18'' east longitude, and the soil pH is 6.1.
The Leica CM1900 cryostat was adjusted to –22˚C and the root and rhizome specimen of E. Purpurea was rapidly cut down at ambient temperature and fixed on a precooled station, and embedded in the medium (Jung Tissue Freeezing Medium) specific for the cryostat, placed on the quick freeze shelf for five minutes, and then cut to 30 µm sections. The sections were mainly for fresh fluorescent histochemical observation.
Neu’s solution [1% 2-amino-ethyldiphe-nylborinate (Sigma-Aldrich) absolute methanol solution] was dropped to the fresh sections. After reacting for 30 s, the sections were mounted in 10% glycerol aqueous solution [
The root and rhizome of the E. purpurea after flowering was cut and rapidly fixed in a solution of 4% glutaric dialdehyde mixed with 4% paraformaldehyde dissolved in 0.1 mol/L sodium phosphate buffer (pH 7.2) overnight at 4˚C, rinsed with phosphate buffer for three times, postfixed in 2% (w/v) osmium tetroxide aqueous solution for 3 h, rinsed with phosphate buffer for three times also, dehydrated in an ethanol series, cleared by propylene oxide and embedded in Epon 812. The specimens were cut to sections of 1 - 2 µm thickness by Leica RM2155 microtome. The sections were stained in 0.05% toluidine blue, mounted in neutral resin, observed and photographed by Leica DMLB microscope.
The specimen prepared as described in 1.2.3 were cut to 70 - 80 nm sections by Leica EM UC6 microtome. The sections were stained by uranyl acetate and lead citrate for 10 - 15 min, then observed and photographed by FEITecnai 12 transmission electron microscopy.
Preparation of immuno-gold probes: 100 µg Laccase (from Rhus vernificera, Siama-Aldrich) was mixed with 10 ml colloidal gold solution, and 1% (v/v) PEG20000 was added to stabilize the gold probe, then the mixture was centrifugated for 60 min at 12000 rpm. The precipitate was resuspended in 0.5 ml PBS (pH 6.0; contained 0.2 mg∙ml–1 PEG20000) and stored at 4˚C until use.
Cytochemical marker: Ultra-thin sections were prepared as described in 3.2.3 and 2.2.4 (no post-fixed inosmium tetroxide aqueous solution). The sections were picked up by Ni grids and suspended in a drop of PBSPEG mixture (pH 6.0, containing 0.2 mg∙ml–1 PEG20000) for 5 - 10 min, then transferred to a drop of Laccasecolloidal gold complex in a moist chamber for 30 min at room temperature. Then the sections were rinsed with PBS solution of pH 7.2 for 2 - 3 times, and rinsed with aseptic water for 3 - 5 times, stained by uranyl acetate and lead citrate individually for 10 - 15 min, then observed and photographed by FEI-Tecnai 12 transmission electron microscope. Colloidal gold solution mixed with 100 µg PBS of pH 6.5 was used as control.
A two-year old root and rhizome of E. purpurea had all characteristics of a secondary structure for accumulating phenolic compounds. The main root was made up of periderm, cortex, secondary phloem, vascular cambium, secondary xylem, primary xylem. The structure of the rhizome resembled that of root (
Neu’s solution is the standard test solution for localization of phenolic compounds in plants, in which borate will react with the phenolic compounds and form a complex, which will generate fluorescence of special color under certain conditions [17,18]. Astandard cichoric acid emits yellow green fluorescence (
The cortex parenchyma cells of a two-year old rhizome treated with Neu’s solution (
parenchyma cells, and xylem parenchyma cells. By way of comparing with the strong fluorescence resulting from cichoric acid (Sigma) treated with Neu’s solution for 30 seconds and lit by 365 nm ultraviolet light (
Based on results of optical microscopy, the main distribution sites of the phenolic compounds were parenchyma cells of cortex, phloem, xylem and pith, and very little phenolic compounds were distributed in the intercellular spaces of the parenchyma cells.
Because the phenolic compounds would be strongly stained by osmium tetroxide [
Under electron microscope, there was light cytoplasm and a typical centric big vacuole in the mature of cortex parenchyma cell of the two-year old rhizome. Alongside the tonoplast, dark osmiophilic droplets of high density were present (
In the cortex parenchyma cells of the root, the distribution features of the phenolic droplets largely resembled that in rhizome (
The distribution features of the phenolic droplets indicated that most of them were present in the vacuoles, intercellular spaces and the surrounding cell wall. None of them was present in the cytoplasm or organelle. The images we acquired suggested that a great number of black flocculent phenolic droplets were transferred from the tonoplast into the vacuole. But why was it not found in the cytoplasm? We reasoned that the black flocculent phenolic droplets would not be recognized in the cytoplasm or the cytoplasm was not a site of the final synthesis of phenolic compounds. To study that question, immunocytochemical technique was employed to locate the phenolic compounds.
Laccase is a polyphenol oxidase, which will catalyze the oxidization of substrates like polyphenols, aromatic polyamines, etc. It will form laccase-gold complex with
colloidal gold solution and signals the precise localization of phenolic compounds in the cells. The distribution and storage sites of the phenolic compounds could be located by observing the distribution and amount of the gold granules under the transmission electron microscope [16,21].
Observed by the immunocytochemical localization, gold granules were found scattered or gathered in the vacuoles of the parenchyma cells of the rhizome (
Also, no gold labeling was found in the cytoplasm, peroxisomes and mitochondria (
Immunocytochemical localization results indicated that the cytoplasm was not the site for the synthesis of the final phenols compounds.
Study for distribution, synthesis site, transfer pathway and storage site of the medicinal constituents in the medicinal plant will provide scientific basis for the identification of medicinal materials and selection of optimal harvest time, optimal storage method and optimal extraction method of
active constituents. Histochemical localization technique is an effective means to find out the distribution and accumulation patterns of medicinal constituents in organs and tissues [
With the Hoepfner-Vorsatz reaction treatment, [
The phenolic compounds in higher plants are synthesized via shikimate pathway. Phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate: coenzyme A ligase (4CL) were three key enzymes influencing that pathway [24,25], and the PAL and C4H were the two key enzymes closely related to the synthesis of phenolic compounds. PAL is the key and rate-limiting enzyme in shikimate pathway, and is the key factor influencing the accumulative level of the phenolic compounds [
In this study, observations at the cytological level revealed that in the parenchyma cell of E. purpurea storing phenolic compounds, the phenolic droplets initially formed small particles on the inner membrane of vacuoles, then gradually diffused into the vacuoles and gathered to form larger spheres. At the same time, black phenolic droplets were found on the cell wall outside the cytoplasm membrane. Although abundant mitochondria, peroxisomes, etc. were present in the cytoplasm, no phenolic droplets were observed in the cytoplasm and those organelles. The immunocytochemical loalization results showed that the vacuole constituents were strongly labeled, while the cytoplasm was not, and the big cellular spaces and their surrounding cell walls were labeled but not as strong as the vacuoles. Peroxisomes, Golgi apparatus and mitochondria were not labeled. Few plastids were labeled, only those containing starch grains were marked with some gold granules, and the gold granules gathered around the starch grains or directly on the starch grains. These results were consistent with the immune gold labeling results of polyphenols in rhizome of Polypodium vulgare L. studied by [
Based on our results as a whole and taking into account of current literature, we propose that in the cytoplasm and membrane organelles (mainly peroxisomes and mitochondria), only the precursors of phenolic compounds were synthesized, as they would not be fixed by osimic acid or react with the Laccase. the precursors enter the vacuoles by active transport energized by mitochondria, and at the same time, are converted to the phenolic compounds by specific enzymes in the tonoplast. The phenolic compounds gradually gathered to form bigger spherical phenolic droplets in vacuoles and stored. The enzymes converting the precursors to phenolic compound were not only present in the tonoplast, but also in the cell membrane. The precursor were converted by the enzymes in the cell membrane and the product was transported to outside the membrane, through the cell wall and accumulated in the intercellular spaces. Further cytological and biochemical study is needed to delineate the enzymes in the tonoplast and cell membrane performing the biochemical conversions.
To resist the attack from herbivores and pathogens, a complex defense mechanism is formed in plants, and the root systems of plants play important role in this process. Commonly, the parenchyma cells of plants will actively synthesize, store and modify phenolic substances, and release the stored phenolic compounds to perform their protective effect when plant gets encounter attacks [20, 30] (Franceschi et al. 1998, 2000). Plenty of phenolic compounds are stored in the vacuoles of parenchyma cells of the E. purpurea root and rhizome, it will improved themself protective effect, especially inhibit herbivores utilization rate of protein, enzyme activity, integrity maintaining of the cell membrane, etc. [
In addition, [
We thank Dr. Jitao Zou for critically reading the manuscript. This work was supported by grants from the Funds for Industry-University-Research Cooperation from the Guang-dong Provincial Government and the Ministry of Education of the PR China (2008B090500250) and Science and Technology Planning Project of Guangdong Province, China (2011B031700026) for financial support.