Effect of drought on photosynthesis, total antioxidant capacity, bioactive component accumulation, and the transcriptome of Atractylodes lancea.

BACKGROUND: Atractylodes lancea (Thunb.) DC, a medicinal herb belonging to the Asteraceae family, often faces severe drought stress during its growth. Until now, there has been no research on the effect of drought stress on the quality formation of A. lancea. Therefore, the present study aimed to study the effects of drought stress on A. lancea through physical and chemical analysis, and to reveal the related molecular mechanisms via transcriptome analysis. RESULTS: The photosynthesis was markedly inhibited under drought stress. There were alterations to photosynthetic parameters (Pn, Gs, Ci) and chlorophyll fluorescence (Fv/Fm, NPQ), and the chlorophyll content decreased. Twenty genes encoding important regulatory enzymes in light and dark reactions, including the Rubisco gene of the Calvin cycle, were significantly downregulated. After exposure to drought stress for more than 4 days, the activities of four antioxidative enzymes (SOD, POD CAT and APX) began to decrease and continued to decrease with longer stress exposure. Meanwhile, most of the genes encoding antioxidative enzymes were downregulated significantly. The downregulation of 21 genes related to the respiratory electron transport chain indicated that the blocked electron transfer accelerated excessive ROS. The MDA content was significantly elevated. The above data showed that 15 days of drought stress caused serious oxidative damage to A. lancea. Drought stress not only reduced the size and dry weight of A. lancea, but also lowered the amount of total volatile oil and the content of the main bioactive components. The total volatile oil and atractylodin content decreased slightly, whereas the content of atractylon and ß-eudesmol decreased significantly. Moreover, ten significantly downregulated genes encoding sesquiterpene synthase were mainly expressed in rhizomes. CONCLUSIONS: After exposed to drought stress, the process of assimilation was affected by the destruction of photosynthesis; stress tolerance was impaired because of the inhibition of the antioxidative enzyme system; and bioactive component biosynthesis was hindered by the downregulation of sesquiterpene synthase-related gene expression. All these had negative impacts on the quality formation of A. lancea under drought stress.

Microbial diversity and community structure changes in the rhizosphere soils of Atractylodes lancea from different planting years.

Atractylodes lancea is a type of typical traditional Chinese medicinal (TCM) herb that is economically important in China. The traditional planting method of A. lancea is to plant in situ continuously for many years, which often leads to impediments for its growth and development and soil-borne diseases. The root-associated microbiome is believed to play an important role in plant resistance and the quality of products from the plant. This study aims to reveal detailed changes in the populations of rhizosphere microorganisms, and providing theoretical guidance for the prevention and control of soil-borne diseases in A. lancea. A high-throughput sequencing approach was utilized to illustrate changes in the microbial community from different planting years. Results and conclusions: The results show that the diversity and composition of the root-associated microbiome was significantly impacted by the consecutive monoculture of A. lancea. At the level of the comparisons of the phyla, Bacteroidetes, Proteobacteria, Ascomycota, and Basidiomycota declined significantly. In contrast, the relative abundance of Actinobacteria, Acidobacteria, and Mortierellomycota distinctly increased. Comparisons at the genus level indicated that Sphingomonas, Flavobacterium, Pseudomonas, Pedobacter, and Tausonia decreased significantly, whereas Mortierella, Cylindrocarpon, Dactylonectria, and Mucor distinctly increased. In conclusion, this study helps to develop an understanding of the impediments involved in the consecutive monoculture of A. lancea.

Identification of autotoxic compounds from Atractylodes macrocephala Koidz and preliminary investigations of their influences on immune system.

Atractylodes macrocephala Koidz (A. macrocephala) is a traditional Chinese medicine that has been widely used in China, Japan, and Korea due to its health benefits. Autotoxicity, as one of the major problems hindering continuous cultivation of A. macrocephala, has been reported to inhibit plant growth by various means. However, the impact of autotoxicity on the plant immune system is rarely reported. In this study, 2, 4-Ditertbutyl phenol (2,4-DP), an autotoxic compound, isolated from root exudates and rhizosphere soil of A. macrocephala was identified by gas chromatography-mass spectrometry (GC-MS). The results of germination trials showed that 2,4-DP had a significant inhibitory effect on seed germination. In addition, in non-inoculated seedlings, three concentrations of 2,4-DP (0.1, 1 and 10 mmol/L) affected indicators of systemic acquired resistance (SAR): accumulation of salicylic acid (SA), activities of protective enzymes, atractylenolides contents, and increased the disease index (DI). In inoculated seedlings, 2,4-DP decreased indicators of SAR and increased the DIs at low and high concentrations but increased indicators of SAR and decreased the DI at a moderate concentration. These results suggest that 2,4-DP has an inhibitory effect on the plant immune system, but it can induce the SAR at a certain concentration by controlling the pathogenic fungi.

Consequences of antagonistic interactions between endophytic fungus and bacterium on plant growth and defense responses in Atractylodes lancea.

Many studies have examined pair-wise interactions between plants and endophytes, while overlooking the interplays among multiple endosymbionts and their combined impacts on hosts. In this study, Atractylodes lancea plantlets were inoculated with endophytic fungus Acremonium strictum AL16, or endophytic bacterium Acinetobacter sp., or both, to investigate the impacts of the three-way symbiosis on the host. Our results showed that defense-related responses of the co-inoculated plantlets were delayed and weakened relative to plantlets with single inoculants, but no detrimental effects on phyto-physiology (growth, photosynthesis) were observed after combined inoculations. Quantitative PCR analysis verified a decrease in AL16 colonization density within plants after co-inoculation with the endobacteria. An in vitro assay was then performed to elucidate the suppressed plant defense responses and reduced fungal colonization by dual inoculation. The data showed that the presence of Acinetobacter sp. reduced AL16 colony diameter and spore germination rate without negatively affecting fungal morphology. Additionally, direct hyphal attachment of the bacterium to AL16 in vitro was visualized by scanning electronic microscopy. Therefore, we propose that a balanced and compatible symbiosis might require constraints conferred by the antagonistic endophyte Acinetobacter sp. on the fungus AL16 in the tripartite endophytic bacterium-fungus-plant system.

Involvement of abscisic acid and salicylic acid in signal cascade regulating bacterial endophyte-induced volatile oil biosynthesis in plantlets of Atractylodes lancea.

The enormous biological diversity of endophytes, coupled with their potential to enhance the production of bioactive metabolites in plants, has driven research efforts focusing on endophytes. However, limited information is available on the impacts of bacterial endophytes on plant secondary metabolism and signaling pathways involved. This work showed that an endophytic Acinetobacter sp. ALEB16, capable of activating accumulation of plant volatile oils, also induced abscisic acid (ABA) and salicylic acid (SA) production in Atractylodes lancea. Pre-treatment of plantlets with biosynthetic inhibitors of ABA or SA blocked the bacterium-induced volatile production. ABA inhibitors suppressed not only the bacterium-induced volatile accumulation but also the induced ABA and SA generation; nevertheless, SA inhibitors did not significantly inhibit the induced ABA biosynthesis, implying that SA acted downstream of ABA production. These results were confirmed by observations that exogenous ABA and SA reversed the inhibition of bacterium-induced volatile accumulation by inhibitors. Transcriptional activities of genes in sesquiterpenoid biosynthesis also increased significantly with bacterium, ABA and SA treatments. Mevalonate pathway proved to be the main source of isopentenyldiphosphate for bacterium-induced sesquiterpenoids, as assessed in experiments using specific terpene biosynthesis inhibitors. These results suggest that Acinetobacter sp. acts as an endophytic elicitor to stimulate volatile biosynthesis of A. lancea via an ABA/SA-dependent pathway, thereby yielding additional insight into the interconnection between ABA and SA in biosynthesis-related signaling pathways.

[Pollen morphological characteristics, viability test and storage of endangered medicinal plant Atractylodes lancea].

OBJECTIVE: To study the pollen morphological characteristics, viability test and storage character of the endangered plant Atractylodes lancea. METHOD: Pollen grains morphologies of A. lancea were observed by scanning electron microscope. The optimum culture medium and viability determination methods were screened out by liquid culture and dyeing methods, and then the pollen germination capacities in different storage conditions were detected. RESULT: The pollen grains are quasi-spherical, with tricolpate and spinous sculpture. The optimal culture medium was ME3 + 16% PEG4000 + 10% sucrose, in which the pollen germination capacity reached to 62.1%, while the other three dyeing methods were not able to be applied to detecting the pollen viability of A. lancea. The low storage temperature could significantly prolong the storage time of pollen of A. lancea. At -80 degrees C, pollen viability could be maintained for 60 days. CONCLUSION: Liquid culture method is suitable for the determination of pollen germination of A. lancea, and the rate of pollen germination is closely related to the storage time and temperature. At last, this study provides a foundation for the artificial pollination and cultivating in wildness of A. lancea.

Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets.

BACKGROUND: Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. RESULTS: Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. CONCLUSIONS: Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway.

[Effect of endophytic fungal elicitors on growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea].

OBJECTIVE: To investigate the effects of endophytic fungal elicitors on the growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea. METHOD: The endophytic fungal elicitors were added to the medium with different concentrations and culture period. Their effects on biomass, atractylodin content and relevant enzyme activities in suspension cultured cells were studied. RESULT: The cell growth was not affected by elicitors at low concentration and obviously inhibited at high concentration. Inhibition rate reached 46.7% by 100 mg L(-1) elicitor. In addition, six strains from A. lancea, among which Rhizoctonia SP1 activity was higher, had distinctly promoted the accumulation of atractylodin. Atractylodin biosynthesis was notably promoted by 20-60 mg L(-1) Rhizoctonia SP1 elicitor. When 40 mg L(-1) Rhizoctonia SP1 elicitor was added in the medium at the 12 day, the maximum content of atractylodin was 28.06 microg L(-1) at the 21 day with 48.3% higher than that of the control and PPO, POD and CAT activities remarkably increased. CONCLUSION: Adding the endophytic elicitors to the medium is able to be effective approaches to enhance atractylodin yield in the suspension culture cell of A. lancea.

[The change rules and correlation between specific leaf weight and dry weight of rhizoma of Atractylodes macrocephala].

OBJECTIVE: To study the change rules of specific leaf weight, dry weight of rhizoma and the drying rate of annual and biennial Atractylodes macrocephala. METHODS: We measured the specific leaf weight and dry weight of rhizoma, and accounted the drying rate and correlation by dugging the annual and biennial plants each 15 days. There were 3 rhizoma types of biennial Atractylodes macrocephala with Drumstick/Frog type, Er-cha type and Wu-zi type. RESULTS: It showed the curve of specific leaf weight of annual plant was "N" style, which of biennial plant was linear. The dry weight and the drying rate of all rhizoma types increased point-blank during the whole growth period. CONCLUSION: The correlation between specific leaf weight and dry weight of rhizoma is obvious in biennial Atractylodes macrocephala but is contrary in annual Atractylodes macrocephala.

[Study on correlation between Atractylodes macrocephala viability and germination rate].

OBJECTIVE: To screen the optimized methods for detection seed viability and germination rate determination of Atractylodes macrocephala, and determine the relationship between seed viability and germination rate. METHOD: There were four methods, which including 2,3,5-triphynel tetrazolilum chloride (TTC) staining, red ink staining, BTB staining and Nongjia method, to evaluate the 12 A. macrocephala local varieties'seed viability and measure their germination rate. RESULT: Seed viability of A. macrocephala using TTC staining ranked the first compared to that of other three methods. Seed viability was significantly related with germination rate using TTC method. Their correlation coefficient reached 0.915 and regression equation was also found out between seed viability (X) and germination rate (Y), which was Y = -0.083 4 + 0.995 4X. CONCLUSION: TTC staining was the optimal method to determine A. macrocephala seed vitality. Furthermore, seed viability was significant related with germination rate of A. macrocephala.

[Study on effect of drought stress on physiological adaptation of seedlings of Atractylodes lancea].

OBJECTIVE: To study the effect of drought stress on the changes of physiological adaptation of Atractylodes lancea seedlings. METHOD: Investigation was carried out on content changes of MDA, soluble protein, and activities of SOD, POD, CAT, APX in A. lancea seedlings under polyethylene glycol (PEG-6000)-simulated drought stress. RESULT: In A. lancea seedlings treated with 15% and 25% PEG, the content of MDA increased significantly with the stress time, and increased more significantly at a higher concentration of PEG. The content of soluble protein increased significantly after treatment on the day one and day three; activities of SOD, POD, CAT and APX increased at first and decreased later, increasing rates rose at high concentration of PEG moreover, activities of POD, APX reached the maximum after three days, and the time of maximum activities changed with concentration of PEG. CONCLUSION: A. lancea seedlings adapted to drought stress by increasing the content of soluble protein to decrease water potential, and by improving activities of protective enzymes to enhance anti-oxidative ability under drought stress.