Physiologia Plantarum 2014
© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Involvement of abscisic acid and salicylic acid in signal cascade regulating bacterial endophyte-induced volatile oil biosynthesis in plantlets of Atractylodes lancea Xiao-Mi Wang, Bo Yang, Cheng-Gang Ren, Hong-Wei Wang, Jin-Yan Wang and Chuan-Chao Dai∗ Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
Correspondence *Corresponding author, e-mail: [email protected] Received 25 January 2014; revised 4 May 2014 doi:10.1111/ppl.12236
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.
Introduction Plants fend off pathogen attack or respond to abiotic stresses via a combination of constitutive and inducible defense mechanisms (De Vleesschauwer et al. 2013). The accumulation of bioactive secondary metabolites, which is regulated by cross-communicating signaling cascades, plays key roles in fine-tuning the innate
immune responses of plants to different stimuli (Yang et al. 2012). Atractylodes lancea is a traditional Chinese medicinal plant. The accumulation of volatile oils, including characteristic sesquiterpenoids (atractylone, 𝛽-eudesmol and hinesol) and polyacetylenes (atractylodin), has been reported to be related to symbiotic processes
Abbreviations – ABA, abscisic acid; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; EF1𝛼, elongation factor 1 alpha; Flu, fluridone; FOS, fosmidomycin sodium salt; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HPLC, high-performance liquid chromatography; IPP, isopentenyl pyrophosphate; JA, jasmonate acid; MEP, 2C-methyl-D-erythritol 4-phosphate; MEV, mevinolin; MS, Murashige and Skoog; MVA, mevalonate; NAA, naphthaleneacetic acid; PAC, paclobutrazol; SA, salicylic acid.
Physiol. Plant. 2014
between endophytic fungus AL12 and A. lancea. Signal molecules, such as nitric oxide (NO), salicylic acid (SA), jasmonate acid (JA) and brassinolide, have been revealed to be involved in AL12-induced volatile accumulation in A. lancea (Wang et al. 2011, Ren and Dai 2012, 2013). SA regulates not only systemic acquired resistance toward stresses in plants but also plant development progress (Vlot et al. 2009). Its role in regulating the secondary metabolite accumulation of plants induced by biotic (endophytic and arbuscular mycorrhizal fungi) and abiotic factors (i.e. ammonium) has also been noted (Xu et al. 2009, Zhou and Zhong 2011, Zhang et al. 2013). Abscisic acid (ABA) predominantly functions as a key signal in regulating plant processes relevant to seed germination, stomatal closure and abiotic stress tolerance (Lee and Luan 2012). ABA has also been noted to act as an abiotic elicitor for plant biosynthesis of bioactive compounds, such as anthocyanin and puerarin (Gagné et al. 2011, Sun et al. 2012). Although endogenous ABA has been reported to be required for flavonoid and catharanthine accumulation elicited by endophyte Sphaeropsis sp. and pathogen Phytophthora boehmeriae in Ginkgo biloba (Hao et al. 2010) and Catharanthus roseus (Chen et al. 2013), respectively, studies related to the involvement of ABA in microorganism-activated plant secondary metabolism have largely remained obscure. Interplays between hormone signaling pathways have emerged as an important regulatory mechanism by which plants tailor their responses to varied stress. Studies have revealed antagonistic or synergistic regulatory relationships between SA and JA of the immune network when plants are challenged by pathogens (Thaler et al. 2012). Xu et al. (2009) and Ren and Dai (2012) showed that signaling crosstalk between SA and JA results in a complementary action in mediating endophyte-induced secondary metabolite accumulation of plants. The ability of ABA to interact with the SA/JA pathways under stress is also remarkable (Robert-Seilaniantz et al. 2011). This hormonal crosstalk has been thought to optimize plant immune responses against attackers that stimulate both SA and JA pathway or prioritize one pathway over the other when plants are simultaneously or sequentially attacked by different enemies (Van der Does et al. 2013). However, compared with the well-studied functions of SA and JA, the roles played by the ‘abiotic stress’ hormone ABA and its interplay with other hormones in plant secondary metabolism are less well understood. Endophytes can improve plant performance by inducing mild but effective defenses without causing visible disease symptoms (Kusari et al. 2012). Unlike the slight growth inhibition by pathogenic elicitors, endophytic fungi stimulate both plant biomass and
secondary metabolism (Wang et al. 2011) and therefore are regarded as prospective microbial resources for producing bioactive chemicals (Kusari et al. 2012). Contrary to non-host elicitors (i.e. heavy metal ions and yeast) that have mainly been applied in suspension and hairy roots cultures (Zhao et al. 2010, Gandi et al. 2012), the balanced endophyte–host symbiosis provides a sustainable way to improve desirable natural products of medicinal herbs, especially at the whole-plant level and in their natural habitats. Although reports have noted that the inoculation of endophytic fungi and plant growth-promoting rhizobacteria is an effective strategy to improve the production of secondary metabolites in plants (Cappellari et al. 2013, Ming et al. 2013), only a few studies have examined these aspects in endosymbiotic bacteria. For example, the production of bisindole alkaloids and artemisinin could be enhanced by the inoculation of endophytic Micrococcus sp. and actinobacterium Pseudonocardia sp. in glasshouse experiments (Li et al. 2012, Tiwari et al. 2013). Nevertheless, studies have rarely considered the underlying signaling mechanisms whereby endophytic bacteria may affect the secondary metabolism of plants. Owing to the concurrent presence of endosymbionts, a given endophyte is believed to directly or indirectly interact with other associated endophytes within plants (Wang et al. 2013). Cross-communication between endophytes within their hosts has been attributed to a significantly higher diversity of natural products than those reported for individual, axenic cultures in laboratory work (Kusari et al. 2012). Considering the reciprocal interplays between endosymbionts, we are not aware of any relationships or distinctions between signaling pathways elicited by cross-species endophytes. Despite numerous studies on the elicitation of plant secondary metabolism by endophytic fungi, the cellular relationships between the host secondary metabolism and endophytic colonization by bacteria still needs to be elucidated. Therefore, this work investigated the effects of endophyte Acinetobacter sp. ALEB16 on the volatile biosynthesis of A. lancea plantlets and the cross-communication between SA and ABA signaling involved in the elicitation process, which has been scarcely studied.
Materials and methods Plant materials and culture conditions Meristem cultures of A. lancea were established as previously described (Wang et al. 2011). Briefly, sterilized plantlets were maintained in Murashige and Skoog (MS) medium [supplemented with 0.3 mg l−1 naphthaleneacetic acid (NAA), 2.0 mg l−1 Physiol. Plant. 2014
6-benzyladenine, 30 g l−1 sucrose and 8% agar] in 100-ml Erlenmeyer flasks. The meristem cultures were then divided and transplanted into rooting medium (1/2 MS, 0.25 mg l−1 NAA). The cultures were maintained in a growth chamber (25/18∘ C day/night, with a light intensity of 80 μmol m−2 s−1 and a photoperiod of 12 h) and sub-cultured every 30 days. Endophytic bacterium and treatments The endophytic bacterium ALEB16 (Acinetobacter sp.) was isolated from the leaves of surface-sterilized A. lancea, cultured in Luria-Bertani liquid medium, and incubated for 24 h at 30∘ C on a shaker at 200 rpm. The bacterial cells were collected by centrifugation at 4000 g for 15 min and re-suspended in sterile distilled water. The bacterial suspensions were then diluted to 0.7 OD600 , which corresponded to a concentration of 106 cells ml−1 of suspension. The density of bacterial cell suspensions was determined with a spectrophotometer. Thirty-day-old rooting plantlets were bacterized with a 200-μl aliquot of bacterial culture. The control plantlets were inoculated with an equal volume of distilled water. The successful colonization by ALEB16 on A. lancea was verified in our previous report (Wang et al. 2013). Chemicals and treatments The solutions of specific inhibitors and signaling molecules, including paclobutrazol (PAC, 0.5, 3 or 9 mM), fluridone (Flu, 0.02, 0.1 or 1 mM), fosmidomycin sodium salt (FOS, 200 μM), mevinolin (MEV, 30 μM), ABA (200 μM) and SA (2 mM) were dissolved in water or 0.2% DMSO. MEV was previously converted to water-soluble sodium salts, as described by Hagen and Grunewald (2000). FOS was purchased from Toronto Research Chemicals Inc. (North York, Canada), and all other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO). All of the above solutions were sterilized by filtering through 0.22-μm sterile filters. Inhibitors were sprayed on the seedling leaves until dripping for a 1-day infiltration period as the inhibitor pre-treatment (Towler and Weathers 2007, Ren and Dai 2012). The reagents and their dosages used here were chosen based on our preliminary study. An equal volume of vehicle solvent was added to plantlets as the control. Three replicates were performed for each treatment. Extraction of volatile oils and gas chromatography analysis After the treatments, harvested plantlets were removed from agar and dried in a heating and drying oven at Physiol. Plant. 2014
36∘ C (Zhang et al. 2009, Ren and Dai 2012, 2013). After drying for at least 48 h, the plantlets were weighed every 2 h until a constant weight (DW) was reached. The method of Zhang et al. (2009) was employed to extract volatile oils from whole plantlets of A. lancea. The dried plants (1 g) were ground in liquid nitrogen and extracted in 4 ml cyclohexane for 10 h. After sonication and centrifugation, the supernatant was dried with anhydrous sodium sulfate and filtered with 0.22-μm microporous membranes before determination. Gas chromatography was carried out using an Agilent 7890A gas chromatograph (Santa Clara, CA) equipped with a flame ionization detector. The temperature program and quantitative analyses of the main volatile components (𝛽-eudesmol, atractylone, hinesol and atractylodin) were essentially performed as previously described with minor modifications (Ren and Dai 2012). An Agilent DB-1HT (30 m × 0.32 mm × 0.10 μm) column was used with the following temperature program: the column was held at 100∘ C for 4 min after injection, and the temperature was increased by 10∘ C min−1 to 140∘ C, held for 10 min, increased by 10∘ C min−1 to 220∘ C, held for 10 min and increased by 10∘ C min−1 to 260∘ C, held for 2 min. Nitrogen was used as a carrier at a flow rate of 0.8 ml min−1 . To measure the volatile oils, SA and ABA, the extraction and the quantification were repeated three times per sample. Measurement of SA SA was extracted following the method of Verberne et al. (2002) with some modifications. Five grams of whole plantlets collected 25 days after inoculation were ground in liquid nitrogen and extracted in 5 ml methanol by sonication. After centrifugation, the supernatant was rotary evaporated, and the residue was re-suspended in 250 μl of 5% trichloroacetic acid. The mixture was re-extracted with 800 μl acetic acid ester:cyclohexane (1:1, v/v). The organic phase was rotary evaporated until dry, dissolved in 600 μl of mobile phase (methanol:2% acetic acid:H2 O, 50:40:10, v: v: v) and filtered with a 0.22-μm microporous membrane for determination. The SA samples were quantified via high-performance liquid chromatography (HPLC) using a reverse-phase column (Hedera Packing Material Lichrospher 5-C18, 4.6 × 200 mm, 5 μm; Bonna-Agela Technologies, Wilmington, DE). The flow rate was 0.5 ml min−1 . SA was detected at 290 nm at 25∘ C (Wang et al. 2011). An Agilent 1290 Infinity with ultraviolet detectors and AGILENT CHEMSTATION Software were used for the quantitative and qualitative analysis of SA and ABA.
ABA extraction and quantification
Results
The free ABA content was determined using the method described by Gagné et al. (2011), with some modifications. Two grams of whole plantlets collected 25 days after inoculation were ground in liquid nitrogen and extracted in 15 ml 80% methanol (v/v) at 4∘ C overnight. After centrifugation, the extract was rotary evaporated to remove methanol and adjusted to pH 3.0 (±0.05). The residue was subjected to three consecutive liquid–liquid extractions with acetic acid ester (1:1, v/v). Finally, the combined organic phase was rotary evaporated until dry, dissolved in 600 μl of HPLC mobile phase (acetonitrile: 1.8% acetic acid, 1:1, v/v), and filtered with a 0.22-μm microporous membrane for determination. The ABA samples were quantified by HPLC using a reverse-phase column (Hedera Packing Material Lichrospher). The flow rate was 0.5 ml min−1 and the column was maintained at 25∘ C. The detection was carried out at 260 nm.
Bacterium-induced volatile oil accumulation
RNA extraction and quantitative RT-PCR The total leaf RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA) as described by Xu et al. (2013). First-strand cDNA was synthesized from 1 μg of total RNA (PrimeScript One Step RT Reagent Kit; Takara, Dalian, China). Real-time qPCR was performed using the DNA Engine Opticon 2 Real-time PCR Detection System (Bio-Rad, Hercules, CA) and SYBR green probe (SYBR Premix Ex Taq system; Takara). Elongation factor 1 alpha (EF1𝛼), a constitutively expressed gene (Zhang et al. 2007), was used as a reference gene for normalization according to Ren and Dai (2012, 2013). The 2−ΔΔCt method was employed for the relative quantification of DXR and HMGR (Livak and Schmittgen 2001). The RT-PCR results were based on the average of triplicates and the standard deviation (SD) is shown. The thermal profile used consisted of an initial denaturation step at 95∘ C for 90 s, followed by 40 cycles of 95∘ C for 30 s, 57∘ C for 30 s and 72∘ C for 30 s. The nucleotide sequences of all primers used are listed in Table S1. Statistical analysis Experiments were performed at least in triplicate, and the data are expressed as the mean ± SD. Statistical significance was determined via a one-way ANOVA followed by Duncan’s multiple range test with the SPSS 13.0 software (SPSS Inc., Chicago, IL). Differences in means were considered to be significant for P-values < 0.05. A Student’s t-test was used for statistical comparisons of two means.
Fig. 1A showed that the maximum contents of hinesol, 𝛽-eudesmol, atractylone and atractylodin in bacterium-treated plantlets increased 0.72-, 0.24-, 0.82and 0.42-fold over the control levels, respectively. The total production of volatile oils of A. lancea plantlets also significantly increased, being approximately 0.30-fold higher than the control levels 25 days after bacterium treatment (Fig. 1B). However, the content ratios of the four constituents remained almost unchanged after bacterial inoculation. Because the endophytic bacterium ALEB16 exerted no effects on plantlet growth (Fig. 2), the results indicated that the bacterium might stimulate the secondary metabolite biosynthesis of the plantlets. Involvement of ABA in bacterium-induced volatile oil production Fig. 3A showed that the ABA contents of the plantlets significantly increased over time after ALEB16 treatment, suggesting that the bacterial strain may trigger the biosynthesis of ABA in A. lancea. To investigate whether ABA was involved in the bacterium-elicited accumulation of volatile oils, the effects of ABA inhibitors on the production of bacterium-induced secondary metabolites in A. lancea were determined. Flu is an inhibitor of phytoene desaturase, which converts phytoene to phytofluene in the pathway of carotenoids (precursors of ABA) biosynthesis and is applied to prevent ABA biosynthesis in G. biloba, Pueraria thomsnii and Psychotria brachyceras (Yoshioka et al. 1998, Hao et al. 2010, Sun et al. 2012, do Nascimento et al. 2013). As shown in Fig. 3B, the application of Flu suppressed not only the bacterium-induced ABA production but also the bacterium-induced volatile oil accumulation. The volatile concentrations of plantlets treated with 0.02 and 0.1 mM Flu were 15.69 and 23.92% lower than that of the bacterium-treated groups, respectively, while the plantlets treated with 1 mM Flu were visibly browned and withered. Therefore, the medium concentration of Flu (0.1 mM) was chosen for the follow-up experiments. These results indicated that the generation of ABA played an essential role in bacterium-induced volatile oil biosynthesis of A. lancea. Involvement of SA in bacterium-induced volatile oil accumulation The results showed that in addition to ABA, SA was also involved in the ALEB16-induced volatile oil production Physiol. Plant. 2014
Fig. 1. Time course of volatile oil accumulation of Atractylodes lancea plantlets. (A) Bacterium-induced production of four main volatile oils (hinesol, 𝛽-eudesmol, atractylone and atractylodin) in A. lancea plantlets. (B) Total contents of the four main volatile oils produced by bacterized plantlets. Thirty-day-old plantlets treated with 200 μl of bacterial suspension (Bacterium) or with an equal volume of sterile distilled water (Control) were harvested, and the volatile oil contents were measured after various time periods. Values are the means of three independent experiments ± SD. The lowercase letters indicate significant differences (P < 0.05).
of hosts (Fig. 4). The SA contents of the bacterium-treated plantlets gradually increased after bacterium treatment, reaching a maximum of 3.71 μg g−1 at 25 days and then decreased (Fig. 4A). To investigate whether SA signaling was involved in the bacterium-elicited volatile oil accumulation, the SA-inhibitor PAC was applied to determine its effects on Physiol. Plant. 2014
the volatile oil contents of A. lancea. PAC is an effective SA biosynthesis-related benzoic acid hydroxylase inhibitor (Leon et al. 1995) and has been applied in many plants (i.e. Pisum sativum and Taxus chinensis) to abolish SA accumulation (Liu et al. 2006, Zhou and Zhong 2011). In our previous study, PAC has been demonstrated to effectively block SA generation and volatile oil
ALEB16-induced volatile accumulation by Flu and PAC further confirmed our results. Dependence of ABA-induced volatile oil accumulation on SA
Fig. 2. Effects of bacterium on growth of Atractylodes lancea plantlets at 30-day intervals. Thirty-day-old plantlets treated with 200 μl of bacterial suspension were harvested at the times indicated in the figure to determine the plantlet biomass (DW). Plantlets that received the same volume of distilled water were used as a control. Values are the means of three independent experiments ± SD.
accumulation when plantlets are inoculated with fungal endophyte AL12 (Ren and Dai 2012). Fig. 4B shows that PAC suppressed both the increases in SA and volatile oil production induced by the bacterium. The contents of volatile oils in Bacterium + 3 mM PAC and Bacterium + 9 mM PAC treatments both decreased to the control levels. Nevertheless, the application of PAC only slightly promoted the accumulation of volatile oils, and no significant differences were observed. Therefore, 3 mM PAC was selected for the follow-up experiments. These data implied that the volatile oil accumulation induced by bacterium ALEB16 likely depended on the endogenous ABA and SA biosynthesis of A. lancea. Interplays between bacterium-induced volatile oil production on ABA generation and SA biosynthesis The results above showed that ABA generation and SA biosynthesis were two signaling events involved in the bacterium-induced volatile accumulation of A. lancea plantlets. However, their relationship and involvement in this process were not yet clear. We found that the bacterium-induced SA generation was strongly inhibited by the ABA inhibitor Flu, whereas the SA biosynthesis inhibitor did not significantly affect the bacterium-induced ABA production (Fig. 5). This finding indicated that the ABA and SA signaling pathways were closely linked and that ABA might act as an upstream signaling molecule of endophyte-induced SA biosynthesis and volatile accumulation in A. lancea plantlets. The data showing that the application of exogenous ABA and SA could reverse the inhibition of
To investigate if the exogenous ABA-induced volatile oil accumulation also depended on SA signaling, the effects of exogenous ABA, SA and their inhibitors on the generation of volatile oils, ABA and SA were determined. As shown in Fig. 6, both exogenous ABA and SA effectively induced volatile oil accumulation in A. lancea plantlets. The increased volatile production induced by ABA was completely abolished by PAC, whereas the increased volatile contents induced by SA were almost unaffected by Flu. Additionally, exogenous ABA could promote the generation of SA; SA application did not appear to influence ABA content in plantlets. The application of 0.1 mM Flu or 3 mM PAC itself did not exert significantly adverse effects on the accumulation of volatile oils and hormones in plantlets compared to the control, which could be due to the relatively low concentration of inhibitors employed (Xu et al. 2009, Hao et al. 2010). These findings further demonstrated that ABA was localized upstream of SA and controlled by the production of SA in the signaling pathway for ALEB16-induced volatile oil biosynthesis of A. lancea. Effect of MEV and FOS on bacterium-induced volatile sesquiterpenoid production The three sesquiterpenoids (atractylone, hinesol and 𝛽-eudesmol) of A. lancea are biosynthesized mainly via two pathways, MVA (mevalonate) and MEP (2C-methyl-D-erythritol 4-phosphate), and MEV and FOS are two specific inhibitors of the two pathways, respectively (Fig. 8A). To further investigate the regulatory mechanism involved in the biosynthesis of volatile sesquiterpenoids induced by ALEB16, the effects of FOS and MEV on bacterium-induced volatile oil accumulation were studied. The enhanced volatile sesquiterpenoid production by the bacterium was mainly inhibited by MEV and only slightly inhibited by FOS (Fig. 7). The contents of atractylone, hinesol and 𝛽-eudesmol in Bacterium + MEV treatment were reduced to the control levels (by 47.06, 52.59 and 20.15%, respectively), whereas the contents of these components showed only relatively slight decreases of 20.20, 43.77 and 9.03%, respectively, in Bacterium + FOS treatment compared to the bacterium treatment. These results suggested that ALEB16 treatment activated both the MVA and MEP pathways and that the MVA pathway may primarily contribute to the biosynthesis of volatile sesquiterpenoids. Physiol. Plant. 2014
Fig. 3. Involvement of ABA in endophytic bacterium-induced volatile oil accumulation in Atractylodes lancea plantlets. (A) Bacterium-induced ABA production in plantlets. Thirty-day-old plantlets treated with 200 μl bacterial suspensions were harvested to determine the ABA content at the times indicated in the figure. The control received sterile water. Values are the means of three independent experiments. Bars represent standard errors. Asterisks denote significant differences from the control (t-test; *P < 0.05; **P < 0.01). (B) Effects of Flu (ABA inhibitor) on bacterium-induced volatile oil accumulation after 25 days. Inhibitors (0.02, 0.1 or 1 mM Flu) were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
Fig. 4. Involvement of SA in endophytic bacterium-induced volatile oil accumulation of Atractylodes lancea plantlets. (A) Bacterium-induced SA production of the plantlets. Thirty-day-old plantlets treated with 200 μl bacterial suspensions were harvested to determine the SA content at the times indicated in the figure. The control received vehicle solvent. Values are the means of three independent experiments. Bars represent standard errors. Asterisks denote significant differences from the control (t-test; *P < 0.05; **P < 0.01). (B) Effects of PAC (SA inhibitor) on bacterium-induced volatile oil accumulation after 25 days. Inhibitors (0.5, 3 or 9 mM PAC) were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
Transcriptional responses of the sesquiterpenoid-related biosynthetic genes to bacterium, ABA and SA treatments The transcriptional expression levels of the sesquiterpenoid-related genes HMGR and DXR (Fig. 8A) were studied in order to further reveal the transcriptional regulation of terpenoid biosynthesis in A. lancea by bacterium ALEB16, ABA and SA application. As shown in Fig. 8B, ALEB16, ABA and SA treatments significantly upregulated the levels of HMGR gene expression by Physiol. Plant. 2014
19.60-, 24.36- and 28.33-fold over the control levels. The transcriptional levels of DXR in bacterium, ABA and SA treatments increased by 3.57-, 5.07- and 11.24-fold compared with the control. Moreover, Flu and PAC predominantly inhibited the upregulation of the two key biosynthetic genes induced by bacterium and ABA treatments. Conversely, the SA-induced gene expression of HMGR was notably suppressed by PAC, while no substantial decrease was noted in SA + Flu treatment, consistent with our observations described
Fig. 5. Interactions between ABA and SA signaling pathways for bacterium-induced volatile oil accumulation in Atractylodes lancea plantlets. Thirty-day-old plantlets treated with 200 μl bacterial suspensions, 0.1 mM Flu, 3 mM PAC, 200 μM ABA and 2 mM SA were harvested after 25 days to determine the volatile oil, ABA and SA contents. Inhibitors were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
Fig. 7. Effects of 200 μM FOS and 30 μM MEV on bacterium-induced volatile oil accumulation in Atractylodes lancea plantlets. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
depended on SA and that ABA was upstream of the SA signaling.
Discussion
Fig. 6. Dependence of ABA-induced volatile oil accumulation on SA in Atractylodes lancea plantlets. Thirty-day-old plantlets treated with 200 μM ABA, 2 mM SA, 3 mM PAC and 0.1 mM Flu were harvested 25 days later to determine the volatile oil, ABA and SA contents. Inhibitors were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
above (Fig. 6). Although the expression levels of DXR gene decreased by 30.41 and 9.48% compared to SA-treated plantlets in response to SA + PAC and SA + Flu treatment, respectively, the three groups did not significantly differ. Accordingly, these results indicated that both the MVA and MEP pathways were involved in the sesquiterpenoid biosynthesis in A. lancea induced by the three elicitors and that the MVA pathway primarily contributed to the biosynthetic process. These results further confirmed that ALEB16- and ABA-induced volatile accumulation
This study showed that the beneficial bacterium Acinetobacter sp. ALEB16 promoted the biosynthesis of volatile oils in A. lancea (Fig. 1) without notably affecting plantlet biomass (Fig. 2). The involvement of ABA and SA in bacterium-induced volatile oil production was demonstrated in our subsequent experiments: (1) the bacterium activated the endogenous generations of ABA and SA in A. lancea plantlets (Figs 3A and 4A). (2) ABA- and SA-specific inhibitors blocked the bacterium-induced volatile oil accumulation (Figs 3B and 4B). (3) Pre-treatment of plantlets with the ABA inhibitor suppressed not only the ALEB16-induced ABA production but also the induced SA generation (Fig. 5), indicating that ABA and SA were at least partially interrelated in the signaling pathway within which ABA was localized upstream of SA. (4) The SA inhibitor mostly abolished the ABA-induced endogenous SA and volatile oil accumulations, which further demonstrated that SA was involved in transducing the signal from ABA to induce volatile accumulation (Fig. 6). To the best of our knowledge, this study is the first to report the signaling crosstalk involved in bacterial endophyte-induced secondary metabolite accumulation of its host plant, especially at the whole-plant level. The stress hormone ABA regulates plant physiological and developmental processes, thereby allowing plants to adapt to stress conditions (Lee and Luan 2012). Consistent with this work (Figs 6 and 8), exogenous Physiol. Plant. 2014
Fig. 8. Proposed pathways of sesquiterpenoid biosynthesis (A) and expression levels of HMGR and DXR in Atractylodes lancea plantlets after 25 days (B). HMGS, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase; HMGR, HMG-CoA reductase; MEP, 2C-methyl-D-erythritol 4-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Adapted from Moses et al. (2013). Solid lines indicate established pathways; dashed lines represent less well-characterized pathways. Lines ending with a bar indicate the repression of enzymatic activities by inhibitors (MEV, FOS). In (B), the gene expression levels in 30-day-old plantlets treated with 200 μl of bacterial suspension, 200 μM ABA, 2 mM SA, 3 mM PAC and 0.1 mM Flu are shown. The relative expression levels were obtained using the 2−ΔΔCt method (see section Materials and methods). Data are presented as mean values of relative gene expression ± SD (n = 3) which are normalized against the reference gene EF1𝛼. Bars with different lowercase letters are significantly different (P < 0.05).
ABA can act as an elicitor of secondary metabolism that upregulates the transcriptional and enzymatic activities of biosynthesis genes (Lacampagne et al. 2010, do Nascimento et al. 2013). Although Salomon et al. (2013) reported that rhizobacterial inoculation alleviates water stress by eliciting the synthesis of ABA and defense terpenes in Vitis vinifera, these terpenoids are induced via an ABA-independent mechanism. Our work reveals the involvement of ABA in endophyte-induced accumulation of host secondary metabolites, whereas other reports mainly focus on the implication of ABA in abiotic stress-regulated biosynthesis of plants (Yang et al. 2012). These findings reveal multifaceted roles for ABA at the crossroads of biotic and abiotic stress responses. Physiol. Plant. 2014
Hormone crosstalk provides fine-tuned regulations for plants to respond to varied environmental cues (Lee and Luan 2012). Most of the information on biochemical interactions between hormones has arisen from studies of plant defense responses. SA is known to activate distinct sets of defense-related genes in plant immunity. Antagonisms exist between SA and JA/ethylene-dependent defenses that prioritize one hormone-dependent pathway over another when plants are confronted with pathogens (Vlot et al. 2009). Jiang et al. (2010) demonstrated that ABA suppresses SA-induced resistance responses, accompanied by an enhanced susceptibility to pathogen infection in rice. However, our experiment showed that ABA was
localized upstream of SA in bacterium-induced volatile accumulation in A. lancea and that no obvious antagonism or complementary interplays were found between the two hormones. Similarly, Yang et al. (2012) showed that drought and exogenous ABA triggered methyl jasmonate accumulation by activating ABA signaling pathway to stimulate tanshinone production. Hossain et al. (2011) demonstrated the involvement of endogenous ABA in methyl jasmonate-induced stomatal closure. Additionally, NO burst is another common event that precedes SA accumulation in many plant defense responses (Vlot et al. 2009). We previously reported that NO mediates fungus-induced volatile accumulation via a SA-dependent signaling pathway (Wang et al. 2011). However, NO bursts were not detected at this 5-day interval measurement (data not shown), probably on account of the early and rapid generation of NO just after bacterial inoculation. Plant terpenoid biosynthesis begins with the generation of isopentenyl pyrophosphate, the principal precursor, via the cytosolic MVA pathway or the plastidial MEP pathway (Moses et al. 2013). 3-Hydroxy-3-methyl glutaryl-CoA reductase (HMGR) is the rate-limiting enzyme in the MVA pathway, and 1-deoxy-D-xylulose 5phosphate reductoisomerase (DXR) is the second enzyme in the MEP pathway. The amplification and quantification of DXR in A. lancea were first reported. The results showed that bacterium-induced volatile accumulation was mostly abolished by MEV and only partially inhibited by FOS (Fig. 7), consistent with the observation that the HMGR-dependent MVA pathway was the main contributor to bacterium-, ABA- and SA-induced volatile accumulation. Similarly, Mansouri et al. (2009) reported that ABA application induces the biosynthesis of secondary terpenoids via the MEP pathway, while it downregulates the biosynthesis of primary terpenoid metabolites from the MEP and MVA pathways in Cannabis sativa. Moreover, the transcriptional level of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the rate-limiting enzyme in the MEP pathway, requires further study. These findings indicate that crosstalk exists between the two biosynthetic pathways for the bacterium-induced sesquiterpenoid accumulation, consistent with studies on diterpenes (Yang et al. 2012). Endophytes can enhance host fitness benefits by activating mild immune responses and promoting the production of host secondary metabolites (Van Wees et al. 2008). Endophytes have also emerged as an intrinsic factor that contributes to the superior quality of geo-authentic medicinal herbs in natural habitats in addition to environmental factors (i.e. topography, soil and climate) (Jia et al. 2009), which is in line with our
previous studies. As biotic elicitors, bacteria have many advantages compared with fungi, such as a short period for culture and elicitation (Jung et al. 2003). We also noted that bacterium ALEB16 only enhanced volatile biosynthesis, whereas endophytic fungus AL12 stimulated both plant biomass and volatile production in A. lancea (Wang et al. 2011). These discrepancies might be due to the differing colonization densities and host sensitivities to invaders of different species. Moreover, the total volatile contents in A. lancea induced by fungus AL12 increased by 74.97% (Ren and Dai 2012), which is a larger increase than that induced by bacterium ALEB16. Previous studies found that ALEB16 triggers weaker defense responses than fungus AL16 (Wang et al. 2013). This might be attributed to the relatively low invasive ability and small size of bacteria, allowing them to occupy smaller niches and trigger weaker host responses than fungi. Additionally, AL12 specifically boosted the ratios of hinesol and atractylone; whereas the concentrations of each volatile constituent equally increased in ALEB16 treatment (Fig. 1). Li et al. (2012) reported that elicitors can regulate gene expressions of enzymes for side chain modifications during the later stages of biosynthesis and thereby alter the constituent ratios of plant secondary metabolite. Thus, our results imply that the promotional model depends on the characteristics of diverse endophytes and that the simultaneous inoculation of endophytes with a variety of beneficial models might facilitate an oriented or synergistic biosynthesis of targeted bioactive products of plants. Signal perception by plant receptors is the first committed step of the elicitor signal transduction pathway. Fungal cell constituents (i.e. chitin, oligosaccharides and lipid derivatives) exhibit elicitor activity across different plant species (Ming et al. 2013). Flagellin and lipopolysaccharides fractions have been thought to act as the elicitor activity domain of invasive bacteria (Zhao et al. 2005, 2010). Because the Gram-negative Acinetobacter is non-motile due to a lack of flagellin, lipopolysaccharides might act as the active component responsible for triggering phytohormone and volatile biosynthesis of A. lancea. In addition to the promoting effect of bacterium ALEB16 on the biosynthetic ability of A. lancea to produce endogenous ABA and SA, changes in the hormone content of bacterized plantlets might be also a result of the exogenous application of hormones produced by ALEB16 itself which requires our further investigation (Arkhipova et al. 2005, Salomon et al. 2013). This work, along with our previous studies, demonstrates that the balanced host–endophyte (bacteria and fungi) symbiosis serves as a promising means to enhance Physiol. Plant. 2014
the production of natural products and that a novel interaction between ABA and SA exists at the biosynthetic level in bacterized plants. Additionally, microbial interactions also play a role in the onset of plant metabolism (Kusari et al. 2012). The signaling cascades and crosstalks involved in endosymbiont-elicited plant secondary metabolism should be more complicated when plants are colonized by multiple symbionts in natural situations. Thus, the chemical ecological interaction between endophytes and the underlying signaling pathways needs to be elucidated in order to fully exploit and modulate their high potential of natural product biosynthesis in plants. Acknowledgements – This work is financially supported by National Natural Science Foundation of China (grant number 31070443), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Research and Innovation Project for College Graduates of Jiangsu Province (grant number CXZZ13_0414) and Integration of Production and Research Projects of Nanjing Science and Technology Commission (grant number 201306019).
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Edited by R. Terauchi
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Specific primer pair sequences used for cDNA amplification of key genes of the terpenoid biosynthetic pathway of Atractylodes lancea.
© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Involvement of abscisic acid and salicylic acid in signal cascade regulating bacterial endophyte-induced volatile oil biosynthesis in plantlets of Atractylodes lancea Xiao-Mi Wang, Bo Yang, Cheng-Gang Ren, Hong-Wei Wang, Jin-Yan Wang and Chuan-Chao Dai∗ Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
Correspondence *Corresponding author, e-mail: [email protected] Received 25 January 2014; revised 4 May 2014 doi:10.1111/ppl.12236
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.
Introduction Plants fend off pathogen attack or respond to abiotic stresses via a combination of constitutive and inducible defense mechanisms (De Vleesschauwer et al. 2013). The accumulation of bioactive secondary metabolites, which is regulated by cross-communicating signaling cascades, plays key roles in fine-tuning the innate
immune responses of plants to different stimuli (Yang et al. 2012). Atractylodes lancea is a traditional Chinese medicinal plant. The accumulation of volatile oils, including characteristic sesquiterpenoids (atractylone, 𝛽-eudesmol and hinesol) and polyacetylenes (atractylodin), has been reported to be related to symbiotic processes
Abbreviations – ABA, abscisic acid; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; EF1𝛼, elongation factor 1 alpha; Flu, fluridone; FOS, fosmidomycin sodium salt; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HPLC, high-performance liquid chromatography; IPP, isopentenyl pyrophosphate; JA, jasmonate acid; MEP, 2C-methyl-D-erythritol 4-phosphate; MEV, mevinolin; MS, Murashige and Skoog; MVA, mevalonate; NAA, naphthaleneacetic acid; PAC, paclobutrazol; SA, salicylic acid.
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between endophytic fungus AL12 and A. lancea. Signal molecules, such as nitric oxide (NO), salicylic acid (SA), jasmonate acid (JA) and brassinolide, have been revealed to be involved in AL12-induced volatile accumulation in A. lancea (Wang et al. 2011, Ren and Dai 2012, 2013). SA regulates not only systemic acquired resistance toward stresses in plants but also plant development progress (Vlot et al. 2009). Its role in regulating the secondary metabolite accumulation of plants induced by biotic (endophytic and arbuscular mycorrhizal fungi) and abiotic factors (i.e. ammonium) has also been noted (Xu et al. 2009, Zhou and Zhong 2011, Zhang et al. 2013). Abscisic acid (ABA) predominantly functions as a key signal in regulating plant processes relevant to seed germination, stomatal closure and abiotic stress tolerance (Lee and Luan 2012). ABA has also been noted to act as an abiotic elicitor for plant biosynthesis of bioactive compounds, such as anthocyanin and puerarin (Gagné et al. 2011, Sun et al. 2012). Although endogenous ABA has been reported to be required for flavonoid and catharanthine accumulation elicited by endophyte Sphaeropsis sp. and pathogen Phytophthora boehmeriae in Ginkgo biloba (Hao et al. 2010) and Catharanthus roseus (Chen et al. 2013), respectively, studies related to the involvement of ABA in microorganism-activated plant secondary metabolism have largely remained obscure. Interplays between hormone signaling pathways have emerged as an important regulatory mechanism by which plants tailor their responses to varied stress. Studies have revealed antagonistic or synergistic regulatory relationships between SA and JA of the immune network when plants are challenged by pathogens (Thaler et al. 2012). Xu et al. (2009) and Ren and Dai (2012) showed that signaling crosstalk between SA and JA results in a complementary action in mediating endophyte-induced secondary metabolite accumulation of plants. The ability of ABA to interact with the SA/JA pathways under stress is also remarkable (Robert-Seilaniantz et al. 2011). This hormonal crosstalk has been thought to optimize plant immune responses against attackers that stimulate both SA and JA pathway or prioritize one pathway over the other when plants are simultaneously or sequentially attacked by different enemies (Van der Does et al. 2013). However, compared with the well-studied functions of SA and JA, the roles played by the ‘abiotic stress’ hormone ABA and its interplay with other hormones in plant secondary metabolism are less well understood. Endophytes can improve plant performance by inducing mild but effective defenses without causing visible disease symptoms (Kusari et al. 2012). Unlike the slight growth inhibition by pathogenic elicitors, endophytic fungi stimulate both plant biomass and
secondary metabolism (Wang et al. 2011) and therefore are regarded as prospective microbial resources for producing bioactive chemicals (Kusari et al. 2012). Contrary to non-host elicitors (i.e. heavy metal ions and yeast) that have mainly been applied in suspension and hairy roots cultures (Zhao et al. 2010, Gandi et al. 2012), the balanced endophyte–host symbiosis provides a sustainable way to improve desirable natural products of medicinal herbs, especially at the whole-plant level and in their natural habitats. Although reports have noted that the inoculation of endophytic fungi and plant growth-promoting rhizobacteria is an effective strategy to improve the production of secondary metabolites in plants (Cappellari et al. 2013, Ming et al. 2013), only a few studies have examined these aspects in endosymbiotic bacteria. For example, the production of bisindole alkaloids and artemisinin could be enhanced by the inoculation of endophytic Micrococcus sp. and actinobacterium Pseudonocardia sp. in glasshouse experiments (Li et al. 2012, Tiwari et al. 2013). Nevertheless, studies have rarely considered the underlying signaling mechanisms whereby endophytic bacteria may affect the secondary metabolism of plants. Owing to the concurrent presence of endosymbionts, a given endophyte is believed to directly or indirectly interact with other associated endophytes within plants (Wang et al. 2013). Cross-communication between endophytes within their hosts has been attributed to a significantly higher diversity of natural products than those reported for individual, axenic cultures in laboratory work (Kusari et al. 2012). Considering the reciprocal interplays between endosymbionts, we are not aware of any relationships or distinctions between signaling pathways elicited by cross-species endophytes. Despite numerous studies on the elicitation of plant secondary metabolism by endophytic fungi, the cellular relationships between the host secondary metabolism and endophytic colonization by bacteria still needs to be elucidated. Therefore, this work investigated the effects of endophyte Acinetobacter sp. ALEB16 on the volatile biosynthesis of A. lancea plantlets and the cross-communication between SA and ABA signaling involved in the elicitation process, which has been scarcely studied.
Materials and methods Plant materials and culture conditions Meristem cultures of A. lancea were established as previously described (Wang et al. 2011). Briefly, sterilized plantlets were maintained in Murashige and Skoog (MS) medium [supplemented with 0.3 mg l−1 naphthaleneacetic acid (NAA), 2.0 mg l−1 Physiol. Plant. 2014
6-benzyladenine, 30 g l−1 sucrose and 8% agar] in 100-ml Erlenmeyer flasks. The meristem cultures were then divided and transplanted into rooting medium (1/2 MS, 0.25 mg l−1 NAA). The cultures were maintained in a growth chamber (25/18∘ C day/night, with a light intensity of 80 μmol m−2 s−1 and a photoperiod of 12 h) and sub-cultured every 30 days. Endophytic bacterium and treatments The endophytic bacterium ALEB16 (Acinetobacter sp.) was isolated from the leaves of surface-sterilized A. lancea, cultured in Luria-Bertani liquid medium, and incubated for 24 h at 30∘ C on a shaker at 200 rpm. The bacterial cells were collected by centrifugation at 4000 g for 15 min and re-suspended in sterile distilled water. The bacterial suspensions were then diluted to 0.7 OD600 , which corresponded to a concentration of 106 cells ml−1 of suspension. The density of bacterial cell suspensions was determined with a spectrophotometer. Thirty-day-old rooting plantlets were bacterized with a 200-μl aliquot of bacterial culture. The control plantlets were inoculated with an equal volume of distilled water. The successful colonization by ALEB16 on A. lancea was verified in our previous report (Wang et al. 2013). Chemicals and treatments The solutions of specific inhibitors and signaling molecules, including paclobutrazol (PAC, 0.5, 3 or 9 mM), fluridone (Flu, 0.02, 0.1 or 1 mM), fosmidomycin sodium salt (FOS, 200 μM), mevinolin (MEV, 30 μM), ABA (200 μM) and SA (2 mM) were dissolved in water or 0.2% DMSO. MEV was previously converted to water-soluble sodium salts, as described by Hagen and Grunewald (2000). FOS was purchased from Toronto Research Chemicals Inc. (North York, Canada), and all other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO). All of the above solutions were sterilized by filtering through 0.22-μm sterile filters. Inhibitors were sprayed on the seedling leaves until dripping for a 1-day infiltration period as the inhibitor pre-treatment (Towler and Weathers 2007, Ren and Dai 2012). The reagents and their dosages used here were chosen based on our preliminary study. An equal volume of vehicle solvent was added to plantlets as the control. Three replicates were performed for each treatment. Extraction of volatile oils and gas chromatography analysis After the treatments, harvested plantlets were removed from agar and dried in a heating and drying oven at Physiol. Plant. 2014
36∘ C (Zhang et al. 2009, Ren and Dai 2012, 2013). After drying for at least 48 h, the plantlets were weighed every 2 h until a constant weight (DW) was reached. The method of Zhang et al. (2009) was employed to extract volatile oils from whole plantlets of A. lancea. The dried plants (1 g) were ground in liquid nitrogen and extracted in 4 ml cyclohexane for 10 h. After sonication and centrifugation, the supernatant was dried with anhydrous sodium sulfate and filtered with 0.22-μm microporous membranes before determination. Gas chromatography was carried out using an Agilent 7890A gas chromatograph (Santa Clara, CA) equipped with a flame ionization detector. The temperature program and quantitative analyses of the main volatile components (𝛽-eudesmol, atractylone, hinesol and atractylodin) were essentially performed as previously described with minor modifications (Ren and Dai 2012). An Agilent DB-1HT (30 m × 0.32 mm × 0.10 μm) column was used with the following temperature program: the column was held at 100∘ C for 4 min after injection, and the temperature was increased by 10∘ C min−1 to 140∘ C, held for 10 min, increased by 10∘ C min−1 to 220∘ C, held for 10 min and increased by 10∘ C min−1 to 260∘ C, held for 2 min. Nitrogen was used as a carrier at a flow rate of 0.8 ml min−1 . To measure the volatile oils, SA and ABA, the extraction and the quantification were repeated three times per sample. Measurement of SA SA was extracted following the method of Verberne et al. (2002) with some modifications. Five grams of whole plantlets collected 25 days after inoculation were ground in liquid nitrogen and extracted in 5 ml methanol by sonication. After centrifugation, the supernatant was rotary evaporated, and the residue was re-suspended in 250 μl of 5% trichloroacetic acid. The mixture was re-extracted with 800 μl acetic acid ester:cyclohexane (1:1, v/v). The organic phase was rotary evaporated until dry, dissolved in 600 μl of mobile phase (methanol:2% acetic acid:H2 O, 50:40:10, v: v: v) and filtered with a 0.22-μm microporous membrane for determination. The SA samples were quantified via high-performance liquid chromatography (HPLC) using a reverse-phase column (Hedera Packing Material Lichrospher 5-C18, 4.6 × 200 mm, 5 μm; Bonna-Agela Technologies, Wilmington, DE). The flow rate was 0.5 ml min−1 . SA was detected at 290 nm at 25∘ C (Wang et al. 2011). An Agilent 1290 Infinity with ultraviolet detectors and AGILENT CHEMSTATION Software were used for the quantitative and qualitative analysis of SA and ABA.
ABA extraction and quantification
Results
The free ABA content was determined using the method described by Gagné et al. (2011), with some modifications. Two grams of whole plantlets collected 25 days after inoculation were ground in liquid nitrogen and extracted in 15 ml 80% methanol (v/v) at 4∘ C overnight. After centrifugation, the extract was rotary evaporated to remove methanol and adjusted to pH 3.0 (±0.05). The residue was subjected to three consecutive liquid–liquid extractions with acetic acid ester (1:1, v/v). Finally, the combined organic phase was rotary evaporated until dry, dissolved in 600 μl of HPLC mobile phase (acetonitrile: 1.8% acetic acid, 1:1, v/v), and filtered with a 0.22-μm microporous membrane for determination. The ABA samples were quantified by HPLC using a reverse-phase column (Hedera Packing Material Lichrospher). The flow rate was 0.5 ml min−1 and the column was maintained at 25∘ C. The detection was carried out at 260 nm.
Bacterium-induced volatile oil accumulation
RNA extraction and quantitative RT-PCR The total leaf RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA) as described by Xu et al. (2013). First-strand cDNA was synthesized from 1 μg of total RNA (PrimeScript One Step RT Reagent Kit; Takara, Dalian, China). Real-time qPCR was performed using the DNA Engine Opticon 2 Real-time PCR Detection System (Bio-Rad, Hercules, CA) and SYBR green probe (SYBR Premix Ex Taq system; Takara). Elongation factor 1 alpha (EF1𝛼), a constitutively expressed gene (Zhang et al. 2007), was used as a reference gene for normalization according to Ren and Dai (2012, 2013). The 2−ΔΔCt method was employed for the relative quantification of DXR and HMGR (Livak and Schmittgen 2001). The RT-PCR results were based on the average of triplicates and the standard deviation (SD) is shown. The thermal profile used consisted of an initial denaturation step at 95∘ C for 90 s, followed by 40 cycles of 95∘ C for 30 s, 57∘ C for 30 s and 72∘ C for 30 s. The nucleotide sequences of all primers used are listed in Table S1. Statistical analysis Experiments were performed at least in triplicate, and the data are expressed as the mean ± SD. Statistical significance was determined via a one-way ANOVA followed by Duncan’s multiple range test with the SPSS 13.0 software (SPSS Inc., Chicago, IL). Differences in means were considered to be significant for P-values < 0.05. A Student’s t-test was used for statistical comparisons of two means.
Fig. 1A showed that the maximum contents of hinesol, 𝛽-eudesmol, atractylone and atractylodin in bacterium-treated plantlets increased 0.72-, 0.24-, 0.82and 0.42-fold over the control levels, respectively. The total production of volatile oils of A. lancea plantlets also significantly increased, being approximately 0.30-fold higher than the control levels 25 days after bacterium treatment (Fig. 1B). However, the content ratios of the four constituents remained almost unchanged after bacterial inoculation. Because the endophytic bacterium ALEB16 exerted no effects on plantlet growth (Fig. 2), the results indicated that the bacterium might stimulate the secondary metabolite biosynthesis of the plantlets. Involvement of ABA in bacterium-induced volatile oil production Fig. 3A showed that the ABA contents of the plantlets significantly increased over time after ALEB16 treatment, suggesting that the bacterial strain may trigger the biosynthesis of ABA in A. lancea. To investigate whether ABA was involved in the bacterium-elicited accumulation of volatile oils, the effects of ABA inhibitors on the production of bacterium-induced secondary metabolites in A. lancea were determined. Flu is an inhibitor of phytoene desaturase, which converts phytoene to phytofluene in the pathway of carotenoids (precursors of ABA) biosynthesis and is applied to prevent ABA biosynthesis in G. biloba, Pueraria thomsnii and Psychotria brachyceras (Yoshioka et al. 1998, Hao et al. 2010, Sun et al. 2012, do Nascimento et al. 2013). As shown in Fig. 3B, the application of Flu suppressed not only the bacterium-induced ABA production but also the bacterium-induced volatile oil accumulation. The volatile concentrations of plantlets treated with 0.02 and 0.1 mM Flu were 15.69 and 23.92% lower than that of the bacterium-treated groups, respectively, while the plantlets treated with 1 mM Flu were visibly browned and withered. Therefore, the medium concentration of Flu (0.1 mM) was chosen for the follow-up experiments. These results indicated that the generation of ABA played an essential role in bacterium-induced volatile oil biosynthesis of A. lancea. Involvement of SA in bacterium-induced volatile oil accumulation The results showed that in addition to ABA, SA was also involved in the ALEB16-induced volatile oil production Physiol. Plant. 2014
Fig. 1. Time course of volatile oil accumulation of Atractylodes lancea plantlets. (A) Bacterium-induced production of four main volatile oils (hinesol, 𝛽-eudesmol, atractylone and atractylodin) in A. lancea plantlets. (B) Total contents of the four main volatile oils produced by bacterized plantlets. Thirty-day-old plantlets treated with 200 μl of bacterial suspension (Bacterium) or with an equal volume of sterile distilled water (Control) were harvested, and the volatile oil contents were measured after various time periods. Values are the means of three independent experiments ± SD. The lowercase letters indicate significant differences (P < 0.05).
of hosts (Fig. 4). The SA contents of the bacterium-treated plantlets gradually increased after bacterium treatment, reaching a maximum of 3.71 μg g−1 at 25 days and then decreased (Fig. 4A). To investigate whether SA signaling was involved in the bacterium-elicited volatile oil accumulation, the SA-inhibitor PAC was applied to determine its effects on Physiol. Plant. 2014
the volatile oil contents of A. lancea. PAC is an effective SA biosynthesis-related benzoic acid hydroxylase inhibitor (Leon et al. 1995) and has been applied in many plants (i.e. Pisum sativum and Taxus chinensis) to abolish SA accumulation (Liu et al. 2006, Zhou and Zhong 2011). In our previous study, PAC has been demonstrated to effectively block SA generation and volatile oil
ALEB16-induced volatile accumulation by Flu and PAC further confirmed our results. Dependence of ABA-induced volatile oil accumulation on SA
Fig. 2. Effects of bacterium on growth of Atractylodes lancea plantlets at 30-day intervals. Thirty-day-old plantlets treated with 200 μl of bacterial suspension were harvested at the times indicated in the figure to determine the plantlet biomass (DW). Plantlets that received the same volume of distilled water were used as a control. Values are the means of three independent experiments ± SD.
accumulation when plantlets are inoculated with fungal endophyte AL12 (Ren and Dai 2012). Fig. 4B shows that PAC suppressed both the increases in SA and volatile oil production induced by the bacterium. The contents of volatile oils in Bacterium + 3 mM PAC and Bacterium + 9 mM PAC treatments both decreased to the control levels. Nevertheless, the application of PAC only slightly promoted the accumulation of volatile oils, and no significant differences were observed. Therefore, 3 mM PAC was selected for the follow-up experiments. These data implied that the volatile oil accumulation induced by bacterium ALEB16 likely depended on the endogenous ABA and SA biosynthesis of A. lancea. Interplays between bacterium-induced volatile oil production on ABA generation and SA biosynthesis The results above showed that ABA generation and SA biosynthesis were two signaling events involved in the bacterium-induced volatile accumulation of A. lancea plantlets. However, their relationship and involvement in this process were not yet clear. We found that the bacterium-induced SA generation was strongly inhibited by the ABA inhibitor Flu, whereas the SA biosynthesis inhibitor did not significantly affect the bacterium-induced ABA production (Fig. 5). This finding indicated that the ABA and SA signaling pathways were closely linked and that ABA might act as an upstream signaling molecule of endophyte-induced SA biosynthesis and volatile accumulation in A. lancea plantlets. The data showing that the application of exogenous ABA and SA could reverse the inhibition of
To investigate if the exogenous ABA-induced volatile oil accumulation also depended on SA signaling, the effects of exogenous ABA, SA and their inhibitors on the generation of volatile oils, ABA and SA were determined. As shown in Fig. 6, both exogenous ABA and SA effectively induced volatile oil accumulation in A. lancea plantlets. The increased volatile production induced by ABA was completely abolished by PAC, whereas the increased volatile contents induced by SA were almost unaffected by Flu. Additionally, exogenous ABA could promote the generation of SA; SA application did not appear to influence ABA content in plantlets. The application of 0.1 mM Flu or 3 mM PAC itself did not exert significantly adverse effects on the accumulation of volatile oils and hormones in plantlets compared to the control, which could be due to the relatively low concentration of inhibitors employed (Xu et al. 2009, Hao et al. 2010). These findings further demonstrated that ABA was localized upstream of SA and controlled by the production of SA in the signaling pathway for ALEB16-induced volatile oil biosynthesis of A. lancea. Effect of MEV and FOS on bacterium-induced volatile sesquiterpenoid production The three sesquiterpenoids (atractylone, hinesol and 𝛽-eudesmol) of A. lancea are biosynthesized mainly via two pathways, MVA (mevalonate) and MEP (2C-methyl-D-erythritol 4-phosphate), and MEV and FOS are two specific inhibitors of the two pathways, respectively (Fig. 8A). To further investigate the regulatory mechanism involved in the biosynthesis of volatile sesquiterpenoids induced by ALEB16, the effects of FOS and MEV on bacterium-induced volatile oil accumulation were studied. The enhanced volatile sesquiterpenoid production by the bacterium was mainly inhibited by MEV and only slightly inhibited by FOS (Fig. 7). The contents of atractylone, hinesol and 𝛽-eudesmol in Bacterium + MEV treatment were reduced to the control levels (by 47.06, 52.59 and 20.15%, respectively), whereas the contents of these components showed only relatively slight decreases of 20.20, 43.77 and 9.03%, respectively, in Bacterium + FOS treatment compared to the bacterium treatment. These results suggested that ALEB16 treatment activated both the MVA and MEP pathways and that the MVA pathway may primarily contribute to the biosynthesis of volatile sesquiterpenoids. Physiol. Plant. 2014
Fig. 3. Involvement of ABA in endophytic bacterium-induced volatile oil accumulation in Atractylodes lancea plantlets. (A) Bacterium-induced ABA production in plantlets. Thirty-day-old plantlets treated with 200 μl bacterial suspensions were harvested to determine the ABA content at the times indicated in the figure. The control received sterile water. Values are the means of three independent experiments. Bars represent standard errors. Asterisks denote significant differences from the control (t-test; *P < 0.05; **P < 0.01). (B) Effects of Flu (ABA inhibitor) on bacterium-induced volatile oil accumulation after 25 days. Inhibitors (0.02, 0.1 or 1 mM Flu) were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
Fig. 4. Involvement of SA in endophytic bacterium-induced volatile oil accumulation of Atractylodes lancea plantlets. (A) Bacterium-induced SA production of the plantlets. Thirty-day-old plantlets treated with 200 μl bacterial suspensions were harvested to determine the SA content at the times indicated in the figure. The control received vehicle solvent. Values are the means of three independent experiments. Bars represent standard errors. Asterisks denote significant differences from the control (t-test; *P < 0.05; **P < 0.01). (B) Effects of PAC (SA inhibitor) on bacterium-induced volatile oil accumulation after 25 days. Inhibitors (0.5, 3 or 9 mM PAC) were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
Transcriptional responses of the sesquiterpenoid-related biosynthetic genes to bacterium, ABA and SA treatments The transcriptional expression levels of the sesquiterpenoid-related genes HMGR and DXR (Fig. 8A) were studied in order to further reveal the transcriptional regulation of terpenoid biosynthesis in A. lancea by bacterium ALEB16, ABA and SA application. As shown in Fig. 8B, ALEB16, ABA and SA treatments significantly upregulated the levels of HMGR gene expression by Physiol. Plant. 2014
19.60-, 24.36- and 28.33-fold over the control levels. The transcriptional levels of DXR in bacterium, ABA and SA treatments increased by 3.57-, 5.07- and 11.24-fold compared with the control. Moreover, Flu and PAC predominantly inhibited the upregulation of the two key biosynthetic genes induced by bacterium and ABA treatments. Conversely, the SA-induced gene expression of HMGR was notably suppressed by PAC, while no substantial decrease was noted in SA + Flu treatment, consistent with our observations described
Fig. 5. Interactions between ABA and SA signaling pathways for bacterium-induced volatile oil accumulation in Atractylodes lancea plantlets. Thirty-day-old plantlets treated with 200 μl bacterial suspensions, 0.1 mM Flu, 3 mM PAC, 200 μM ABA and 2 mM SA were harvested after 25 days to determine the volatile oil, ABA and SA contents. Inhibitors were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
Fig. 7. Effects of 200 μM FOS and 30 μM MEV on bacterium-induced volatile oil accumulation in Atractylodes lancea plantlets. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
depended on SA and that ABA was upstream of the SA signaling.
Discussion
Fig. 6. Dependence of ABA-induced volatile oil accumulation on SA in Atractylodes lancea plantlets. Thirty-day-old plantlets treated with 200 μM ABA, 2 mM SA, 3 mM PAC and 0.1 mM Flu were harvested 25 days later to determine the volatile oil, ABA and SA contents. Inhibitors were added 1 day prior to bacterial inoculation. The control received vehicle solvent. Values are the means of three independent experiments ± SD. Bars with different lowercase letters are significantly different (P < 0.05).
above (Fig. 6). Although the expression levels of DXR gene decreased by 30.41 and 9.48% compared to SA-treated plantlets in response to SA + PAC and SA + Flu treatment, respectively, the three groups did not significantly differ. Accordingly, these results indicated that both the MVA and MEP pathways were involved in the sesquiterpenoid biosynthesis in A. lancea induced by the three elicitors and that the MVA pathway primarily contributed to the biosynthetic process. These results further confirmed that ALEB16- and ABA-induced volatile accumulation
This study showed that the beneficial bacterium Acinetobacter sp. ALEB16 promoted the biosynthesis of volatile oils in A. lancea (Fig. 1) without notably affecting plantlet biomass (Fig. 2). The involvement of ABA and SA in bacterium-induced volatile oil production was demonstrated in our subsequent experiments: (1) the bacterium activated the endogenous generations of ABA and SA in A. lancea plantlets (Figs 3A and 4A). (2) ABA- and SA-specific inhibitors blocked the bacterium-induced volatile oil accumulation (Figs 3B and 4B). (3) Pre-treatment of plantlets with the ABA inhibitor suppressed not only the ALEB16-induced ABA production but also the induced SA generation (Fig. 5), indicating that ABA and SA were at least partially interrelated in the signaling pathway within which ABA was localized upstream of SA. (4) The SA inhibitor mostly abolished the ABA-induced endogenous SA and volatile oil accumulations, which further demonstrated that SA was involved in transducing the signal from ABA to induce volatile accumulation (Fig. 6). To the best of our knowledge, this study is the first to report the signaling crosstalk involved in bacterial endophyte-induced secondary metabolite accumulation of its host plant, especially at the whole-plant level. The stress hormone ABA regulates plant physiological and developmental processes, thereby allowing plants to adapt to stress conditions (Lee and Luan 2012). Consistent with this work (Figs 6 and 8), exogenous Physiol. Plant. 2014
Fig. 8. Proposed pathways of sesquiterpenoid biosynthesis (A) and expression levels of HMGR and DXR in Atractylodes lancea plantlets after 25 days (B). HMGS, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase; HMGR, HMG-CoA reductase; MEP, 2C-methyl-D-erythritol 4-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Adapted from Moses et al. (2013). Solid lines indicate established pathways; dashed lines represent less well-characterized pathways. Lines ending with a bar indicate the repression of enzymatic activities by inhibitors (MEV, FOS). In (B), the gene expression levels in 30-day-old plantlets treated with 200 μl of bacterial suspension, 200 μM ABA, 2 mM SA, 3 mM PAC and 0.1 mM Flu are shown. The relative expression levels were obtained using the 2−ΔΔCt method (see section Materials and methods). Data are presented as mean values of relative gene expression ± SD (n = 3) which are normalized against the reference gene EF1𝛼. Bars with different lowercase letters are significantly different (P < 0.05).
ABA can act as an elicitor of secondary metabolism that upregulates the transcriptional and enzymatic activities of biosynthesis genes (Lacampagne et al. 2010, do Nascimento et al. 2013). Although Salomon et al. (2013) reported that rhizobacterial inoculation alleviates water stress by eliciting the synthesis of ABA and defense terpenes in Vitis vinifera, these terpenoids are induced via an ABA-independent mechanism. Our work reveals the involvement of ABA in endophyte-induced accumulation of host secondary metabolites, whereas other reports mainly focus on the implication of ABA in abiotic stress-regulated biosynthesis of plants (Yang et al. 2012). These findings reveal multifaceted roles for ABA at the crossroads of biotic and abiotic stress responses. Physiol. Plant. 2014
Hormone crosstalk provides fine-tuned regulations for plants to respond to varied environmental cues (Lee and Luan 2012). Most of the information on biochemical interactions between hormones has arisen from studies of plant defense responses. SA is known to activate distinct sets of defense-related genes in plant immunity. Antagonisms exist between SA and JA/ethylene-dependent defenses that prioritize one hormone-dependent pathway over another when plants are confronted with pathogens (Vlot et al. 2009). Jiang et al. (2010) demonstrated that ABA suppresses SA-induced resistance responses, accompanied by an enhanced susceptibility to pathogen infection in rice. However, our experiment showed that ABA was
localized upstream of SA in bacterium-induced volatile accumulation in A. lancea and that no obvious antagonism or complementary interplays were found between the two hormones. Similarly, Yang et al. (2012) showed that drought and exogenous ABA triggered methyl jasmonate accumulation by activating ABA signaling pathway to stimulate tanshinone production. Hossain et al. (2011) demonstrated the involvement of endogenous ABA in methyl jasmonate-induced stomatal closure. Additionally, NO burst is another common event that precedes SA accumulation in many plant defense responses (Vlot et al. 2009). We previously reported that NO mediates fungus-induced volatile accumulation via a SA-dependent signaling pathway (Wang et al. 2011). However, NO bursts were not detected at this 5-day interval measurement (data not shown), probably on account of the early and rapid generation of NO just after bacterial inoculation. Plant terpenoid biosynthesis begins with the generation of isopentenyl pyrophosphate, the principal precursor, via the cytosolic MVA pathway or the plastidial MEP pathway (Moses et al. 2013). 3-Hydroxy-3-methyl glutaryl-CoA reductase (HMGR) is the rate-limiting enzyme in the MVA pathway, and 1-deoxy-D-xylulose 5phosphate reductoisomerase (DXR) is the second enzyme in the MEP pathway. The amplification and quantification of DXR in A. lancea were first reported. The results showed that bacterium-induced volatile accumulation was mostly abolished by MEV and only partially inhibited by FOS (Fig. 7), consistent with the observation that the HMGR-dependent MVA pathway was the main contributor to bacterium-, ABA- and SA-induced volatile accumulation. Similarly, Mansouri et al. (2009) reported that ABA application induces the biosynthesis of secondary terpenoids via the MEP pathway, while it downregulates the biosynthesis of primary terpenoid metabolites from the MEP and MVA pathways in Cannabis sativa. Moreover, the transcriptional level of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the rate-limiting enzyme in the MEP pathway, requires further study. These findings indicate that crosstalk exists between the two biosynthetic pathways for the bacterium-induced sesquiterpenoid accumulation, consistent with studies on diterpenes (Yang et al. 2012). Endophytes can enhance host fitness benefits by activating mild immune responses and promoting the production of host secondary metabolites (Van Wees et al. 2008). Endophytes have also emerged as an intrinsic factor that contributes to the superior quality of geo-authentic medicinal herbs in natural habitats in addition to environmental factors (i.e. topography, soil and climate) (Jia et al. 2009), which is in line with our
previous studies. As biotic elicitors, bacteria have many advantages compared with fungi, such as a short period for culture and elicitation (Jung et al. 2003). We also noted that bacterium ALEB16 only enhanced volatile biosynthesis, whereas endophytic fungus AL12 stimulated both plant biomass and volatile production in A. lancea (Wang et al. 2011). These discrepancies might be due to the differing colonization densities and host sensitivities to invaders of different species. Moreover, the total volatile contents in A. lancea induced by fungus AL12 increased by 74.97% (Ren and Dai 2012), which is a larger increase than that induced by bacterium ALEB16. Previous studies found that ALEB16 triggers weaker defense responses than fungus AL16 (Wang et al. 2013). This might be attributed to the relatively low invasive ability and small size of bacteria, allowing them to occupy smaller niches and trigger weaker host responses than fungi. Additionally, AL12 specifically boosted the ratios of hinesol and atractylone; whereas the concentrations of each volatile constituent equally increased in ALEB16 treatment (Fig. 1). Li et al. (2012) reported that elicitors can regulate gene expressions of enzymes for side chain modifications during the later stages of biosynthesis and thereby alter the constituent ratios of plant secondary metabolite. Thus, our results imply that the promotional model depends on the characteristics of diverse endophytes and that the simultaneous inoculation of endophytes with a variety of beneficial models might facilitate an oriented or synergistic biosynthesis of targeted bioactive products of plants. Signal perception by plant receptors is the first committed step of the elicitor signal transduction pathway. Fungal cell constituents (i.e. chitin, oligosaccharides and lipid derivatives) exhibit elicitor activity across different plant species (Ming et al. 2013). Flagellin and lipopolysaccharides fractions have been thought to act as the elicitor activity domain of invasive bacteria (Zhao et al. 2005, 2010). Because the Gram-negative Acinetobacter is non-motile due to a lack of flagellin, lipopolysaccharides might act as the active component responsible for triggering phytohormone and volatile biosynthesis of A. lancea. In addition to the promoting effect of bacterium ALEB16 on the biosynthetic ability of A. lancea to produce endogenous ABA and SA, changes in the hormone content of bacterized plantlets might be also a result of the exogenous application of hormones produced by ALEB16 itself which requires our further investigation (Arkhipova et al. 2005, Salomon et al. 2013). This work, along with our previous studies, demonstrates that the balanced host–endophyte (bacteria and fungi) symbiosis serves as a promising means to enhance Physiol. Plant. 2014
the production of natural products and that a novel interaction between ABA and SA exists at the biosynthetic level in bacterized plants. Additionally, microbial interactions also play a role in the onset of plant metabolism (Kusari et al. 2012). The signaling cascades and crosstalks involved in endosymbiont-elicited plant secondary metabolism should be more complicated when plants are colonized by multiple symbionts in natural situations. Thus, the chemical ecological interaction between endophytes and the underlying signaling pathways needs to be elucidated in order to fully exploit and modulate their high potential of natural product biosynthesis in plants. Acknowledgements – This work is financially supported by National Natural Science Foundation of China (grant number 31070443), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Research and Innovation Project for College Graduates of Jiangsu Province (grant number CXZZ13_0414) and Integration of Production and Research Projects of Nanjing Science and Technology Commission (grant number 201306019).
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ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis. Appl Microbiol Biotechnol 90: 1027–1036
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Specific primer pair sequences used for cDNA amplification of key genes of the terpenoid biosynthetic pathway of Atractylodes lancea.
