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Bidirectional movement of tunicamycin in Arabidopsis thaliana

New Phytologist2023Vol. 241(1), pp. 10–16

Abstract

Various environmental factors are linked to endoplasmic reticulum (ER) stress, which is caused by an accumulation of misfolded proteins in the ER and membrane lipid disturbance (Xu & Taubert, 2021). The cellular response to ER stress, termed the ER stress response, including the unfolded protein response (UPR), has been extensively studied in a number of model organisms including higher plants (Yu et al., 2021). Plants as a model organism for ER stress allow for investigating organ-specific, tissue-specific, and cell type-specific ER stress responses. In addition, an emerging issue of the systemic ER stress responses in a whole living organism may be investigated, which significantly advances our current understanding of the cell biology of ER stress response (Cho & Kanehara, 2017; Lai et al., 2018). To induce ER stress, a variety of chemicals such as dithiothreitol and thapsigargin have been using, among which tunicamycin is the most widely and frequently applied one regardless of the model organism. Tunicamycin inhibits the first step in the biosynthesis of N-linked glycan in protein glycosylation, thus resulting in the accumulation of misfolded proteins in the ER (Olden et al., 1979). For plants, tunicamycin can be supplemented in liquid or solid-agar medium (McCormack et al., 2015). A whole plant such as an Arabidopsis thaliana seedling is exposed to tunicamycin in liquid medium, which is supposed to trigger the ER stress response locally, whereas a root is the major organ exposed to tunicamycin on solid-agar medium. A number of studies using solid-agar medium have reported a sensitivity of whole seedlings including shoot, which show hyper- or hypo-sensitivity to tunicamycin-induced ER stress (Mishiba et al., 2013; Lin et al., 2019). These studies raise a question of whether tunicamycin can be transported from root to shoot because root but not shoot is supposed to be exposed to tunicamycin on solid-agar medium. In fact, a recent report showed that the UPR in A. thaliana acts systemically in the shoot-ward direction independent of tunicamycin transport because tunicamycin is not transported from root to shoot (Lai et al., 2018). However, information is limited on how tunicamycin is absorbed and transported within a plant body despite the increase in number of studies using tunicamycin in various plant species including Zea mays (Srivastava et al., 2018) and Oryza sativa (Liu et al., 2020). The lack of information may be due to absence of an established method to quantify tunicamycin extracted from plants. Thus, the aim of our present work was to: establish a UPLC-MS/MS method to quantify tunicamycin: and assess the directional movement of tunicamycin in plants by using seedlings of A. thaliana. We first optimized a UPLC-MS/MS condition to quantify commercially available tunicamycin (Merck, Burlington, MA, USA, Cat. No. 654380, Batch no.: 3315820, MW 844.9) as a standard by reference to a recent study that validated the method for determining tunicamycin content in rat plasma (Gabani et al., 2019). The UPLC-MS/MS involved an ACQUITY Ultra Performance LC (UPLC) system (Waters, Milford, MA, USA) coupled to an LTQ-Orbitrap Elite (Thermo Scientific, Waltham, MA, USA) mass spectrometer with an ACQUITY UPLC BEH Phenyl column (1.7 μm, 2.1 × 100 mm; Waters). Using the UPLC flow-binary gradient operating-time program shown in Table 1, the total chromatographic run time was 6 min. We detected tunicamycin with a retention time of 2.58 min (Fig. 1a). According to the previous study (Gabani et al., 2019), the fragmentation pattern of tunicamycin (Fig. 1a) was similar to that of homolog C, whose multiple reaction monitoring transition (m/z) was 845.2→624.2. Homolog C is identified as the PubChem Compound for Tunicamycin B (PubChem Identifier: CID 71458815; Fig. 1b). Hence, we used the transition (m/z) 845.4407→624.3502 (Fig. 1a) for quantifying tunicamycin in our further experiments. To test whether tunicamycin is detected in shoot or root by root-specific or shoot-specific tunicamycin treatment, respectively, we took advantage of the shoot–root split-culture system reported previously (Lai et al., 2018) with minor modifications. We first grew A. thaliana on half-strength solid MS medium for 14 d. Ten 14-d-old seedlings were then transferred to a two-compartment Petri plate with liquid MS media, which contained tunicamycin or dimethyl sulfoxide (DMSO, mock). We prepared different treatment conditions (Figs 1c, 2a): both shoot and root in DMSO (Mock, D/D); shoot in medium with tunicamycin and root in mock (Shoot-specific TM treatment, T/D); shoot in mock and root in medium with tunicamycin (Root-specific TM treatment, D/T). To reach a steady state, we tested a 24 h period for culturing plants in the Petri plate. After the treatment, shoots and roots were washed with distilled water three times by pipetting carefully to avoid contamination of media between two compartments. The roots and shoots were then harvested separately. Tunicamycin was extracted from roots or shoots detached from 30 of the 14-d-old seedlings as described in Materials and Methods. Next, we quantified tunicamycin extracted by the above conditions with three biologically independent samples, each with three technical replicates. A standard curve was drawn for tunicamycin at different concentrations (0, 0.1, 1, 5, 10, and 20 ppm in 80% (v/v) methanol) with three technical replicates for normalization (Supporting Information Table S1; Fig. S1). Our quantification revealed that with shoot-specific tunicamycin treatment, a mean of 1.754 and 0.640 μg tunicamycin was detected in shoots and roots, respectively, from 30 of the 14-d-old seedlings (2.394 μg in total; Table 2). In the root-specific TM treatment (D/T), a mean of 2.138 μg tunicamycin was detected from the 30 whole seedlings. Of note, the total tunicamycin was divided nearly equally in shoots and roots (a mean of 1.009 and 1.129 μg, respectively; Table 2). For the mock condition, the mean tunicamycin content was −0.042 and −0.041 μg in shoots and roots, respectively (Table 2). Thus, with shoot-specific TM treatment (T/D), 73.2 ± 2.8% tunicamycin was detected in shoots and the remaining 26.8 ± 2.8% was in roots (Fig. 1c). By contrast, with root-specific tunicamycin treatment (D/T), 52.7 ± 5.0% tunicamycin was detected in roots and the remaining 47.3 ± 5.0% was in shoots (Fig. 1c). Hence, our results suggest the bidirectional movement of tunicamycin in both shoot-ward and root-ward directions in A. thaliana (Fig. 1c). To confirm the route of tunicamycin movement, we quantified tunicamycin in media after 24 h treatment. As shown in Table 3, tunicamycin was not detected in mock compartment of all the treatments in Fig. 2(a), suggesting that tunicamycin is transported within plant body rather than a mere diffusion across the medium. To examine whether the movement of tunicamycin from tunicamycin-treated organs to tunicamycin-untreated organs is sufficient to systemically induce the UPR, we treated tunicamycin in shoot (T/D) or in root (D/T) of 14-d-old seedlings using shoot–root split-culture system (Lai et al., 2018) for 24 h (Figs 1c, 2a). After the treatment, we harvested root and shoot samples separately for RNA isolation then analyzed the transcript level of the four UPR-responsive genes by a semi-quantitative polymerase chain reaction (qPCR), including three BINDING PROTEINS (BiP1, BiP2, and BiP3), CALNEXIN (CNX), and CALRETICULIN (CRT; Noh et al., 2003), which aid the proper folding of proteins in the ER and thus used as the marker genes for ER stress response. Our data showed that transcript levels of BiP1/2, BiP3, CNX, and CRT were significantly induced in root of the shoot-specific TM treatment (T/D) and in shoot of the root-specific TM treatment (D/T), compared with those of the mock (D/D; Fig. 2b). These results indicate that the movement of tunicamycin is sufficient to induce UPR genes in non-treated tissues. To further confirm whether ER stress occurs upon root-to-shoot movement of tunicamycin, we detected aggregated protein, a molecular feature of unfolded protein in ER stress, in leaves of seedlings in the root-specific TM treatment (D/T) by using a commercially available aggresome detection kit (Proteostat® Aggresome Detection Kit (Enzo:ENZ-51035, Farmingdale, NY, USA); Cho & Kanehara, 2017). As shown in Fig. 3, when we treated 14-d-old plants with a proteasome inhibitor MG-132 as a positive control for 16 h, the signal of aggregated protein was detected in seedling leaves of the MG-132 treatment (Fig. 3f–j, magenta indicated aggregated protein detection), but not in leaves of mock treatment (D/D; Fig. 3a–e). Of note, we found an increase in aggregated protein signal in leaves of seedlings in the root-specific treatment (D/T; Fig. 3k–o). Taken together, these results suggest that the tunicamycin's root-to-shoot movement is sufficient to induce ER stress in the leaves of the root-TM specific treatment (D/T). Tunicamycin has been widely used for the UPR study in mammalian cells and microbes especially in the context of medical research. However, these studies mostly involve cell biology, providing no opportunity to investigate the mobility of tunicamycin across the tissues. Here, plant is an excellent model to study the systemic effect of tunicamycin across the tissues. In this study, we found that tunicamycin can be transported not only from shoot to root but also from root to shoot (Fig. 1b; Table 2). Our observation shed light on the novel mobile feature of the well-established chemical compound, which may advance the UPR research beyond plant science. A significant aspect of the findings toward the future development is what is the molecular mechanism of movement associated with the transfer of bioactivity to a distant tissue. Since the movement of tunicamycin from root to shoot systemically induced UPR genes and aggresomal protein in the shoot of plant (Figs 2b, 3), it is likely under control rather than a spontaneous diffusion of the compound. A recent study showed that tunicamycin can be transported by a plant Nitrate Transporter1/Peptide Transporter Family (NPF1/NPF) protein NPF2.13 in plant–microbe interaction (Liu et al., 2023). Also, tunicamycin is produced by Streptomyces species which are abundant in soil microorganisms (Takatsuki et al., 1971). Since root is known as the frontier to perceive environmental stresses that trigger ER stress (Cho & Kanehara, 2017), we speculate that plant root may be equipped to take up and transport naturally occurring tunicamycin for environmental responses. In conclusion, we established a method to quantify tunicamycin in plants by using UPLC-MS/MS, which shows the bidirectional movement of tunicamycin from roots up to shoots or shoots down to roots (Fig. 1d). This method allowed us to demonstrate that the movement of tunicamycin systemically induced the ER stress in a distant tissue (Figs 2b, 3k–o). Arabidopsis thaliana plants (Columbia-0 ecotype) were grown on Murashige and Skoog (MS)-agar plates under continuous light at 22°C for 14 d. MS medium was used at half-strength concentration for all plant cultures (Murashige & Skoog, 1962). For tunicamycin treatment, 14-d-old wild-type seedlings were treated with DMSO or 5 μg ml−1 tunicamycin (Merck, Cat. No. 654380, Batch no.: 3315820, MW 844.9) in half-strength MS liquid medium for 24 h in the shoot–root split-culture system as described (Lai et al., 2018) with 10 seedlings per plate. The shoots and roots were then washed with distilled water three times and harvested separately for further experiments. Tunicamycin was extracted by reference to two previous studies (Takatsuki et al., 1971; Stracke et al., 2007). Shoots or roots detached from 30 of the 14-d-old seedlings were ground in liquid N2 until a fine powder and then 1.2 ml of ice-cold 80% (v/v) methanol was added. The extract was incubated at 70°C for 30 min with manual inversion every 5 min. The extract was then centrifuged at 12 000 g at 4°C for 10 min. The supernatant was transferred to a new microtube and dried by SpeedVac (Vacufuge Plus; Eppendorf, Hamburg, Germany) at 30°C. The pellet was suspended with 120 μl of 80% (v/v) methanol, centrifuged to remove debris, and 10 μl of the supernatant was injected for UPLC-MS/MS analysis. To determine tunicamycin content in the media, after 24 h of treatment, 100 μl media from each compartment was collected and dried by SpeedVac (Vacufuge Plus; Eppendorf) at 30°C. The pellet was then suspended with 100 μl of 80% (v/v) methanol and 10 μl of the supernatant was injected for UPLC-MS/MS analysis. Tunicamycin quantification was optimized by reference to the method in a previous study(Gabani et al., 2019). An ACQUITY Ultra Performance LC (UPLC) system (Waters) coupled with a LTQ-Orbitrap Elite (Thermo Scientific) mass spectrometer was used for the UPLC-MS/MS analysis by an HESI interface. The chromatographic separation for samples involved using an ACQUITY UPLC BEH Phenyl Column (1.7 μm, 2.1 × 100 mm; Waters). The column was maintained at 40°C, and 10 μl sample was injected per run. The mobile phase A was 20% (v/v) acetonitrile with 8 mM ammonium formate, pH 3.0, and mobile phase B was 100% (v/v) acetonitrile. The gradient elution had a flow rate of 0.4 ml min−1 with a total analysis time of 6 min. The gradient included 1% phase B at 0 min, 99.5% phase B at 4 min, holding at 99.5% phase B until 5 min, 1% phase B at 5.01 min, and holding at 1% phase B until 6 min (Table 1). General instrument conditions were sheath gas, auxiliary gas, and sweep gas 35, 15, and 1 arbitrary units, respectively; ion transfer tube temperature 360°C; vaporizer temperature 350°C; and spray voltage 3200 V for positive mode. For analysis, a full MS scan mode with a scan range (m/z) 90–1000, resolution 15 000, was used. Xcalibur 4.1 (Thermo Scientific) was used for data processing. A standard curve was drawn with tunicamycin at different concentrations (0, 0.1, 1, 5, 10, and 20 ppm in 80% (v/v) methanol) for normalization. The experiment involved three biologically different samples, each with three technical replicates. Samples after TM treatment as described in ‘Tunicamycin treatment’ were used for total RNA isolation by the SV Total RNA Isolation System (Promega) according to the manufacturer's instructions. The first-strand cDNA was synthesized by using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara, Kusatsu, Shiga, Japan). RT-qPCR was performed with an Applied Biosystems 7500 Real-Time PCR system (Thermo Scientific). Reactions (10 μl total volume) were performed in a 96-well plate containing 2.5 μl 4× CAPITAL qPCR Green Master Mix (Biotechrabbit, Berlin, Germany), 50 ng cDNA, and 200 nM of each gene-specific primer under the following amplification regime: 50°C for 2 min, 95°C for 10 min; 40 cycles of 95°C for 15 s, and 60°C for 1 min. The comparative threshold cycle method was used to determine the relative amount of target transcripts as compared with ACTIN2 (At3g18780) level. At least three biologically independent experiments were performed, each with three technical replicates. The oligonucleotide primers used are reported (Kanehara et al., 2015). Detection of aggregated protein was conducted as described (Cho & Kanehara, 2017). Fourteen-day-old wild-type seedlings were treated with DMSO or 5 μg/ ml tunicamycin (Merck, Cat. No. 654380, Batch no.: 3315820, MW 844.9) in half-strength MS liquid medium for 24 h in the shoot–root split-culture system as described (Lai et al., 2018) with 10 seedlings per plate. Seedlings treated with MG-132 (5 μM) for 16 h were used as the positive control. After chemical treatment, leaf were detached and stained with Proteostat® Aggresome Detection Kit (Enzo:ENZ-51035) according to the manufacturer's instruction. Briefly, leaf was fixed with 4% paraformaldehyde in the Assay Buffer (provided by Proteostat® Aggresome Detection Kit) for 30 min at room temperature and then were washed by phosphate-buffered saline (PBS) three times. Leaf was then transferred into a permeabilizing solution (0.5% TritonX-100, 3 mM EDTA pH 8.0 in 1× Assay Buffer) for 30 min at 4°C. After washing three times with PBS, leaf was incubated with Proteostat® Aggresome dye (1 μl ml−1) and Hoechst 33342 (1 μl ml−1) in Assay Buffer for 30 min in the dark at room temperature. Aggregated protein was observed using Zeiss LSM 880 Airyscan FAST confocal microscopy. The rhodamine filter set for imaging aggresome signal and a DAPI filter set for imaging the nuclear signal. We thank Dr Kanehara Kazue (Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan) for her hosting and advising AN during the period of the Special Project for Research Scholar (110-2811-B-001-520). We also thank the Metabolomics Core Facility at the Agricultural Biotechnology Research Center of Academia Sinica for the mass spectrometry analysis. This work was supported by the Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-108-108) to YW, and Special Project for Research Scholar (110-2811-B-001-520), the Ministry of Science and Technology, Taiwan and JSPS fellowship, and JSPS Grant-in-Aid, the Japan Society for the Promotion of Science, Japan to AN. None declared. AN and YN conceived the research. AN and YW designed and performed experiments, and analyzed data. AN, YW, and YN wrote the manuscripts; All authors commented on the manuscript and approved the contents. All data are included in the main article or in the Supporting Information. Fig. S1 Standard curve used in this study. Table S1 Raw data for drawing the standard curve for tunicamycin at different concentrations. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract

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