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Jun 5, 2015 - ABSTRACT: The peptide elicitor PIP-1 can induce various immune responses in tobacco cells. Previously, we showed that types of responses...
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Photocontrol of Elicitor Activity of PIP‑1 to Investigate Temporal Factors Involved in Phytoalexin Biosynthesis Yonghyun Kim, Masahiro Miyashita,* and Hisashi Miyagawa Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan S Supporting Information *

ABSTRACT: The peptide elicitor PIP-1 can induce various immune responses in tobacco cells. Previously, we showed that types of responses induced by PIP-1 are different depending on its stimulation periods; short-term stimulation induces weak responses, whereas long-term stimulation leads to strong responses including production of the phytoalexin capsidiol. However, key components that directly regulate the initiation of capsidiol biosynthesis in response to continuous stimulation with PIP-1 remain unclear. In this study, we designed a photocleavable PIP-1 analog containing 3-amino-3-(2-nitrophenyl)propionic acid as a photocleavable residue. The activity of the analog can be “switched off” using ultraviolet (UV) irradiation without undesired side effects. This analog induced a significant level of capsidiol production unless UV-irradiated, whereas no capsidiol production was observed when tobacco cells were UV-irradiated 1 h after treatment. Using this analog, we found that the elicitor-inducible 3hydroxy-3-methylglutaryl-CoA reductase activity is regulated based on the duration of the stimulation with PIP-1, which could be associated with the initiation of capsidiol biosynthesis. KEYWORDS: peptide, photocleavable analog, phytoalexin, MAP kinase, plant immunity

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INTRODUCTION Plants have evolved a unique immune system to protect themselves against pathogen attacks.1,2 This immune system consists of a series of plant immune responses such as oxidative burst, reinforcement of cell walls, phytoalexin biosynthesis, and hypersensitive response (HR).3−5 These responses can be characteristically invoked after recognizing different types of pathogen-derived molecules, known as elicitor or pathogenassociated molecular patterns.6 Elicitors with diverse chemical properties have been described to date, including peptides, proteins, lipids, and oligosaccharides, which are considered key to understanding pathogen-recognition mechanisms in plants.7,8 To this end, some elicitors have been shown to interact with specific domains of the receptors located in the plasma membrane of plant cells to transmit signals via mitogen-activated protein (MAP) kinase cascades leading to the expression of immune responses to counteract pathogens.9−12 Previously, we discovered a peptide elicitor (YGIHTH-NH2, PIP-1) by screening a library of synthetic combinatorial random hexapeptides (Figure 1).13 PIP-1 induces various immune responses in tobacco cells; however, the types of responses induced by PIP-1 are different depending on its concentration. Phytoalexin production (a late-phase immune response) requires concentrations approximately 10- to 50-fold higher than those required to trigger oxidative burst (an early-phase immune response).14 We found that this difference is attributed to a rapid decrease in the PIP-1 concentration in the medium due to enzymatic hydrolysis in the presence of tobacco cells. This implies that continuous stimulation with PIP-1 is necessary to induce phytoalexin biosynthesis, which is associated with long-lasting MAP kinase activity.14 However, key components that directly regulate the initiation of © 2015 American Chemical Society

phytoalexin biosynthesis in response to continuous stimulation with PIP-1 remain unclear. In a previous study, the stimulation period required for phytoalexin biosynthesis was determined by removing PIP-1 from the culture medium at a specific time point. In the experiment, the cells were thoroughly washed with fresh media to remove treated PIP-1. However, it was difficult to precisely control the level of stimulation in terms of temporal scales, and more importantly, the washing operation could mechanically stress the cells to induce some undesired responses. All these issues in the cell-washing procedure could have biased the results in an unknown fashion. Thus, quicker and less aggressive methods to “switch off” the activity of PIP-1 are essential to analyze temporal factors regulating plant immune responses induced in response to continuous stimulation with the elicitor. Over the past two decades, several lines of research have attempted to integrate photosensitive substructures into biologically active molecules to design probes whose activity could be controlled temporally.15−19 For peptides, photocleavable amino acids such as 3-amino-3-(2-nitrophenyl)propionic acid (Anp) and (2-nitro)phenylglycine (Npg) have been successfully developed. These amino acids incorporated in a peptide undergo structural changes when exposed to ultraviolet (UV) irradiation, followed by a rapid degradation, modifying the activity of the peptides.20 For example, Anp was effectively used by Parker et al. to study yeast peptide pheromone α-factor.21 In their study, a photocleavable α-factor analog containing an Anp residue actively stopped the cell cycle Received: Revised: Accepted: Published: 5894

January 27, 2015 May 27, 2015 June 5, 2015 June 5, 2015 DOI: 10.1021/acs.jafc.5b01910 J. Agric. Food Chem. 2015, 63, 5894−5901

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Journal of Agricultural and Food Chemistry

nitro)phenylglycine was synthesized as described by Rodenko et al.20 (Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), cycloheximide (CHX), and 3-hydroxy-3methylglutaryl-coenzyme A (HMG-CoA) were purchased from Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and Santa Cruz Biotechnology (Dallas, TX), respectively. Synthesis of Photocleavable Analogs. An Fmoc solid-phase peptide synthesis strategy was used to synthesize photocleavable analogs. Deprotection of the Fmoc protecting group was conducted using 20% piperidine in DMF 3 times for 3, 3, and 20 min. Coupling of Fmoc-protected amino acids (3 equiv) was performed in the presence of O-(benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3 equiv), 1-hydroxybenzotriazole (HOBt) (3 equiv), and N,N-diisopropylethylamine (DIPEA) (6 equiv) in N,Ndimethylformamide (DMF). Completion of the reaction was checked by Kaiser test.22 After completion of all coupling reactions, the resin was washed with DMF, diethyl ether, and methanol and subsequently vacuum-dried. Peptide cleavage and removal of side-chain protecting groups were conducted using cleavage cocktails [TFA/triisopropylsilane/1,3-dimethoxybenzene (92.5:2.5:5) for Fmoc-SAL-resin attached peptides and TFA/triisopropylsilane/H2O (95:2.5:2.5) for Cl-Trt(2-Cl)-resin attached peptides] for 2 h at room temperature. The reaction mixture was filtered, and the filtrate was mixed with cold diethyl ether to precipitate the peptides. The peptides were then vacuum-dried. For analogs containing (2-nitro)phenylglycine, dichloromethane was used instead of DMF throughout the process. Peptide purification was conducted by RP-HPLC using a 250 mm × 10 mm i.d., 5 μm, protein and peptide C18 semipreparative column (Grace, Columbia, MD), and lyophilized. Purified peptides were identified by electrospray ionization liquid chromatography−mass spectrometry (ESI-LC/MS) on an LCMS-2010 instrument (Shimadzu, Kyoto, Japan). Measurement of H2O2 Generation. H2O2 generation was measured as described previously.23 Suspension-cultured tobacco cells (0.2 g) were resuspended in 1 mL of assay buffer (175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4, 2.0 mM MES, pH 5.75) and incubated for 1 h at 25 °C using a tube rotator. Peptide samples were added to tobacco cell suspensions, and the cells were reincubated for 15 min at 25 °C. The supernatants (60 μL) were added to a 96-well microplate containing an H2O2 indicator solution (140 μL, 100 mM DA-64, 1 U/mL horseradish peroxidase, 100 mM PIPES, pH 7.0). This microplate was incubated for 10 min at 37 °C. The absorbance at 727 nm was observed using a Benchmark microplate reader (Bio-Rad, Hercules, CA). The amount of H2O2 was calculated using a calibration curve based on pure H2O2. As an index of activity, the concentration required for half-maximal responses (EC50) was calculated using PRISM (GraphPad Software, La Jolla, CA). Peptide Photolysis. The photocleavable analogs were dissolved in distilled water at 100 μM of final concentration. The solution was irradiated with UV light (365 nm, 10 mW/cm2) using a Blak-ray B100A UV lamp (UVP, Upland, CA). The UV-irradiated solution was analyzed with LCMS-2020 and LCMS-IT-TOF (Shimadzu) equipped with a 150 mm × 2.1 mm i.d., 3.6 μm, Aeris PEPTIDE XB-C18 reverse phase column (Phenomenex, Torrance, CA). Cell Death Assay. Cell death was measured as described previously with modifications.24 Cells (0.2 g) were treated with peptide samples and incubated for 24 h at 25 °C using a tube rotator. After incubation, the cell suspension was reincubated in Evans blue solution (Wako Pure Chemical Industries) at a final concentration of 0.01% for 15 min and then washed with distilled water 3 times to remove any unabsorbed dye. The washed cells were transferred into a new microtube containing 50% methanol and 1% SDS to extract the dye in dead cells and incubated for 1 h at 60 °C. The solution was centrifuged for 15 min at 1900g. Absorbance of the supernatant at 595 nm was measured on a microplate reader (Bio-Rad). Quantitation of Capsidiol. Capsidiol content was quantitated as described previously.13 Tobacco cells (0.6 g) resuspended in 3 mL of LS medium were incubated for 1 h. The cells were then treated with peptide samples and incubated for 24 h at 25 °C on a rotary shaker under continuous illumination. After incubation, capsidiol was

Figure 1. Structure of PIP-1 and its analogs. Anp, (R)-3-amino-3-(2nitrophenyl)propionic acid; Npg, (RS)-(2-nitro)phenylglycine. Values in parentheses indicate EC50 for H2O2 generation.

at G1 phase and was rapidly inactivated by UV irradiation. This inactivation reactivated the cell cycle in all cells in a homogeneous and synchronous manner, allowing analysis of the cell cycle progress in detail. In the present study, we designed a photocleavable PIP-1 analog to investigate the mechanisms underlying the stimulation period-dependent induction of late immune responses such as phytoalexin biosynthesis. The introduction of a photocleavable amino acid into PIP-1 successfully provided an analog, the activity of which can be “switched off” by UV irradiation. Using this analog, we explored key components that are associated with the initiation of phytoalexin biosynthesis in response to continuous stimulation with PIP-1.

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MATERIALS AND METHODS

Plant Cell Culture. Tobacco cells (Nicotiana tabacum cv. Xanthi) were maintained as suspension cultures in 25 mL of liquid Linsmaier and Skoog (LS) medium at pH 5.75 containing 1-naphthalenacetic acid (10 μM), 5-benzyladenine (1 μM), and 3% of sucrose in a 100 mL Erlenmeyer flask. The cells were grown at 25 °C under continuous illumination on a rotary shaker (100 rpm) and subcultured every 2 weeks. The experiments were conducted using 9- and 10-day old subcultured tobacco cells. Chemicals. N-(9-Fluorenylmethoxycarbonyl) (Fmoc)-NH-SAL resin, Cl-Trt(2-Cl)-Resin, and general Fmoc-protected amino acids were purchased from Watanabe Chemical Industries (Hiroshima, Japan). Fmoc-(R)-3-amino-3-(2-nitrophenyl)propionic acid was purchased from PepTech Corporation (Bedford, MA) and Fmoc-(RS)-(25895

DOI: 10.1021/acs.jafc.5b01910 J. Agric. Food Chem. 2015, 63, 5894−5901

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Journal of Agricultural and Food Chemistry extracted from the culture medium using 4 mL of ethyl acetate twice and vacuum-dried. The residue was then dissolved in 1 mL of ethyl acetate and subjected to QP-2010 GC/MS analysis (Shimadzu), equipped with a 30 m × 0.25 mm, 0.25 μm phase thickness, DB-5MS capillary column (J & W Scientific, Folsom, CA). Other GC/MS conditions were described previously.14 The amount of capsidiol was quantitated from the peak areas obtained in the total ion chromatogram, using a calibration curve based on an authentic standard purified from Capsicum annuum fruit.25 In-Gel Kinase Assay. In-gel kinase assay was performed as described previously with modifications.26 Tobacco cells (0.6 g) resuspended in 3 mL of LS medium were incubated for 1 h at 25 °C. The cells were then treated with peptide samples and reincubated for 3 h. After all medium was removed, tobacco cells were ground in liquid nitrogen using a mortar and pestle. Powdered tobacco cells were homogenized using 1.5 mL of extraction buffer [50 mM HEPES, pH 7.4, 5 mM EDTA, 5 mM EGTA, 5 mM DTT, 10 mM NaF, 10 mM Na3VO4, 50 mM β-glycerophosphate, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 2 μg/mL antipain, 2 μg/mL aprotinin, and 2 μg/mL leupeptin] and centrifuged at 16 000g for 30 min at 4 °C. Protein extracts (10 μg) were electrophoresed on 12% SDSpolyacrylamide gels embedded with 0.25 mg/mL myelin basic protein (MBP) (Sigma, St. Louis, MO) as a kinase substrate. After electrophoresis, the gel was washed with washing buffer (20% 2propanol, 50 mM Tris, pH 8.0) three times for 20 min each at room temperature to remove SDS and incubated with buffer A (50 mM Tris, pH 8.0, 5 mM DTT) for 1 h. The proteins in the gel were denatured using denaturation buffer (50 mM Tris−HCl, pH 8.0, 6 M guanidine· HCl, 20 mM DTT, 2 mM EDTA) twice for 30 min each. The kinases were allowed to renature by incubations in buffer A containing 0.04% of Tween 20 overnight at 4 °C with 5 changes of buffer. After equilibration in assay buffer (50 mM HEPES, pH 8.0, 2.0 mM DTT, 0.1 mM EGTA, and 20 mM MgCl2) for 30 min at room temperature, the gel was incubated in 20 mL of assay buffer containing 30 μM ATP and 1.85 MBq of [γ-32P]-ATP (111 TBq/mmol) (PerkinElmer, Waltham, MA) for 1 h at room temperature. The reaction was stopped by transferring the gel into a solution containing 10% trichloroacetic acid (w/v) and 1% sodium phosphate (w/v). The gel was washed five times with the same solution for 20 min each to remove any unincorporated [γ-32P]-ATP and dried on filter paper using a vacuum gel-dryer. The dried gel was then exposed to BAS-MS 2040 imaging plates (Fujifilm, Tokyo, Japan) and analyzed using BAS-2500 (Fujifilm). Prestained size markers (Nacalai Tesque) were used to estimate the size of the kinases. Gene Expression. Tobacco cells (0.6 g) suspended in 3 mL of LS medium were treated with peptide samples and incubated for 4 h at 25 °C. Incubated cells were collected by filtration and ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted from powdered tobacco cells using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The RNA was then incubated with 10 U of RQ1 RNase-Free DNase (Promega, Madison, WI) for 30 min at 37 °C to degrade the DNA. cDNA was synthesized from 1 μg of total RNA using a PrimeScript RT reagent kit (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed by Thermal Cycler Dice (Takara Bio) with amplification buffer (25 μL of final volume) containing 2 μL of cDNA template, SYBR Premix Ex Taq II (Takara Bio), and 0.4 μM of the following forward and reverse primer; HMGR2: 5′-GAGGGGAGAGGAAAGTCTGTAG-3′ and 5′-TCTCTATGTTCTGAGCTGGGTC-3′. Actin (5′-CCATTGGCTCAGAGAGGTTC-3′ and 5′GTTGGAAGGTGCTGAGAG-3′) was used as a reference gene.27 Enzyme Assay. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was measured as described previously.28 In brief, tobacco cells (0.6 g) treated with peptide samples for 4 h were used for crude enzyme extraction. Tobacco cells were ground in liquid nitrogen using a mortar and pestle and homogenized using extraction buffer (50 mM Tris-HCl, 10 mM β-mercaptoethanol, 1% (w/v) PVP, pH 7.5). The mixture was centrifuged at 16 000g at 4 °C for 30 min, and the supernatant was used for measurement of HMGR activity after

determining protein concentrations using a Bio-Rad protein assay reagent. The crude extract was added to the assay buffer (50 mM TrisHCl, 0.3 mM HMG-CoA, 0.2 mM NADPH, and 4 mM DTT, pH 7.0), and NADPH oxidation was measured by monitoring a decrease in absorbance at 340 nm. One unit of HMGR enzyme is equivalent to the oxidation of 1 mM NADPH per minute. Elicitor-inducible HMGR activity was estimated by subtracting the constitutive activity in the absence of the elicitor from the activity in the presence of the elicitor.

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RESULTS AND DISCUSSION Design of Photocleavable PIP-1 Analogs and Evaluation of Their Elicitor Activity. In a previous contribution, we reported that PIP-1 can be rapidly hydrolyzed from its Cterminal end in the presence of tobacco cells and that no elicitor activity was observed when the C-terminal two residues were deleted.14 The degradation-resistant analog MePIP-1 was obtained by attaching an N-methyl group to the amide bond between Thr and Ala residues (Figure 1). This analog exhibited elicitor activity higher than that of PIP-1 in terms of phytoalexin production. To obtain photocleavable PIP-1 analogs whose activity can be switched off by UV irradiation, the analogs must be stable against enzymatic degradation unless UV-irradiated. In this regard, MePIP-1 was suitable as a starting compound for development of photocleavable PIP-1 analogs. Therefore, we first substituted the Thr residue of MePIP-1 with a photocleavable amino acid (Anp) (Figure 1, [Anp5]MePIP-1) because cleavage of the amide bond between His and Anp by UV irradiation was expected to produce inactive peptides. However, this analog did not show any measurable activity, even before UV irradiation (Figure 1). This lack of activity could be associated with the β-amino acid structure of Anp, which adds an additional carbon atom to the main peptide chain, possibly causing unfavorable structural changes preventing the interaction between the peptide and the receptor. Subsequently, we attempted to substitute the Thr residue with a photocleavable α-amino acid (Npg) (Figure 1, [Npg5]MePIP1), although substitution also resulted in loss of biological activity (Figure 1). In this case, the steric interaction between the 2-nitrobenzyl group and the neighboring terminal N-methyl group might have influence on the activity by changing the overall molecular conformation into the inactive form. Therefore, we explored different strategies to obtain new degradation-resistant PIP-1 analogs that could be suitable for designing photocleavable analogs. While studying the relationship between structure and enzymatic degradation of PIP-1, it became apparent that the basic character of the C-terminal His residue could be important for the recognition by proteases derived from tobacco cells (data not published). Accordingly, we substituted the C-terminal His residue with an acidic amino acid, Asp, and replaced the C-terminal amide group with a carboxy group, which is known to increase PIP-1 elicitor activity (Figure 1, [Asp6]PIP-1).13 As expected, [Asp6]PIP-1 displayed higher degradation resistance compared with that of PIP-1 without affecting elicitor activity (Figure 1 and Figure S1 in the Supporting Information). On the basis of the structure of [Asp6]PIP-1, we designed a photocleavable analog as shown above. After substitution of the Thr residue of [Asp6]PIP-1 with Anp (Figure 1, PcPIP-1), the resulting compound retained the elicitor activity and was resistant to degradation (Figures 1 and 2). In the following sections, we examine whether photocontrol of the elicitor activity of this analog is possible. 5896

DOI: 10.1021/acs.jafc.5b01910 J. Agric. Food Chem. 2015, 63, 5894−5901

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sophenyl)-3-oxopropanoyl aspartic acid, the expected photolyzed products of PcPIP-1. On the basis of these results, we assumed that these two unknown peaks observed after photolysis can be the structural isomers of an intermediate produced during photolysis of PcPIP-1. Previous studies have proposed that the photolysis of 2-nitrobenzyl compounds is a multistep process, and the decay of aci-nitro intermediates (Figure S3 in the Supporting Information) is considered to be rate-limiting.29,30 By analogy, the photolysis of PcPIP-1 might involve the formation of relatively long-lived α-hydroxybenzyl intermediates;31−33 therefore, it seems likely that the unknown peaks detected were associated with the 3-hydroxy-3-(2nitrosophenyl) intermediate product, as shown in Figure S3 in the Supporting Information. The possible epimerization of this intermediate can account for the occurrence of two peaks with the same molecular mass. Photocontrol of Activity of the Photocleavable PIP-1 Analog. To confirm that PcPIP-1 can be inactivated by UV irradiation, we first compared the defense-inducing activity of PcPIP-1 with and without UV irradiation. Regarding earlyphase immune responses, treating cells with intact PcPIP-1 at 50 or 100 μM significantly increased the level of H2O2 generated (Figure 4A). Exposing PcPIP-1 to UV irradiation in the absence of tobacco cells reduced markedly its elicitor activity. When cells were treated with UV-irradiated PcPIP-1 at 50 and 100 μM, the level of H2O2 generated decreased by 6and 2-fold, respectively. Exposing PcPIP-1 to UV irradiation also significantly affected the induction of the late-phase immune responses, such as HR-like cell death and phytoalexin production, compared with that by intact PcPIP-1. The levels of cell death induced by UV-irradiated PcPIP-1 were evidently lower than those induced by intact PcPIP-1 (Figure 4B). The production of capsidiol, the major phytoalexin in tobacco, also markedly differed between UV-irradiated and intact PcPIP-1; in this case, the activity was nearly entirely repressed by UV irradiation both at 50 and 100 μM (Figure 4C). These results indicated that UV-irradiated PcPIP-1, a mixture of the photolyzed products and intermediates, presents a level of elicitor activity sufficiently lower than that of intact PcPIP-1. We then examined whether PcPIP-1 can be also inactivated by UV irradiation in the presence of tobacco cells. As shown in Figure 5A, when tobacco cells were UV-irradiated for 5, 10, or 15 min following treatment with PcPIP-1 at 100 μM for 1 h, a time-dependent decrease in capsidiol production was observed,

Figure 2. Degradation resistance of PcPIP-1 in the presence of tobacco cells. Relative amounts of PcPIP-1 in the cell culture medium after treatment are plotted (n = 2).

Photolysis of the Photocleavable PIP-1 Analog. We evaluated the reactivity of the photocleavable analog PcPIP-1 in aqueous solution under UV irradiation. ESI-LC/MS was used to monitor the degradation of PcPIP-1. When PcPIP-1 (m/z observed for [M+2H]2+, 398.68; retention time, 15.1 min) was irradiated at 365 nm for 5 min, approximately 10% of the initial concentration was detected (Figure 3). Instead, several new peaks appeared in the chromatogram. The molecular masses of two peaks detected at 3.7 min (m/z 488.27 for [M + H]+) and 6.3 min (m/z 489.26 for [M + H]+) of retention time corresponded with those of YGIH- NH2 and YGIH-OH, respectively, identified by comparing with synthesized standards. These peptides were considered to be produced by photolysis of the Anp residue, although the formation mechanism of the latter was not clear. The molecular mass of the other two peaks detected at 13.0 and 13.5 min of retention times was similar to that of the original peptide (m/z observed for [M+2H]2+, 398.67; calculated, 398.66). These peaks were still detected after irradiation with UV light for an additional 10 min; however, only marginal levels of the original peptide were detected at that stage. MS/MS analysis showed that whereas fragment ions related to the amino acid sequence were regularly observed for the case of the original peptide, the two unknown peaks gave only two major fragment ions (Figure S2 in the Supporting Information). MS3 analysis suggested that these fragment ions correspond to YGIH-NH2 and 3-(2-nitro-

Figure 3. HPLC profile of photolyzed products of PcPIP-1 at 100 μM. 5897

DOI: 10.1021/acs.jafc.5b01910 J. Agric. Food Chem. 2015, 63, 5894−5901

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consistent with those from a previous study in which wash-out of PIP-1 from the cells 1 h after treatment resulted in no capsidiol production.14 Therefore, our results confirmed that exposing PcPIP-1 to UV irradiation for 15 min was effective to inactivate it to entirely repress its action on tobacco cells. Capsidiol production after treatment with 100 μM of PIP-1 was not affected by 15 min of UV irradiation, indicating that UV irradiation per se had no negative effect on tobacco cells in relation to phytoalexin production. Next, we investigated the stimulation period required to initiate capsidiol production in tobacco cells using PcPIP-1. Tobacco cells were treated with PcPIP-1 at 100 μM and irradiated with UV light for 15 min after 1, 3, 6, and 9 h. Capsidiol production was measured 24 h after treatment with PcPIP-1. Significant levels of capsidiol were detected when the cells were UV-irradiated 6 or 9 h after elicitation. By contrast, no capsidiol was detected when UV-irradiated 1 or 3 h after elicitation (Figure 5B). These results indicated that the continuous stimulation with PcPIP-1 for a time period between 3 and 6 h is required to induce phytoalexin production. These results were consistent with those from a previous study that used the cell-washing method to remove treated PIP-1 from the culture medium, where 3−6 h of continuous stimulation was required for capsidiol production.14 However, the amount of capsidiol detected when PcPIP-1 was inactivated 6 or 9 h after elicitation by UV irradiation was much larger than that observed in the experiments using the cell-washing method. This is probably due to the difference in the degree of stresses placed on the tobacco cells. Cell-washing is more likely to damage cells than irradiation with UV light, possibly affecting negatively capsidiol biosynthesis. Moreover, the amount of the capsidiol accumulated in the extracellular fluid up to the time of cell-washing could have been lost, resulting in an underestimation of capsidiol production. Thus, our results showed that photocleavable PcPIP-1 presents several advantages as a tool to study phytoalexin induction mechanisms by elicitors because of its ability to stop the stimulation of the immune system rapidly but not aggressively, without damaging the cells or losing the phytoalexin accumulated in the medium. Effect of PIP-1 Stimulation Period on Activation of Signaling Pathways and Biosynthesis Enzymes Important for Capsidiol Production. Following the recognition of pathogens or elicitors, MAP kinase cascades mediate the

Figure 4. Comparisons of immune responses induced by PcPIP-1 between those with and without UV irradiation in the presence of tobacco cells. (A) H2O2 generation, (B) HR-like cell death, and (C) capsidiol production. Asterisks indicate significant difference at P < 0.05 according to the Student’s paired t-test. Vertical bars indicate ±SD of three independent replicates. N.D.: not detected.

indicating that photolysis of PcPIP-1 occurred in the presence of tobacco cells similar to the process observed for aqueous solution in the absence of tobacco cells. These results were

Figure 5. Comparisons of capsidiol production after treatment with PIP-1 and PcPIP-1 at 100 μM. (A) Effect of UV-irradiation period. UV irradiation for 0−15 min was applied 1 h after sample treatment, and 0 min was used as a control. (B) Effect of activation period. UV irradiation (15 min) was applied 1−9 h after sample treatment. Asterisks indicate significant difference compared with their control according to Tukey’s multiple comparison test (*P < 0.01; **P < 0.001). Vertical bars indicate ±SD of three independent replicates. N.D.: not detected. 5898

DOI: 10.1021/acs.jafc.5b01910 J. Agric. Food Chem. 2015, 63, 5894−5901

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Journal of Agricultural and Food Chemistry signaling pathway invoking late-phase immune responses.9 In a previous study, we showed that PIP-1 induces activation of a 48 kDa kinase in tobacco cells,14 which corresponds to a salicylic acid-induced protein kinase (SIPK).26 From our previous study on the relationship between SIPK activation and capsidiol production, we concluded that a prolonged activation of SIPK induced by continuous elicitor stimulation is likely to be involved in the induction of capsidiol biosynthesis. However, which key components directly control capsidiol production in response to continuous stimulation with PIP-1 remains unknown. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR) acts as an upstream rate-limiting enzyme in the mevalonate pathway, and it plays an important role in the synthesis of isoprenoids essential for plant growth and development via constitutive activity that is likely to be regulated by HMGR1.34 It was shown that the elicitor-inducible HMGR activity is considered to be responsible for capsidiol biosynthesis in tobacco cells,35,36 which seems to be associated with HMGR2, an elicitorinducible HMGR isogene in Solanaceae plants.37,38 However, the mechanisms underlying the regulation of HMGR activity in response to elicitor treatment are poorly understood. In a previous study, we showed that short-term stimulation with PIP-1 induced the accumulation of HMGR2 transcripts,14 whereas no production of capsidiol was observed. In this regard, it is possible that HMGR activity is “switched on” only after continuous stimulation with PIP-1 via translational and/or post-translational regulatory mechanisms, leading to capsidiol production. Therefore, we investigated the relationship between the duration of stimulation with PIP-1 and the induction of HMGR activity along with the activation of SIPK using PcPIP-1. We first examined the effect of the duration of stimulation with PIP-1 on SIPK activation. As shown in Figure 6A, SIPK activity increased significantly after 3 h of treatment with PcPIP-1. This enhancement of SIPK activity was much weaker when PcPIP-1 was inactivated by UV irradiation 1 h after elicitation. This result was consistent with our previous results obtained using the cell-washing method, where the enhanced SIPK activity decreased after washing out the elicitor.14 However, the decrease in the SIPK activity was much sharper in the present study, compared to that observed using the cellwashing method. In the latter, high levels of the SIPK activity were still detected even after washing out the elicitor, probably due to physical stress or damage caused to the cells during washing operations. Our results here showed that the use of the photocleavable analog can effectively “switch off” elicitormediated MAP kinase activation including downstream responses, such as phosphorylation of its substrates. Thus, this method appears to be particularly advantageous for investigating key components important for capsidiol production in response to continuous stimulation with PIP-1 through MAP kinase signaling. We then examined the effect of the duration of the stimulation with PIP-1 on the induction of HMGR2 expression. Following treatment with either PIP-1 or PcPIP-1, HMGR2 transcripts increased significantly regardless of UV irradiation (Figure 6B). This result clearly indicated that the short-term elicitor stimulation is enough for induction of the expression of HMGR2, as previously observed.14 Treatment of protein synthesis inhibitor (CHX) did not suppress HMGR2 expression induced by PcPIP-1, suggesting that de novo protein synthesis is not involved in this gene expression. Next, the effect of the

Figure 6. Effect of stimulation period on MAP kinase and HMGR activation. (A) Comparison of MAP kinase activity induced by PcPIP1 (100 μM) between those with and without UV irradiation. UV irradiation (15 min) was applied 1 h after treatment with PcPIP-1. The activity of MAP kinase was measured 3 h after sample treatment by the in-gel kinase assay using MBP as a substrate. (B) Comparison of relative quantity of HMGR2 transcripts. Suspension cultured tobacco cells were treated with 100 μM of PIP-1 or PcPIP-1. (C) Comparison of the elicitor-inducible HMGR activity. Suspension cultured tobacco cells were treated with xylanase (Xyl, 0.1 μg/mL), PIP-1 (10 and 100 μM), or PcPIP-1 (100 μM). Distilled water was used as a control. Increases in the activity from that of the control were shown as the inducible HMGR activity. +UV: UV irradiation for 15 min at 1 h after elicitation. +CHX: cycloheximide (10 μg/mL) treatment. The total RNA and crude enzyme were extracted 4 h after elicitation. Asterisks indicate significant difference at P < 0.05 according to the Student’s paired t-test. Vertical bars indicate ±SD of three independent replicates.

duration of the stimulation with PIP-1 on the elicitor-inducible HMGR activity was examined. When a fungal elicitor xylanase, which is known to induce capsidiol production,13,39,40 was treated, significant enhancement of the HMGR activity was observed (Figure 6C). Similar enhancement was observed after treatment with PIP-1 at 100 μM, which induces capsidiol production. This enhancement was not affected by UV irradiation, indicating that UV irradiation at this wavelength has no adverse effect on enhancement of the HMGR activity. Moreover, treatment with PIP-1 at 10 μM, which induces only 5899

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low levels of capsidiol production due to enzymatic degradation by tobacco cells, did not show significant enhancement of the HMGR activity. In the case of PcPIP-1, an increase in the HMGR activity was observed after treatment at 100 μM, where capsidiol production was induced. However, this enhancement was not induced when PcPIP-1 was inactivated by UV irradiation 1 h after treatment, where no capsidiol production was observed. Thus, production of capsidiol correlated well with enhancement of the HMGR activity. This suggests that the elicitor-inducible HMGR activity, which is switched on in response to continuous stimulation with PIP-1, is closely associated with the initiation of capsidiol biosynthesis, although the involvement of other factors cannot be ruled out. In addition, because CHX suppressed the enhancement of the HMGR activity, it is possible that de novo protein synthesis is involved in induction of the HMGR activity through translational or post-translational regulation mechanisms. In a previous study, we showed that transient activation of SIPK induces only the accumulation of HMGR2 transcripts and does not lead to capsidiol production, and that capsidiol production requires an increase in SIPK activity for at least 3 h.14 In the present study, we showed that the increase in HMGR activity was also associated with the prolonged activation of SIPK activity. Therefore, it is possible that SIPK plays a dual role in immune response signaling pathways: induction of defense-related genes at an early phase and translational and/or post-translational activation of enzymes at a late phase. In fact, as shown in Nicotiana benthamiana, the expression of HMGR2 is regulated by MAPK-mediated phosphorylation of WRKY8 transcription factors in response to pathogen inoculation.41 Regarding translational or posttranslational regulation of HMGR activity, recombinant HMGR1 from Arabidopsis has been shown to be inactivated in vitro by Brassica oleracea HMGR kinase A through phosphorylation of serine residues present in the catalytic domain.42 In addition, HMGR activity has been demonstrated to be negatively regulated by protein phosphatase 2A that is considered to be a post-translational regulator of HMGR activity under stress conditions in Arabidopsis, and it was suggested that both active and inactive phosphorylation sites may exist in HMGR.43 Thus, continuous stimulation with PIP-1 might influence phosphorylation of either these regulatory proteins or HMGR via kinase cascades including SIPK. Further investigations on variations in phosphorylation of regulatory proteins through continuous stimulation with PIP-1 would help understanding the regulatory mechanisms involved in phytoalexin biosynthesis in response to elicitors. In summary, we designed photocleavable analogs to investigate which factors are involved in the initiation of phytoalexin production induced by PIP-1. An analog containing 3-amino-3-(2-nitrophenyl)propionic acid as the photocleavable residue exhibited a significant elicitor activity and was able to induce both early- and late-phase immune responses. This analog (PcPIP-1) was highly resistant to enzymatic degradation by tobacco cells, although it was easily inactivated through exposure to UV irradiation. Using this analog, we found that an increase in HMGR activity necessary for capsidiol production was significantly regulated by the stimulation period of PIP-1. To our knowledge, this study is the first report demonstrating that the continuous action of elicitor is required for activation of HMGR necessary for capsidiol production in tobacco cells.

Article

ASSOCIATED CONTENT

S Supporting Information *

Degradation resistance of YGIHTD-OH in the presence of tobacco cells (Figure S1), MS/MS spectra of PcPIP-1 and its photolyzed products (Figure S2), and the structure of possible long-lived intermediate in the photolysis process of PcPIP-1 in aqueous solution (Figure S3). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01910.

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AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +81-75-753-6123. E-mail: [email protected]. jp. Funding

This study was partly supported by MEXT KAKENHI (Grant 23580148), Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Professor Ritsuo Nishida and Dr. Hajime Ono (Kyoto University) for quantitative RT-PCR analysis. REFERENCES

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