Melatonin functions in priming of stomatal immunity in Panax notoginseng and Arabidopsis thaliana

Abstract

Melatonin (MT) plays important roles in plant disease response, but the mechanisms are largely unknown. Here, we show that MT functions in stomatal immunity in Panax notoginseng and Arabidopsis thaliana. Biochemical analyses showed that MT-induced stomatal closure plays a prominent role in preventing invasion of bacteria Pseudomonas syringe pv. tomato (Pst) DC3000 via activation of mitogen-activated protein kinase (MAPK) and NADPH oxidase-mediated reactive oxygen species production in P. notoginseng. The first putative phytomelatonin receptor 1 (PMTR1) is a plasma membrane protein required for perceiving MT signaling in stomatal closure and activation of MAPK. Biochemical and genetic tests found PMTR1 is essential for flg22- and MT-induced MAPK activation in a heterotrimeric GTP-binding protein Gα subunit GPA1-independent manner. GPA1 functions in the same genetic pathways of FLS2/BAK1 (Flagellin Sensing 2/Brassinosteroid Insensitive 1-associated kinase 1)- as well as PMTR1-mediated flg22 and MT signaling in stomatal closure. The stomata in pmtr1 are insensitive to MT and flg22, but the application of MT induces stomatal closure and reduces the bacterial growth in fls2 and bak1 plants, indicating that PMTR1 might be a downstream signaling component in FLS2- and BAK1-mediated stomatal immunity. In summary, our results (i) demonstrate that phytomelatonin functions in the priming of stomatal immunity and (ii) provide insights into the phytomelatonin signaling transduction pathway.

Melatonin regulates plant innate immunity via the putative receptor PMTR1, which mediates the activation of MAPK cascades and GPA1 signaling.

Introduction

Plants have evolved a complex and efficient innate immune system to sense the invasion of external pathogens. Plant immune receptors on the cell surface are responsible for the perception of microorganisms (host-derived immunogenicity molecular model), and are called pattern recognition receptors (PRRs; Dodds and Rathjen, 2010). Two layers of innate immunity are involved in plants fighting against invading pathogens (Jones and Dangl, 2006). The first is the recognition of pathogen- and microbe-associated molecular patterns (PAMP/MAMPs) through PRRs on the cell surface to induce basal resistance and subsequent defense response, which is known as PAMP/MAMPs-triggered immunity (PTI; Nürnberger and Kemmerling, 2006; Li et al., 2016; Saijo et al., 2018). For example, perceiving PAMP/MAMPs by PRRs triggers rapid activation of the heterotrimeric GTP-binding protein (Zhang et al., 2008), mitogen-activated protein kinase (MAPK) cascades (Cheng et al., 2015; Su et al., 2017), reactive oxygen species (ROS) burst (Li et al., 2014), and Ca²⁺ transient influx through the Ca²⁺-permeable channel OSCA1.3 (hyperosmolality-gated calcium-permeable channel; Thor et al., 2020), which are all associated with stomatal closure. The second line of plant defense is effector-triggered immunity (ETI) mediated by activation of plant resistance proteins via intracellularly localized receptors NLRs (nucleotide-binding, leucine-rich repeat proteins) for detecting the pathogenic effectors associated with regulating the expression of resistance (R) genes and the hypersensitive response (HR) cell death (Wang et al., 2020).

The formation of guard cells is a milestone in the evolution of terrestrial plants (Qi et al., 2018). Stomata formed by guard cells are the main gates for gas exchange. However, stomata also provide entry sites for pathogen invasion. Plants have evolved a mechanism to close the stomata to reduce the invasion of pathogens after perceiving PAMP/MAMPs by PRRs, which is called stomatal defense or stomatal immunity (Melotto et al., 2006, 2017). Upon sensing MAMPs (e.g. bacterial flagellins or flg22 peptide, as the biologically active epitope of the bacterial flagellin), Flagellin Sensing 2 (FLS2) interacts with its coreceptor Brassinosteroid Insensitive 1-associated kinase 1 (BAK1) that phosphorylates the heterotrimeric GTP-binding protein Gα subunit (GPA1), resulting in ROS burst and stomatal immunity through regulation of the interaction between GPA1 and NADPH oxidase RbohD (Zhong et al., 2019; Xue et al., 2020). FLS2 could interact with the receptor-like cytoplasmic kinase BIK1 (Botrytis-induced kinase 1) to regulate ROS production and Ca²⁺ signaling associated with stomatal immunity by direct phosphorylation of NADPH oxidase RbohD (Lu et al., 2010; Li et al., 2014). Upon activation, the MAPK cascades also play essential roles in stomatal immunity via regulation of organic acids metabolism (Su et al., 2017), which is a signaling event independent of the GPA1 and NADPH oxidase-mediated ROS burst (Xu et al., 2014; Xue et al., 2020).

Microbes have evolved strategies to overcome stomatal immunity. For example, coronatine (COR), a phytotoxin produced by Pseudomonas syringae, promotes reopening of the closed stomata through hijacking the plant jasmonic acid receptor COI1 and inhibiting flg22-induced ROS production (Melotto et al., 2006; Lee et al., 2013).

Melatonin (MT, N-acetyl-5-methoxytryptamine) is a ubiquitous signaling molecule in plants and animals (Wei et al., 2018). The biosynthesis of plant endogenous MT (referred to as phytomelatonin) begins from tryptophan via four consecutive enzymatic steps catalyzed by tryptophan decarboxylase (TDC), serotonin N-acetyltransferase 1 (SNAT1), caffeate O-methyltransferase 1 (COMT1), and N-acetylserotonin methyltransferase (ASMT; Zhang et al., 2015). The first putative phytomelatonin receptor PMTR1/CAND2 has been identified in Arabidopsis (Arabidopsis thaliana), which is essential for MT-induced stomatal closure through interaction with GPA1 and activation of NADPH oxidase-mediated ROS production (Wei et al., 2018). Additionally, the daily rhythms of phytomelatonin signaling are essential for the maintenance of diurnal stomatal closure via preserving ROS dynamics (Li et al., 2020).

MT is important in plant normal growth and abiotic stress responses. For example, many studies have shown the functions of MT in seed germination and plant growth, rooting and flowering, fruit ripening, modulating primary and secondary metabolisms, and regulating the level and action of several plant hormones (Arnao and Hernández-Ruiz, 2019a, 2020a, 2020b). Exogenous MT improves plant resistance to drought, salt, cold, heat, toxic metal, and UV light stresses (Arnao and Hernández-Ruiz, 2019b; Altaf et al., 2020; Moustafa-Farag et al., 2020a, 2020b). Additionally, MT is also involved in plant biotic stress responses. For example, the application of MT can effectively inhibit the activity of Rhizoctonia solani Kuhn and enhance the resistance of apple trees to brown spot disease (Yin et al., 2013). Shi et al. (2015) found that the virulent pathogen strain of Pseudomonas syringe pv. tomato (Pst) DC3000 rapidly increased endogenous phytomelatonin concentration in Arabidopsis. Exogenous application of MT induced defense signaling such as NO production and expression of the salicylic acid (SA)- and pathogen-related genes (Shi et al., 2015). However, the detailed mechanisms of phytomelatonin regulation of plant immunity are still largely unknown.

Panax notoginseng (Bruk.) FH Chen is a highly valuable herb in Chinese traditional medicine; it has been used for hundreds of years. Leaf diseases caused by Alternaria panax Whetzel and Pseudomonas syringae (Pst) plague the growth and production of P. notoginseng (Zhou et al., 2013). To prevent and control foliar diseases, a variety of fungicides and bactericides have been used during the production process, posing a potential risk to human health. In this study, we investigated whether and how phytomelatonin regulates P. notoginseng biotic resistance using a model pathogen Pst DC3000 (Xin and He, 2013). The results showed that the application of MT significantly increased the P. notoginseng innate immunity via ROS- and MAPK-mediated stomatal defense. Using biochemical and genetic analysis in Arabidopsis, we further found that MAPK and GPA1 are two independent signaling pathways in MT- and flg22-mediated stomatal immunity that occur downstream of PMTR1. Additionally, PMTR1 might be a downstream component of immune receptors in the FLS1 and BAK1 signaling in flg22-induced stomatal immunity. Therefore, our work (1) suggests that MT has a function in priming of stomatal immunity and (2) identifies PMTR1 as a mediator of the plant PTI response via regulation of the MAPK and GPA1 signaling pathways.

Results

MT induces stomatal immunity in P. notoginseng

The pretreatment with MT (2 h/Pst) or flg22 (flg22 2 h/Pst) for 2 h followed by Pst DC3000 inoculation as opposed to having MT together with spray or infiltration inoculation by Pst DC3000 (MT+Pst) significantly reduced the bacterial growth in P. notoginseng leaves (Figure 1, A and B), indicating that MT might be involved in plant pre- and postinfection immunity. Additionally, the pretreatment with MT or flg22 for 2 h showed the lowest bacterial growth under both treatment conditions (Figure 1, A and B); importantly, the application of MT did not reduce the growth of bacteria in the liquid medium (Supplemental Figure S1). These findings indicated MT might function similarly to MAMP flg22 as a signal molecule to initiate plant innate immunity without having direct antibacterial activity.

Figure 1.

MT is involved in plant stomatal immunity in P. notoginseng. Effect of MT (10 μM) or flg22 (5 μM) on bacterial growth in P. notoginseng leaves after being spray- (A) or infiltration-inoculated (B) by Pst DC3000 for 2 and 72 h. DMSO 2 h/Pst indicates plant leaves were spray-pretreated by DMSO (1/1,000, v/v) for 2 h and then spray- or infiltration-inoculated with Pst DC3000. MT (or flg22) 2 h/Pst denotes MT (or flg22) foliar spray pretreatment for 2 h and then spray or infiltration inoculation with Pst DC3000; MT+Pst denotes the MT treatment and bacterial inoculation applied together. C, Images of stomata after the plants were pretreated by MT for 2 h. DMSO (1/1,000, v/v) treatment as the solvent control (Con). Bar = 100 μm. D, The stomatal aperture on P. notoginseng intact leaves measured by the silicon polymer impression method. Leaves were pretreated with MT (10 μM MT) or without (DMSO, 1/1,000, v/v) for 2 h before being surface-inoculated with Pst DC3000 (OD₆₀₀ = 0.2) (0 h). Stomatal aperture was measured after foliar-spray inoculation with Pst DC3000 for 0 [MT (DMSO) 2 h/Pst 0 h], 2 [MT (DMSO) 4 h/Pst 2 h] and 4 h [MT (DMSO) 6 h/Pst 4 h]. Values are means ± se (n = 3 for A and B, and n = 30 for D). Different letters (in A, B, and D for a given treatment duration) indicate significant differences at P < 0.05 (Student’s t test). The experiments were performed three times and yielded similar results.

Stomata are the major entry sites for invasion of foliar pathogens; therefore, we measured the stomatal aperture directly in P. notoginseng leaves using the silicon polymer impression method (Li et al., 2020). The foliar-spray pretreatment with MT on P. notoginseng leaves for 2 h (MT 2 h, Pst 0 h) or Pst DC3000 spray-inoculated for 2 h with (MT 4 h, Pst 2 h) or without MT pretreatment (DMSO 4 h, Pst 2 h) induced stomatal closure in P. notoginseng leaves, but the closed stomata reopened after the leaves were inoculated by Pst DC3000 for 4 h in the presence (MT 6 h, Pst 4 h) or absence of MT pretreatment (DMSO 6 h, Pst 4 h; Figure 1, C and D). COR is a known phytotoxin produced by P. syringae that induces the closed stomata reopening to overcome stomatal immunity. With the presence of COR in the medium bathing leaf epidermal strips, MT failed to induce stomatal closure (Supplemental Figure S2). These results indicate that phytomelatonin induces preinfection immunity via induction of stomatal closure to prevent the entry of bacteria into plant leaves, but phytomelatonin-induced stomatal closure is blocked by the presence of COR.

MT and flg22 induce stomatal closure via NADPH oxidase-mediated ROS production in P. notoginseng

ROS play a central role in plant defense response and stomatal closure. Therefore, we analyzed the stomatal aperture and ROS production after the MT and flg22 treatments with or without CAT (catalase, the H₂O₂ scavenger), DPI (diphenyleneiodonium chloride, an inhibitor of NADPH oxidase) or SHAM (salicylhydroxamic acid, a cell wall peroxidase inhibitor). MT, flg22, and H₂O₂ promoted the closure of stomata (Figure 2, A) and ROS production (Figure 2, B;Supplemental Figure S3), which is consistent with the previous studies (Melotto et al., 2006; Wei et al., 2018). Exogenous addition of CAT and DPI, but not SHAM, significantly diminished the stomatal closure (Figure 2, A) and ROS production (Figure 2, B;Supplemental Figure S3) induced by MT or flg22, indicating that MT- and flg22-induced ROS production and the associated stomatal closure are dependent on the NADPH oxidase.

Figure 2.

NADPH oxidase is required for MT- and flg22-induced stomatal closure and ROS burst in the guard cells of P. notoginseng. Effects of 50 μM H₂O₂, 20 μM SHAM, 20 μM DPI, and 100 U/mL CAT on the MT (10 μM, MT)- or flg22 (5 μM)-induced stomatal closure (A) and the corresponding guard cell H₂O₂ fluorescence intensity (B). The epidermal strips of 1-year-old P. notoginseng leaves were incubated in the medium for 3 h under light (100 μmol m⁻² s⁻¹), and then were transferred into a fresh incubation medium containing the treatment chemicals under light (100 μmol m⁻² s⁻¹) for 2 h. The stomatal aperture was measured as width/length ratio. The data are expressed as means ± se (A, n = 30; B, n = 20). Different letters indicate significant differences at P < 0.05 (Student’s t test). The experiments were performed three times and yielded similar results.

MT induces the expression of pathogen-related genes and activates the MAPK in P. notoginseng

Having ascertained the pretreatment of MT could induce the innate immunity in P. notoginseng, we then analyzed the expression of defense-related genes after the plants were treated with 0 (control) or 10 μM MT for 0, 1 and 2 h or with MT plus surface-inoculated Pst DC3000 for 2 h (+Pst 2 h). The results showed that the MT treatment significantly induced the expression of PnBAK1-3, PnMPK3-1, PnMPK3-2, PnMPK6-2, PnWRKY22-6, PnPR1-2, PnPR5-4, and PnNPR-1, and promoted the Pst DC3000-induced expression of PnFLS2, PnMPK3-1, PnMPK3-2, PnPR1-2, and PnBGL2-1 (Figure 3, A–C; Supplemental Figure S4). In contrast, several genes in P. notoginseng leaves, including PnGPA1, PnRGS1 and PnWRKY22-1, did not show significant differences with or without the MT pretreatment (Figure 3, A–C; Supplemental Figure S4), indicating that the expression of these genes might not be regulated by MT.

Figure 3.

MT regulates expression profiles of defense-related genes and activation of MPK3 and MPK6 in P. notoginseng. Time-course changes in the expression of genes related to immune proteins (A), defense-related (B) and MAPKs (C) without (−MT) or with MT treatment for 0, 1 and 2 h without or with Pst DC3000 surface inoculation (+Pst 2 h). Values are means ± se (n = 3). Asterisks denote significant difference between the indicated treatments at P < 0.05 (Student’s t test). D and E, The MAPK activation assay in 1-year-old P. notoginseng leaves foliar-treated by 10 μM MT. In (D), the MT treatments lasted for 0, 1, and 2 h; in addition, +Pst 2 h denoted that leaves were foliar-treated by 10 μM MT and Pst DC3000 for 2 h. In (E), the MT treatment lasted for 0, 2, 4, 8, and 24 h. MAPK activation was detected by immunoblot analysis using the Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr204) rabbit monoclonal antibodies. Actin was used as an internal loading control. Western blot experiments were performed at least three times and yielded similar results.

Activation of MAPKs is one of the earliest defense responses; hence, we analyzed the phosphorylation of MPK3 and MPK6 in P. notoginseng leaves (Figure 3, D and E). The results showed that foliar spray pretreatment with MT only for 1–2 h substantially induced MPK3 and MPK6 phosphorylation (Figure 3, D). Furthermore, the MT-mediated activation of MPK3 and MPK6 was increasing for at least 8 h in the absence of bacterial infection (Figure 3, E).

Activation of MPK3 and MPK6 is required for MT-induced stomatal immunity in P. notoginseng and A. thaliana

MPK3 and MPK6 are rapidly activated upon plant perception of PAMP/MAMPs and play critical roles in plant defense responses. We analyzed whether activation of MPK3 and MPK6 is also involved in MT-induced innate immunity in P. notoginseng. Similar to MAMP flg22, the MT-only treatment induced the phosphorylation of MPK3 and MPK6 in P. notoginseng leaves, and in the presence of U0126, an inhibitor of MPK cascade, either MT or flg22 failed to activate MPK3 and MPK6 (Figure 4, A). Furthermore, the application of U0126 abolished the MT- or flg22-induced stomatal closure (Figure 4, B) and MT-mediated inhibition of bacterial invasion (Figure 4, C). These results indicate that MT induces stomatal immunity via activation of the MPK3 and MPK6 signaling cascades in P. notoginseng leaves.

Figure 4.

Activation of MPK3 and MPK6 is involved in MT-induced stomatal immunity in P. notoginseng. A, The MAPK activation assay in P. notoginseng leaves treated by MT in the absence or presence of U0126 (an inhibitor of MPK cascade). The leaves were first pretreated without or with 100 μM U0126 for 1 h and then transferred into ddH₂O containing 10 μM MT or 5 μM flg22 for 15 min. Western blot experiments were performed at least three times and yielded similar results. B, Stomatal aperture of P. notoginseng leaf epidermal strips treated by 10 μM MT, 5 μM flg22 or Pst DC3000 (OD = 0.2) for 2 h without or with pretreatment by 100 μM U0126 for 1 h. The epidermal strips of 1-year-old P. notoginseng leaves were incubated in the incubation medium for 3 h under light (100 μmol m⁻² s⁻¹), and then the strips were transferred into a fresh incubation medium without or with 100 μM U0126 for 1 h pretreatment followed by the treatment with MT, flg22 or Pst DC3000 (Pst) for 2 h under light (100 μmol m⁻² s⁻¹). Values are means ± se (n = 30). Different letters indicate significant differences at P < 0.05 (Student’s t test). C, Growth of Pst DC3000 in P. notoginseng leaves treated by MT after the pretreatment without or with U0126. The plant leaves were sprayed by 100 μM U0126, incubated for 1 h, and then were treated by 10 μM MT for 2 h before bacterial inoculation. The bacterial populations were measured immediately (0 dpi) and 3 d after the leaves were inoculated by Pst DC3000 (3 dpi). Values are means ± se (n = 3). Different letters indicate significant differences at P < 0.05 (Student’s t test). The experiments were performed three times and yielded similar results.

We further confirmed that activation of MAPK is involved in MT-mediated plant stomatal immunity using a biochemical/genetic approach in Arabidopsis (A. thaliana). Given that mpk3mpk2 double mutant is lethal, we used MPK3SR (mpk3 mpk6 pMPK3::MPK3^TG) and MPK6SR (mpk3 mpk6 pMPK3::MPK3^YG; Xu et al., 2014; Su et al., 2017; Zhu et al., 2019). MPK3SR or MPK6SR is chemically sensitized MPK3 or MPK6 that can be inhibited by NA-PP1. In the absence of NA-PP1, both MPK3SR and MPK6SR plants showed phenotypes similar to Col-0 regarding bacterial resistance (Figure 5, A and B) and stomatal aperture (Figure 5, C); however, the application of NA-PP1 increased the susceptibility to Pst DC3000 in the MPK3SR and MPK6SR, but not in Col-0 plants (Figure 5, A and B). The MT treatment significantly reduced bacterial growth (Figure 5, A) and induced stomatal closure (Figure 5, C) in Col-0, MPK3SR and MPK6SR in the absence of NA-PP1. In contrast, MT failed to induce bacterial resistance (Figure 5, A and B) and stomatal closure (Figure 5, C) in MPK3SR and MPK6SR in the presence of NA-PP1. These results suggested that activation of MPK3 and MPK6 is required for MT-mediated stomatal immunity.

Figure 5.

MPK3 and MPK6 are required for MT-mediated stomatal immunity in Arabidopsis. Leaf appearance (A, 4 dpi) and bacterial growth (B) in Col-0, MPK3SR and MPK6SR. After the plants (3- to 4-week-old) were foliarly pretreated by NA-PP1 (2 μM) for 2.5 h and then by 10 μM MT (+MT) for further 2 h, the 10 mM MgCl₂ solution containing Pst DC3000 (OD = 0.2) was spray-inoculated on plant leaves, and the bacterial growth was measured immediately after inoculation (0 dpi) and 3 d after (3 dpi). The equivalent concentration of DMSO (1/1,000, v/v) was used as solvent control. For (A), bar = 1 cm. Values are means ± se (n = 3). Different letters indicate significant differences at P < 0.05 (Student’s t test). C, Stomatal apertures of Col-0, MPK3SR and MPK6SR. The leaf epidermal strips from 3- to 4-week-old plants were first pretreated by NA-PP1 (2 μM) under light (100 μmol m⁻² s⁻¹) for 2.5 h, and were then treated by 10 μM MT or 5 μM flg22 for 2 h. The equivalent concentration of DMSO (1/1,000, v/v) was used as a solvent control. Values are means ± se (n = 30). Different letters indicate significant differences at P < 0.05 (Student’s t test). The experiments were performed three times and yielded similar results.

Phytomelatonin receptor 1 is a plasma membrane protein required for perceiving a MT signal in MAPK activation

We previously found that phytomelatonin receptor 1 (PMTR1) is a plasma membrane protein that shows specific and saturable MT binding and is required for perceiving the MT/phytomelatonin signal in stomatal closure (Wei et al., 2018; Li et al., 2020). In this study, we further confirmed that PMTR1 is a membrane protein using confocal microscopy analysis in onion bulb epidermal cells (Supplemental Figure S5). Additionally, the constructs GFP-PMTR1 and PIP2-mCherry (a marker for the plasma membrane localization; Nelson et al., 2007) were transiently coexpressed in Arabidopsis protoplasts. The results showed that fluorescence of GFP-PMTR1 overlapped with that of PIP2-mCherry at the plasma membrane (Figure 6, A). Western blots showed the expression of PMTR1 in the plasma membrane protein fraction, but not in the soluble fractions (similarly to the positive control of the plasma membrane H⁺-ATPase) in transgenic PMTR1-OE Arabidopsis plants (Figure 6, B). The coimmunoprecipitation and Western blot analyses further confirmed the interaction between PMTR1 and GPA1 (Figure 6, C) as we previously reported using the yeast split-ubiquitin system (mbSUS) and biomolecular fluorescence complementation (Wei et al., 2018).

Figure 6.

PMTR1 is a plasma membrane protein and is required for activation of MPK3 and MPK6 in a GPA1-independent manner in Arabidopsis. A, Plasma membrane localization of PMTR1 in Arabidopsis protoplasts. The GFP-PMTR1 signal was merged with that of the PIP2-mCherry (an intrinsic plasma membrane protein) marker in Arabidopsis protoplasts. GFP, fluorescence of green fluorescent protein; RFP, fluorescence of red fluorescent protein indicating expression of mCherry; Chloroplast fluorescence, autofluorescence of chloroplasts; bright field, protoplast image under normal field of view; Merged, overlay of the GFP, RFP, chloroplast fluorescence, and bright field images. 35S: GFP-PMTR1/35S: PIP2-mCherry, coexpression of the pCAMBIA1300-35S-GFP-PMTR1 and pCAMBIA1300-35S-PIP2-mcherry plasmids; 35S: GFP/35S: mCherry, coexpression of the pCAMBIA1300-35S-GFP and pCAMBIA1300-35S-mcherry plasmids. Scale bar = 20 μm. B, Immunoblotting assay showing FLAG antibody recognized FLAG-tagged PMTR1 in the plasma membrane (PM) protein fraction from transgenic PMTR1-OE Arabidopsis plants. H⁺-ATPase, plasma membrane protein control. C, Coimmunoprecipitation assays confirming the interaction between PMTR1 and GPA1. Total protein from leaves of 3-week-old Col-0 and PMTR1-OE-FLAG were immunoprecipitated by anti-FLAG. Blots were probed with anti-GPA1 or anti-FLAG. Input was used to detect the presence of GPA1 or PMTR1 in Col-0 and/or PMTR1-OE-FLAG transgenic plants after protein isolation. Output represented the interaction between PMTR1 and GPA1 in Col-0 and PMTR1-OE-FLAG after immunoprecipitation by anti-FLAG. D, Expression levels of AtPMTR1 gene in wild type Col-0 and pmtr1 mutant plants. E, The MAPK activation assay in Col-0, gpa1-4 and pmtr1 plants in the presence or absence of 10 μM MT for the 15-min treatments. MAPK activation was detected by immunoblot analysis using the Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr204) rabbit monoclonal antibodies. Western blot experiments were performed at least three times and yielded similar results.

Semiquantitative quantitative real-time PCR (RT-PCR) and reverse transcription quantitative PCR (RT-qPCR) showed that the expression of AtPMTR1 was abolished in the pmtr1 mutant plants (Figure 6, D;Supplemental Figure S6), which is in accordance with the results previously reported by us (Wei et al., 2018; Li et al., 2020) and others using the same germplasm (Jin et al., 2012). Western blot analysis found that MT increased the phosphorylation of MPK3 and MPK6 in the Col-0 and gpa1-4, but not in pmtr1 mutant plants (Figure 6, E), indicating that PMTR1 is required for perceiving the MT signal in the activation of MAPK in a GPA1-independent manner.

MAPK and GPA1 are two independent signaling pathways downstream of PMTR1 in stomatal immunity

RT-qPCR analysis (Supplemental Figure S7) found that the expression of AtSNAT1, AtCOMT1 and AtPMTR1 was increased rapidly (e.g. within 0.5-1 h) by MT or flg22 individually or in combination with Pst DC3000, indicating that the phytomelatonin signaling is induced by the bacterial infection in Arabidopsis. Therefore, we analyzed the phytomelatonin signaling in stomatal immunity using pmtr1, PMTR1-OE and gpa1-4. The PMTR1-OE plants showed greater resistance to Pst DC3000 infection as shown by less severe disease symptoms (Figure 7, A) and lower bacterial growth (Figure 7, B) compared with the mutant lines pmtr1 and gpa1-4. The application of MT increased bacterial resistance (Figure 7, A and B) and induced stomatal closure (Figure 7, C) in Col-0 and PMTR1-OE, but not in pmtr1 and gpa1-4. However, MT and flg22 promoted stomatal closure in gpa1-4/GPA1-HA (Supplemental Figure S8), further confirming GPA1 is required for MT- and flg22-induced stomatal closure (Melotto et al., 2006; Wei et al., 2018). Activation of MPK3 and MPK6 was impaired in pmtr1 in the presence or absence of MT or flg22 (Figure 7, D), but not in the gpa1-4 null mutant plants (Figure 7, D). These results indicate that PMTR1 is required for MT- and flg22-induced activation of MPK3 and MPK6 in a GPA1-independent manner.

Figure 7.

PMTR1 is required for flg22- and MT-mediated activation of MAPK in a GPA1-independent manner in Arabidopsis. Disease symptoms (A, 4 dpi) and bacterial growth (B) in Col-0, gpa1-4, pmtr1, and PMTR1-OE plants spray-inoculated by Pst DC3000 (OD = 0.2) with or without 10 μM MT treatment. The equivalent concentration of DMSO (1/1,000, v/v) was used as a solvent control. For A, bar = 1 cm. For (B), values are means ± se (n = 3). Different letters indicate significant differences at P < 0.05 (Student’s t test). C, Stomatal aperture of Col-0, gpa1-4, pmtr1, and PMTR1-OE plants in the absence (Con) or the presence of 10 μM MT or 5 μM flg22. The data are expressed as means ± se (n = 30). Different letters indicate significant differences at P < 0.05 (Student’s t test). D, The MAPK activation assay in Col-0, gpa1-4, pmtr1, and PMTR1-OE plants in the presence or absence of 10 μM MT or 5 μM flg22 for the 15-min treatments. MAPK activation was detected by immunoblot analysis using the Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr204) rabbit monoclonal antibodies. Western blot experiments were performed at least three times and yielded similar results.

MT induces stomatal immunity in fls2 and bak1

The immune receptors FLAGELLIN SENSING 2 (FLS2) and BRASSINOSTEROID INSENSITIVE 1-associated kinase 1 (BAK1) play a central role in sensing the bacterial flagellin signaling and initiating the immunity responses. Exogenous application of MT induced stomatal closure (Figure 8, A) and decreased bacterial growth (Figure 8, B) in fls2 and bak1-4, indicating that MT-induced stomatal immunity is independent of FLS2 and BAK1.

Figure 8.

MT induces stomatal closure and decreases bacterial growth in fls2 and bak1-4. A, Leaf epidermal strips of Col-0, fls2, and bak1-4 plants were floated on the stomatal opening buffer solution under light (100 μmol m⁻² s⁻¹) for 3 h and then were exposed to 10 μM MT or 5 μM flg22 for 2 h before stomatal aperture measurement. The data are expressed as means± se (n = 30). Different letters indicate significant differences at P < 0.05 (Student’s t test). B, Col-0, fls2, and bak1-4 plants were spray-inoculated with Pst DC3000 (OD = 0.2) after 2-h pretreatment with or without 10 μM MT. Bacterial growth was measured at 0 and 3 d after inoculation (dpi). The data are expressed as means ± se (n = 3). Different letters indicate significant differences at P < 0.05 (Student’s t test). The experiments were performed three times and yielded similar results.

Discussion

Phytomelatonin is a putative hormone associated inter alia with plant biotic resistance (Arnao and Hernández-Ruiz, 2019c; Zhao et al., 2021; Moustafa-Farag et al., 2020c). However, the regulatory mechanisms are still largely unknown. Here, we provided strong evidence that MT regulates stomatal immunity in P. notoginseng and A. thaliana via activation of MAPK and heterotrimeric GTP-binding protein Gα subunit (GPA1), two independent signaling pathways regulated by PMTR1. Furthermore, PMTR1 acts downstream of FLAGELLIN SENSING 2 (FLS2)- and BRASSINOSTEROID INSENSITIVE 1-associated kinase 1 (BAK1)- mediated stomatal immunity and might be a mediator of plant PTI response.

MT plays a prominent role in stomatal immunity

Plants have developed two layers of innate immunity consisting of PRRs-mediated PTI and intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs)-mediated ETI to sense and protect themselves against the invading pathogens. The innate immune is initiated after perceiving the PAMP/MAMPs by PRRs on the plant cell surface (Bittel and Robatzek, 2007; Meng and Zhang, 2013). NLRs are intracellular proteins that sense pathogen-derived effector proteins inside the plant cell. PTI and ETI have long been considered as two separate immune signaling pathways; however, recently Yuan et al. (2021) found that PTI and ETI interact. For example, PRR-mediated activation of RBOHD in PTI is required for full activation of RBOHD in ETI. Furthermore, the NLR signaling of ETI could rapidly increase the transcription and translation of the key PTI components (Yuan et al., 2021).

The stomata formed by guard cells play an active role in preventing bacterial invasion (Melotto et al., 2006), which has been extensively studied as a model to understand plant PTI responses and cellular signaling transduction (Melotto et al., 2017). Bacterial flagellin and its active epitope flg22 peptide trigger stomatal closure via FLS2- and BAK1-mediated activation of Gα and NAPDH oxidase-dependent ROS production (Zeng et al., 2010; Liang et al., 2016; Xu et al., 2019) and activation of MAPK signaling cascades (Xu et al., 2014; Su et al., 2017). Similarly to flg22, we showed here that MT is involved in immunity before invasion by bacteria as reflected in the MT pretreatment before spray-inoculation of Pst DC3000 reducing bacterial populations in P. notoginseng leaves (Figure 1, A), which might be due to MT-mediated induction of stomatal closure (Figure 1, C and D), ROS production (Figure 2), MAPK activation, and increased expression of genes associated with PTI (e.g. PnFLS2, PnBAK1, PnMPK3-1, PnMPK3-2, and PnMPK6-2; Figures 1–4; Supplemental Figures S2–S4, S6). Additionally, we also observed a decrease in bacterial growth in P. notoginseng leaves pretreated by MT before infiltration-inoculation by Pst DC3000 (Figure 1); in contrast, the application of MT in liquid growth medium did not affect the bacterial growth (Supplemental Figure S1), indicating that MT-mediated inhibition of bacterial growth is not due to the antimicrobial activity. However, genes associated with both the PTI and ETI signaling (e.g. PnPR1-2, PnPR5-4, PnNPR1, PnBGL2-1, and WRKY22-6) were upregulated by MT (Figure 3;Supplemental Figure S4), which might be related to increased bacterial resistance after infiltration-inoculation due to the interaction between PTI and ETI (cf. Yuan et al., 2021). The MT pretreatment before spray-inoculation by Pst DC3000 reduced the bacterial growth in leaves to a greater extent than in the case of infiltration-inoculation (e.g. 10-fold versus 3.5-fold reduction; Figure 1, A and B), indicating that MT might play a prominent role in the PTI response in stomatal immunity in P. notoginseng.

MT- and flg22-triggered MPK3 and MPK6 activation and stomatal immunity require PMTR1 but not GPA1

MAPK cascades, including MAPK kinases (MAPKKKs or MAP3Ks), MAPK kinases (MKKs or MAP2Ks) and MAPKs (MPK), are conserved signaling components in both plants and animals. In mammals, MAPKs act downstream of G protein signaling (Goldsmith and Dhanasekaran, 2007). In plants, activation of MAPK cascades and G protein signaling are the earliest defense responses. However, activation of MAPK is independent of G protein; they are two independent pathways associated with stomatal immunity via regulating organic acids metabolism (Xu et al., 2014; Su et al., 2017) or activating NAPDH oxidase-mediated ROS production (Xu et al., 2019; Xue et al., 2020). For example, the flg22-induced ROS burst (but not phosphorylation of MPK3 and MPK6) was impaired in gpa1-4, xlg2 and agb1 (Liang et al., 2016; Liang and Zhou, 2018; Xue et al., 2020), all of which are the important G protein signaling components associated with plant innate immunity.

Activation of MPK3 and MPK6 mediated by MKK4 and MKK5 is involved in MT-induced plant defense responses (Lee and Back, 2016). In a recent review article, Zhao et al. (2021) demonstrated that activation of MAPK could be mediated by PMTR1, which might be essential for biotic stress resistance. The data obtained here confirmed their speculation. In the present study, we found that MT induced the expression of MKK4/5 and MPK3/6 and phosphorylation of MPK3/6 in P. notoginseng (Figures 3, D and E, and 4A) and Arabidopsis Col-0 (Figures 6, E, and 7, D). Furthermore, activation of MPK3 and MPK6 is required for MT- and flg22-mediated stomatal immunity in both P. notoginseng (Figure 4, C) and Arabidopsis Col-0 (Figure 5, A–C). Additionally, MT- and flg22-induced phosphorylation of MPK3 and MPK6 is impaired in the pmtr1, but not in gpa1, mutant plants (Figures 6, E, and 7, D), indicating that PMTR1 is required for both MT- and flg22-induced activation of MPK3 and MPK6 in a GPA1-independent manner. Similarly, although interactions between the receptors FLS2, BAK1 and BSK1 (BR-Signaling Kinase 1) are required for plant innate immunity, flg22-induced MAPK is abolished in the fls2 but not in bak1 and bsk1, mutant plants (Shi et al., 2013; Liang et al., 2016; Liang and Zhou, 2018). These results indicated that plants might utilize a fine-tuning mechanism for regulating the GPA1 and MAPK signaling pathways after perceiving input signals (e.g. MT and flg22). The mutant plants with impaired expression of AtMPK3/6 and AtGPA1 were insensitive to flg22- and MT-induced stomatal closure (Figures 5, C, and 7, C; see also Zeng et al., 2010; Xu et al., 2014; Su et al., 2017), indicating that the MAPK cascades and G protein signaling pathways might interact to regulate stomatal closure with yet to be characterized signaling nodes.

PMTR1 and GPA1 act downstream of FLS2- and BAK1-mediated stomatal immunity

Heterotrimeric GTP-binding protein/G-protein coupled receptors (GPCRs) are key regulators of a multitude of signaling pathways in animals and yeast. In Arabidopsis, many seven-transmembrane (7TMs) proteins, including one putative GPCR (GCR1), candidate G-protein coupled receptors (CANDs), GPCR-type G proteins GTG1 and GTG2, with structural similarity to animal GPCRs that suggest interaction with Gα, have been identified (Vilardaga et al., 2003; Pandey and Assmann, 2004; Moriyama et al., 2006; Gookin et al., 2008; Pandey et al., 2009). However, due to plant Gα having low GTPase activity and self-activation via high GDP/GTP exchange activity (Urano et al., 2012a; Bradford et al., 2013), it has traditionally been suggested that the plant G protein signaling does not require GPCR-like receptors (Urano et al., 2013; Urano and Jones, 2013). Instead, accumulating evidence suggests that Gα protein could be regulated via phosphorylation/dephosphorylation by receptor-like kinases (RLKs), including FLS2, BAK1 and RGS1 (Regulator of G Signaling 1; Liang and Zhou, 2018; Tunc-Ozdemir et al., 2017; Urano et al., 2012b). For example, BAK1 could directly interact and phosphorylate GPA1 at Thr19 (Xue et al., 2020), resulting in ROS burst and stomatal immunity (Xu et al., 2019).

PMTR1 is the first putative GPCR-like protein found in plants that binds specifically with MT (Wei et al., 2018). Our previous work suggested that MT induced stomatal closure via GPA1-activated NADPH oxidase-mediated ROS production (Wei et al., 2018). In the study presented here, we further confirmed PMTR1 is a plasma membrane protein (Figure 6, A and B; Supplemental Figure S5) that could interact with GPA1 (Figure 6, C), in contrast to the recent report by Lee and Back (2020). Furthermore, Wang et al., (2021) more recently found that the MT-induced osmotic stress tolerance in Arabidopsis is mediated by PMTR1 using the same plant materials as we previously did (Wei et al., 2018). Additionally, PMTR1 is required for perceiving the exogenous MT and endogenous phytomelatonin signaling, at least in stomatal closure (Figure 7, C; Wei et al., 2018; Li et al., 2020) and MAPK activation (Figure 6, E, and 7, E). Similarly, Kong et al (2021) also found that MT and its homologs (5-methoxytryptamine and 5-methoxyindole) induce stomatal closure and expression of immune genes via trP47363 and trP13076 in Nicotiana benthamiana, two homologous proteins of PMTR1. Biochemical and genetic results presented here (Figure 7, C) and elsewhere (Pandey et al., 2009; Wei et al., 2018) suggested that GPCR-like (G) proteins including PMTR1, GTG1 and GTG2 function in the same genetic pathways as GPA1 in MT-, flg22-, and ABA-induced ROS production and stomatal closure. Therefore, plants might possess similar G-protein components that exist in metazoan systems, but with a different and as yet unknown regulating mechanism (Pandey and Vijayakumar, 2018; Pandey, 2019), which is not surprising because the GPCRs/G protein systems are different in various taxonomic classes and have evolved into two different lineages (Wei et al., 2018; Li et al., 2020). Similar to gpa1-4, pmtr1 is also insensitive to flg22-induced stomatal closure, whereas application of MT induces stomatal closure (Figure 8, A) and reduces bacterial invasion (Figure 8, B) in the fls2 and bak1 mutant plants. It is likely that PMTR1 is required for FLS2- and BAK1-mediated activation of G protein signaling in stomatal immunity, whereas PMTR1-mediated MT signaling in stomatal closure might be independent of FLS2 and BAK1.

In summary, the data presented here provided evidence for MT function in priming stomatal immunity in both P. notoginseng and Arabidopsis. Furthermore, our results suggested that PMTR1 might be a mediator of the plant PTI response and a key effector in activation of MAPK and GPA1 downstream of FLS2- and BAK1-initiated innate stomatal immunity. Although the interaction between PMTR1 and GPA1 might play an important role in MT and flg22 signaling in stomatal closure, the MT- and flg22-induced MAPK activation is dependent on PMTR1 but not GPA1, indicating that not all of the PMTR1-related signaling and functions are dependent on G protein. Therefore, the regulatory mechanisms among the FLS2, BAK1, PMTR1, MAPK, and GPA1 signaling cascades warrant further work.

Materials and methods

Plant materials

Panax notoginseng and Arabidopsis (A. thaliana) were used in this study. One-year-old P. notoginseng plants were cultured in a greenhouse located at Kunming University of Science and Technology (N 24.85°, E 102.87°, altitude 1895 m). Arabidopsis plants used in this study are all in Columbia (Col-0) ecotype background, including Col-0 (wild-type, WT), pmtr1 (cand2-1), PMTR1-OE, MPK6SR, MPK3SR, gpa1-4, gpa1-4/GPA1-HA, bak1-4, and fls2 (see the Acknowledgment section for the sources of seed). The plants were grown in a mixed medium (vermiculite:perlite:peat soil = 1:1:1) at 22°C under cool white light (100 μmol m⁻² s⁻¹) with a 12-h light/12-h dark cycle.

Bacterial infection assays

Due to phytomelatonin rhythms affecting the stomatal aperture (Li et al., 2020), bacterial infection assays were performed at the same time of a day (Zeitgeber time 4 h, ZT4; 12 h light/12 h dark). Pst DC3000 strain was grown on King’s B (peptone 20 g L⁻¹, glycerol 10 mL L⁻¹, K₂HPO₄ 1.5 g L⁻¹, ${\text{MgSO}}_4^{.}$7H₂O 1.5 g L⁻¹), either on 1.5% (w/v) agar plates or in liquid media containing 50 mg/mL rifampicin at 28°C. For foliar spray inoculation, Pst DC3000 suspension (OD₆₀₀ = 0.2) in 10 mM MgCl₂ with 0.02% v/v Silwet L-77 was sprayed onto the leaf surface until completely wet. For infiltration inoculation, bacteria were suspended in 10 mM MgCl₂ to an OD₆₀₀ of 0.002 and infiltrated using a needleless syringe into three leaves on each of three plants per mutant or WT. The experiments were conducted at least three times. Leaf discs were taken using a cork borer (0.48 cm²) after the leaf surface was sterilized with 75% v/v ethanol and sterile water. The samples were ground in 10 mM MgCl₂, diluted, and plated on King’s B plates (1.5% w/v agar) with 50 mg mL⁻¹ rifampicin. Plates were incubated at 28°C, and colonies were counted 2 d later.

Stomatal aperture measurements

Stomatal assay was performed as we described previously (Wei et al., 2018; Li et al., 2020). For measurement of stomatal aperture directly on P. notoginseng intact leaves, the plants were first foliar-spray pretreated by MT for 2 h and were then spray-inoculated by Pst DC3000 (OD₆₀₀ = 0.2). The dimethyl sulfoxide (DMSO) was used as a solvent control for MT. After inoculation by Pst DC3000 for 0 [Pst 0 h with MT (or DMSO) pretreatment for 2 h], 2 h [Pst 2 h with MT (or DMSO) pretreatment for 4 h] and 4 h [Pst 4 h MT (or DMSO) pretreatment for 6 h], the treated leaves of P. notoginseng were covered with silicon impression gel (1:1 freshly prepared mixture of the two components of dental condensation silicone; Zhermack, Italy). After 1 h, the material was removed and clear nail polish was placed on the impression gel for 4 h to obtain a positive impression of the leaf. The impressions were analyzed by a bright-field microscope (Olympus BX60).

To analyze the effect of various chemicals on stomatal aperture, the epidermal strips from 1-year-old P. notoginseng or 3- to 4-week-old Arabidopsis leaves were placed in the incubation medium containing 50 mM KCl, 0.1 mM CaCl₂, and 10 mM MES-KOH (pH 6.15) for 3 h under light (100 μmol m⁻² s⁻¹) to induce stomatal opening. Then, 10 μM MT (Sigma-Aldrich), 5 μM flg22 (QRLSTGSRINSAKDDAAGLQIA, synthesized by Scilight Biotechnology, LLC, Beijing, China), 50 μM H₂O₂, 20 μM salicylhydroxamic acid (SHAM, a cell wall peroxidase inhibitor; Shanghai Macklin Biochemical Co., Ltd., China), 20 μM diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidase; Sigma-Aldrich), 100 U/mL catalase (CAT, the H₂O₂ scavenger; Sigma-Aldrich), and/or 1.5 μM of COR (a phytotoxin produced by Pseudomonas syringae, Sigma-Aldrich) were added for further 2-h incubation under light (100 μmol m⁻² s⁻¹). For MPK6SR and MPK3SR, 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (NA-PP1; 2 μM final concentration) was added at the beginning of illumination. The stocks of these compounds were dissolved in either ddH₂O (flg22, CAT, and H₂O₂), ethanol (SHAM), or DMSO (MT, NA-PP1, DPI, and COR). The controls appropriately contained the same amount of the relevant solvent; the controls did not affect stomatal aperture. Stomatal aperture was evaluated by measuring the width and the length of the stomatal aperture under a microscope (Olympus BX60), and the width/length ratio was calculated as described previously (Wei et al., 2018). The stomatal aperture experiments were performed at least three times and yielded similar results.

H₂O₂ production measurement

We examined H₂O₂ production in guard cells using the H₂O₂-sensitive fluorescent probe H2DCFDA (2ʹ,7ʹ-dichlorodihydrofluorescein diacetate, Sigma-Aldrich) by a confocal microscope as described previously (Wei et al., 2018), excitation 488 nm and emission 535 nm with normal scanning speed. All images were obtained with the same Nikon A1 Elements software scanning settings, including detector gain and laser intensity settings.

Confocal microscopy analysis of subcellular localization of PMTR1

We used onion bulb epidermal cells and Arabidopsis protoplasts for subcellular localization analysis of PMTR1. The Agrobacterium harboring 35S::PMTR1-YFP or 35S::YFP vector was transiently expressed in onion bulb epidermal cells as previously described (Sun et al., 2007). The 35S::PMTR1-YFP (pCAMBIA1300-PMTR1-YFP) and 35S::YFP (pCAMBIA1300-N1-YFP) vectors were constructed as we previously reported (Wei et al., 2018).

The coding region of PMTR1 was amplified using gene-specific primers with the EcoRI and SalI restriction sites (Supplemental Figure S3) and cloned into a pCAMBIA1300-35S-GFP-C vector to generate a GFP-PMTR1 fusion construct driven by the cauliflower mosaic virus 35S promoter (pCAMBIA1300-35S-GFP-PMTR1). The plasma membrane localization marker PIP2-mCherry (pCAMBIA1300-35S-PIP2-mcherry) was generated using primers shown in Supplemental Table S1 and was cotransformed with GFP-PMTR1 into Arabidopsis protoplasts via the PEG-mediated transfection method following Yoo et al. (2007). The transformed Arabidopsis protoplasts were incubated at 25°C for 15 h. Fluorescence was detected by a Nikon A1 laser scanning confocal microscope at the standard scanning speed. Images were captured using the following wavelengths, YFP fluoresence (excitation, 488 nm; emission, 510 nm), chlorophyll autofluoresence (excitation, 640 nm; emission, 675 nm) and mcherry fluoresence (excitation, 587 nm; emission, 645 nm). The experiments were performed three times and showed similar results.

Semiquantitative and quantitative RT-PCR

Semiquantitative and quantitative RT-PCR were performed as described previously (Wei et al., 2018). Leaves were harvested from at least 10 plants of 1-year-old or 3–4-week-old Arabidopsis per sample as a biological replicate. And the experiments were conducted at least three times. Total RNA was extracted from using Trizol reagent (Takara), and 2 μg RNA was reverse transcribed using a Prime Script RT reagent Kit (with gDNA Eraser, Takara). All primers used for RT-PCR in P. notoginseng or Arabidopsis are listed in Supplemental Table S2 or S3, respectively. The relative gene expression level was calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

Protein extraction and Western blot analysis

Total proteins were extracted for MAPK activation analysis. The leaves of 1-year-old P. notoginseng or 3-week-old Arabidopsis were lysed in the extraction buffer containing 10 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% v/v glycerol, 1% v/v Triton-X 100, a proteinase inhibitor cocktail (Roche), and phosphatase inhibitor cocktails 2 and 3 (Sigma). MAPK activation was monitored by Western blot with antibodies that recognize dual phosphorylation of the activation loop of MAPK (pTEpY). The Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr204) rabbit monoclonal antibodies (Cell Signaling, Boston, MA, USA) were used according to the manufacturer’s protocol. Actin (AS132640, Agrisera, Vännäs, Sweden) was used as an internal loading control.

For Western blot analysis of the subcellular location of PMTR1, the total, soluble protein and membrane fractions from Col-0 and PMTR1-OE leaves were extracted as described by Ma et al., (2015). Five grams of leaves from 3-week-old plants were lysed in the membrane protein extraction buffer [20 mM HEPES (pH 7.5), 40 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 10% v/v glycerol, 0.1% v/v Triton-X 100, a proteinase inhibitor cocktail (Roche), and 2 mM PMSF (phenylmethylsulfonyl fluoride)]. After centrifugation at 12,000g at 4°C for 20 min, the supernatant was used as total protein. Then, the supernatant was centrifuged at 100,000g at 4°C for 1 h. The microsomal pellets (membrane fraction) were resuspended in the suspension buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% v/v Triton X-100, a proteinase inhibitor cocktail (Roche), and 2 mM PMSF. The supernatant was used as the soluble protein fraction. The protein concentrations were measured using a BCA kit (Solarbio). Approximately 30 μg of total, soluble and membrane proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were first probed with FLAG antibody (20543-1-AP, Proteintech, Chicago, IL, USA) or plasma membrane H⁺-ATPase (AS07260, Agrisera, Vännäs, Sweden), and then by a goat anti-rabbit IgG conjugated with peroxidase.

Coimmunoprecipitation assay

Approximately 200 μg of total protein was incubated with anti-FLAG antibody at 4°C for 1 h in the protein extraction buffer containing 20 mM HEPES (pH 7.5), 40 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 10% v/v glycerol, 0.1% v/v Triton-X 100, a proteinase inhibitor cocktail (Roche), and 2 mM PMSF. After centrifugation at 1,000g at 4°C for 5 min, the pellets were washed three times with the protein extraction buffer. The pellets were then resuspended in 20 μL of 1×electrophoresis sample buffer, separated by SDS-PAGE, transferred to PVDF membranes and probed with anti-FLAG (20543-1-AP, Proteintech) or Anti-GPA1 (AS122370, Agrisera).

Accession numbers

Sequence data were obtained from TAIR (www.arabidopsis.org) and the National Center for Biotechnology Information with the following accession numbers: PMTR1 (At3G05010), MPK3 (At3G45640), MPK6 (At2G43790), GPA1 (AT2G26300), FLS2 (AT5G46330), and BAK1 (AT4G33430).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Table S1 . Primers used for vector construction.

Supplemental Table S2 . Primers used for RT-qPCR in P. notoginseng.

Supplemental Table S3 . Primers used for RT-PCR and RT-qPCR in Arabidopsis.

Supplemental Figure S1 . Effects of MT on the growth of Pst DC3000 in the liquid medium.

Supplemental Figure S2 . MT failed to induce stomatal closure in the presence of COR.

Supplemental Figure S3 . Effects of 50 μM H₂O₂, 20 μM SHAM, 20 μM DPI, and 100 U mL⁻¹ CAT on MT (10 μM)- or flg22 (5 μM)-induced H₂O₂ production in P. notoginseng.

Supplementaryl Figure S4 . Expression profiles of the well-known defense-related genes in P. notoginseng.

Supplemental Figure S5 . Plasma membrane localization of PMTR1 in onion cells.

Supplemental Figure S6 . The expression level of AtPMTR1 in leaves of 3- to 4-week-old Col-0, pmtr1 and gpa1-4 plants treated by different concentrations of MT.

Supplemental Figure S7 . The expression level of the MT-, MAPKs- and pathogen-related genes in leaves of 3- to 4-week-old Col-0 plants treated by MT or flg22 for 0, 0.5, 1, 3, and 6 h with or without surface-inoculation by Pst DC3000.

Supplemental Figure S8 . MT and flg22 promote stomatal closure in gpa1-4/GPA1-HA.

 

C.Q.: conceived and designed the study, analyzed the data, wrote and revised the manuscript; Y.Q.: performed most of the experiments, analyzed the data, wrote and revised the manuscript; P.Z., M.W., Z.S., H.S., and W.J.: performed or contributed to parts of some experiments; D.S., S.Y., and Y.X.: analyzed the data and provided suggestion for the writing of the manuscript; C.X., H.L., G.W., and R.Z.: analyzed the data, interpreted the results, and revised the manuscript. All authors discussed the results and commented on the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is: Qi Chen (chenq0321@kust.edu.cn or chenq0321@163.com).