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Cellular messengers involved in the inhibition of the Arabidopsis primary root growth by bacterial quorum-sensing signal N-decanoyl-L-homoserine lactone

BMC Plant Biology volume 22, Article number: 488 (2022) Cite this article

Abstract

Background

N-acyl-homoserine lactones (AHLs) are used as quorum-sensing signals by Gram-negative bacteria, but they can also affect plant growth and disease resistance. N-decanoyl-L-homoserine lactone (C10-HSL) is an AHL that has been shown to inhibit primary root growth in Arabidopsis, but the mechanisms underlying its effects on root architecture are unclear. Here, we investigated the signaling components involved in C10-HSL-mediated inhibition of primary root growth in Arabidopsis, and their interplay, using pharmacological, physiological, and genetic approaches.

Results

Treatment with C10-HSL triggered a transient and immediate increase in the concentrations of cytosolic free Ca2+ and reactive oxygen species (ROS), increased the activity of mitogen-activated protein kinase 6 (MPK6), and induced nitric oxide (NO) production in Arabidopsis roots. Inhibitors of Ca2+ channels significantly alleviated the inhibitory effect of C10-HSL on primary root growth and reduced the amounts of ROS and NO generated in response to C10-HSL. Inhibition or scavenging of ROS and NO neutralized the inhibitory effect of C10-HSL on primary root growth. In terms of primary root growth, the respiratory burst oxidase homolog mutants and a NO synthase mutant were less sensitive to C10-HSL than wild type. Activation of MPKs, especially MPK6, was required for C10-HSL to inhibit primary root growth. The mpk6 mutant showed reduced sensitivity of primary root growth to C10-HSL, suggesting that MPK6 plays a key role in the inhibition of primary root growth by C10-HSL.

Conclusion

Our results indicate that MPK6 acts downstream of ROS and upstream of NO in the response to C10-HSL. Our data also suggest that Ca2+, ROS, MPK6, and NO are all involved in the response to C10-HSL, and may participate in the cascade leading to C10-HSL-inhibited primary root growth in Arabidopsis.

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Introduction

Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as signaling molecules for intercellular communication in a process known as quorum sensing (QS) [1,2,3]. To date, numerous AHL derivatives with acyl chains of different lengths (from 4 to 18 carbons) and substitutions of hydroxyl or oxo groups at the γ position of the carbon chain has been identified from more than 70 species of Gram-negative bacteria [2, 4]. AHL-mediated QS plays an essential role in many bacterial physiological processes including symbiosis, virulence, antibiotic production, extracellular polysaccharide production, resistance to oxidative stress, biofilm formation, and motility [5,6,7].

Plants can also sense and respond to bacterial QS signals [8,9,10,11,12]. For example, Mathesius et al. (2003) found that more than 150 proteins in Medicago truncatula changed in abundance after application of 3-oxo-N-(tetrahydro-2-oxo-3-furaryl)-hexadecenamide (3OC16-HSL) isolated from cultured Sinorhizobium meliloti [13]. Schuhegger et al. (2006) demonstrated that N-hexanoyl-homoserine lactone (C6-HSL) induces systemic accumulation of salicylic acid- and ethylene-dependent defense gene transcripts [14]. In Arabidopsis, N-3-oxo-tetradecanoyl-homoserine-lactone (3OC14-HSL) conferred resistance to biotrophic and hemibiotrophic pathogens via an oxylipin/SA-dependent pathway [9]. Whereas long-chain AHLs (side chains with 12–14 carbons) induce defense responses, short-chain AHLs (side chains with fewer than 8 carbons) promote elongation of the plant primary root. The G-protein-coupled receptor (GCR) and the transcription factor AtMYB44 were involved in the regulation of primary root growth by 3-oxo-octanoyl-homoserine lactone (3OC8-HSL) [15, 16].

Medium-chain AHLs, i.e., those with 10 carbon atoms in the carbon chain with or without substitutions at the γ position, have multiple functions in root morphogenesis, plant senescence, and defense responses [11, 17,18,19,20]. Hu et al. (2018) reported that N-decanoyl-homoserine lactone (C10-HSL) activates plant systemic resistance against Botrytis cinerea in tomato, and that C10-HSL-induced resistance is largely dependent on the jasmonic acid (JA) signaling pathway [19]. Ortiz-Castro et al. (2008) compared the effects of seven different AHLs on root architecture in Arabidopsis and found that C10-HSL had the strongest effect to inhibit primary root growth and promote lateral root formation [18]. Bai et al. (2012) found that an analog of C10-HSL, N-3-oxo-decanoyl-homoserine lactone (3OC10-HSL), mediated auxin-dependent adventitious root formation via H2O2- and nitric oxide (NO)-dependent cGMP signals in mung bean (Vigna radiata) seedlings [11]. However, the signaling pathway through which C10-HSL modulates plant primary root growth remains unclear.

Reactive oxygen species (ROS) and NO are important signals that participate in various physiological processes [14, 18, 21,22,23,24,25,26]. Zhang et al. (2017) found that ROS and NO were involved the inhibition of primary root growth by exogenous H2S in Arabidopsis [27]. Treatments with C6-HSL, N-octanoyl-homoserine lactone (C8-HSL), C10-HSL, and N-decanoyl-homoserine-lactone (C12-HSL) led to NO accumulation in the calyptra and elongation zone of barley roots and altered root morphology [28]. Recently, we reported that pretreatment with 3OC8-HSL and subsequent pathogen invasion triggered an augmented ROS burst in Arabidopsis [29].

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that participate in the regulation of growth and development and in responses to environmental stress in plants [30,31,32,33,34]. AtMPK3/6 regulate plant growth, development, and stress tolerance by interacting with Ca2+ and ROS pathways [31, 35,36,37]. Schikora et al. (2011) found that 3OC14-HSL pretreatment activated AtMPK3/6 upon challenge by flg22 in Arabidopsis [9].Calcium ions (Ca2+) are a ubiquitous intracellular second messenger that participate in many signal transduction pathways in plants [38,39,40,41]. In our previous study, we found that C4-HSL induced a transient and immediate increase in the cytosolic free Ca2+ concentration ([Ca2+]cyt) in Arabidopsis [10]. Overall, data from the literature suggest that Ca2+, ROS, NO, and MAPK participate in the plant’s response to bacterial AHLs. However, the details of their involvement and the interplay among them in the regulation of primary root growth by C10-HSL is unknown. In this study, we investigated the involvement of intracellular Ca2+, ROS, NO, and MPKs in C10-HSL-inhibited primary root growth in Arabidopsis. Our data indicate that C10-HSL inhibits primary root growth via a cascade involving Ca2+, ROS, MAPK, and NO in Arabidopsis.

Results

C10-HSL influences Arabidopsis root system architecture

Previously, Ortiz-Castro et al. (2008) reported that C10-HSL is one of the most active AHLs tested to date, in terms of modification of Arabidopsis root system architecture [18]. In this study, we first confirmed the effects of C10-HSL on post-embryonic root development. We found that 10 nM to 1 µM C10-HSL treatment slightly caused a reduction in primary root growth and promoted lateral root formation (Fig. S1a, b). The primary root growth of plants treated with 15 to 75 µM C10-HSL was inhibited by 40–86% (Fig. 1a, b) and treatment 30 µM C10-HSL caused a 74% reduction in primary root length. Treatment with C10-HSL also resulted in significant increases in lateral root density and the density of lateral root primordia (Fig. 1c–e and Fig. S1a). These data confirmed that C10-HSL can inhibit primary root growth and stimulate lateral root formation.

Fig. 1
figure 1

 N-decanoyl-homoserine lactone (C10-HSL) influences root system architecture in Arabidopsis. (a) Root architecture of wild-type (Col-0) Arabidopsis treated with different C10-HSL concentrations (0–75 µM), bar = 1 cm. (b) The effects of different C10-HSL concentration on primary root length, n = 40. (c) The effects of different C10-HSL concentration (0–75 µM) on lateral root density, n = 40. (d) The effects of different C10-HSL concentration (0–75 µM) on lateral root primordia density, n = 40. For the picture a-d, 3-d-old Arabidopsis thaliana seedlings were transferred to half-strength MS medium supplemented with the indicated concentrations of C10-HSL and grown for 5 d. (e) Representative photographs of root hairs formed at the primary root tip region of 3-day-old Arabidopsis seedlings grown in the presence of the indicated concentration of C10-HSL for 5 d, bar = 1 mm. All the error bars represent +/- SD. (Different letters indicate significantly different values, P < 0.05 by Tukey’s test)

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NO is involved in the inhibition of primary root growth by C10-HSL

Nitric oxide is an important gaseous signal that regulates root development [23, 42, 43]. To investigate the role of NO in C10-HSL-induced inhibition of primary root growth, we first measured NO production after C10-HSL treatment in Arabidopsis seedlings. Using the NO specific fluorescent probe diaminofluorescein diacetate (DAF-2DA), we found that C10-HSL elicited NO production in the roots within 15 min of treatment, and this effect lasted for 1 h or even longer (Fig. 2a, b). Supplementation with 100 µM 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO-specific scavenger, markedly alleviated the C10-HSL-induced inhibition of primary root growth (Fig. 2c, d). Consistent with the phenotypic results, NO production induced by C10-HSL treatment was significantly suppressed by cPTIO (Fig. 2e, f). We further analyzed the transcript levels of NOA1, encoding cGTPase [44], and NIA1 and NIA2, encoding nitrate reductase, after treatment with C10-HSL in wild-type Arabidopsis. After C10-HSL treatment, the transcript level of NOA1 increased within 1 h, peaked at 6 h after treatment, and then gradually declined (Fig. S2f). Similarly, the transcript levels of NIA1 and NIA2 increased within 1 h of treatment with C10-HSL in Arabidopsis roots (Fig. S2a, b). Seedlings of the noa1 mutant were less sensitive to C10-HSL treatment with respect to primary root growth (Fig. 2 g). Likewise, nia1 and nia2 single mutants and nia1nia2 double mutants were less sensitive to inhibition of primary root growth by C10-HSL (Fig. S3e). Collectively, these data suggest that NO is involved in the C10-HSL-mediated inhibition of primary root growth.

Fig. 2
figure 2

NO is involved in the inhibition of primary root growth mediated by C10-HSL. (a, b) Detection of NO production in roots of Col-0 seedlings treated with C10-HSL (0–75 µM) for 0–60 min using the NO-specific fluorescent probe DAF-2DA (a) and quantification of NO-specific fluorescence intensities (b), bar = 50 μm, n = 30. (c), Root architecture of wild-type Arabidopsis exposed to 30 µM C10-HSL with or without 100 µM cPTIO, bar = 1 cm. (d) Primary root length of wild-type seedlings exposed 30 µM C10-HSL with or without 100 µM cPTIO, n = 40. (e, f) Detection of NO production in roots of Col-0 seedlings treated as described in (c) using the NO-specific fluorescent probe DAF-2DA (e) and quantification of NO-specific fluorescence intensities (f). (g), Relative primary root growth of wild-type and noa1 exposed to 30 µM C10-HSL, n = 40. For the picture c, d and g, 3-d-old Arabidopsis thaliana seedlings were transferred to half-strength MS medium supplemented with the indicated concentrations of C10-HSL or other chemicals and grown for 5 d. Control or Con refers to solvent control. All the error bars represent +/- SD. (Different letters indicate significantly different values, P < 0.05 by Tukey’s test; *P < 0.05, **P < 0.001, Student’s t test)

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ROS are essential in C10-HSL-induced inhibition of primary root growth

In plants, ROS play an essential role in root growth and development [25, 45, 46]. To investigate the role of ROS in C10-HSL-mediated inhibition of primary root growth, we determined their levels in C10-HSL-treated seedlings using the ROS-specific fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCF-DA). The ROS levels in wild-type seedlings treated with 30 µM C10-HSL increased and peaked at 15 min after treatment (Fig. 3a, b). One important component of ROS is H2O2, which can be scavenged by catalase (CAT). When CAT was added to the medium containing 30 µM C10-HSL, the ROS level was 30% lower than that in the control (solvent-treated) seedlings (Fig. 3c, d). Then, we examined primary root elongation in wild-type seedlings treated with 30 µM C10-HSL in the presence or absence of exogenous H2O2 and CAT. We found that the C10-HSL-induced inhibition of primary root growth was increased by exogenous H2O2, but alleviated by CAT (Fig. 3e, f). The effect of C10-HSL on primary root growth was similar to that of H2O2, because addition of H2O2 alone significantly inhibited primary root growth (Fig. 3e, f). To verify the role of ROS in C10-HSL-mediated primary root growth inhibition, we analyzed the Arabidopsis ROS-biosynthesis-related respiratory burst oxidase homolog (Rboh) NADPH oxidase double mutant rbohD/F. In terms of the inhibition of primary root growth, rbohD/F plants were less sensitive to C10-HSL treatment than were wild-type plants (Fig. 3 g). These data indicate that ROS plays an essential role in the modulation of root growth by C10-HSL.

Fig. 3
figure 3

ROS is involved in C10-HSL-mediated inhibition of primary root growth. (a, b) Detection of ROS level in the roots of wild-type seedlings exposed to 30 µM C10-HSL for 0–60 min using the ROS-specific florescent probe DCF-DA (a), and quantification of ROS-specific fluorescence intensities (b) bar = 50 μm. (c, d) Detection of ROS level in the roots of wild-type seedlings exposed to 30 µM C10-HSL with or without 500 µM CAT for 15 min using the ROS-specific florescent probe DCF-DA (c), and quantification of ROS-specific fluorescence intensities (d), n = 30, bar = 50 μm. (e, f) Relative primary root growth of wild-type seedlings exposed to 30 µM C10-HSL with or without 1.5 mM H2O2 and 500 µM CAT for 5 d, bar = 1 cm, n = 40. (g) Relative primary root growth of Col-0 and noa1 seedlings exposed to 30 µM C10-HSL for 5 d, n = 40. Control or Con refers to solvent control. All the error bars represent +/- SD. (Different letters indicate significantly different values, P < 0.05 by Tukey’s test. *P < 0.05, **P < 0.001, Student’s t test)

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MAPKs participate in the inhibition of primary root growth by C10-HSL

Previous studies have shown that MPK3/6 participate in the induction of the defense response by 3OC14-HSL in Arabidopsis [9], and that MPK4 regulates primary and lateral root growth [47, 48]. To investigate whether MAPKs play roles in the C10-HSL-induced inhibition of primary root growth, we first assessed the effects of U0126, a selective inhibitor of MAPK, on primary root elongation in wild-type Arabidopsis (Columbia-0). Addition of U0126 alleviated the C10-HSL-induced inhibition of primary root growth (Fig. 4a, b) and this effect was dose-dependent (Fig. S3a, b). Western blotting using an anti-phosphate antibody showed that, compared with wild-type seedlings in the control, wild-type seedlings treated with C10-HSL had stronger phosphorylation activity of MPK6, which started at about 5 min and peaked at 45 min after C10-HSL treatment (Fig. 4c, d and Fig. S5 a-d). We also found that MPK3 and MPK4 in Arabidopsis were weakly activated from 30 to 60 min after treatment with 30 µM C10-HSL (Fig. 4c, d).

Fig. 4
figure 4

MAPKs participates in the inhibition of primary root growth by C10-HSL. (a, b) Relative primary root growth of Col-0 and mpk6 seedlings exposed to 30 µM C10-HSL with(+) or without(-) 20 µM U0126 (MAPK inhibitor) for 5 d, n = 40, bar = 1 cm. (c) Phosphorylation status (pMPK6, pMPK3 and pMPK4) and total MPK6 expression level in wild-type seedlings exposed to 30 µM C10-HSL or Control for 0–60 min. (d) Relative expression of pMPK6 in wild type seedlings treated with or without 30 µM C10-HSL for 0–60 min. Control or Con refers to solvent control. All the error bars represent +/- SD (Different letters indicate significantly different values, P < 0.05 by Tukey’s test)

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To confirm the involvement of MAPK in the response to C10-HSL, the effects of C10-HSL on primary root growth were compared between wild-type and mpk6 Arabidopsis seedlings. The inhibitory effect of C10-HSL on primary root growth was significantly alleviated in the mpk6 mutant compared with the wild-type seedlings (Fig. 4a, b). Taken together, these results indicate that MAPKs, especially MPK6, are involved in the inhibition of primary root growth modulated by C10-HSL.

MPK6 mediates the C10-HSL-induced inhibition of primary root growth downstream of ROS and upstream of NO

On the basis of the data presented above, we concluded that ROS, NO, and MPK6 are important intermediate components in the regulation of primary root growth by C10-HSL. To explore the interplay among these components, we first detected the effect of the ROS scavenger CAT on the activation of MPK6 induced by C10-HSL. We found that the addition of CAT significantly decreased the activation of MPK6 by C10-HSL (Fig. 5a and Fig. S6 a-c). Next, we measured ROS and NO production in the mutants noa1 and rbohD/F, respectively, after C10-HSL treatment. We found that C10-HSL-induced NO production was abolished in the rbohD/F mutant, while C10-HSL-induced ROS production was similar between wild-type and noa1 (Fig. 5b, c). In addition, the ROS production induced by C10-HSL treatment was unaffected in the nia1 and nia2 single mutants and in the nia1/2 double mutant (Fig. S2d). Further, qRT-PCR analyses showed that the induction of noa1 expression by C10-HSL in wild-type plants was suppressed in mutant rbohD/F seedlings after exposure to C10-HSL (Fig. 5d). These data suggest that C10-HSL induces ROS production upstream of MAPK6 activation and NO accumulation in Arabidopsis roots.

Fig. 5
figure 5

MPK6 mediates the C10-HSL-induced inhibition of primary root growth downstream of ROS and upstream of NO. (a) Phosphorylation status (pMPK6) and total expression level of MPK6 in wild-type seedlings exposed to 30 µM C10-HSL with or without 500 µM CAT for 45 min. (b) Quantification of NO-specific fluorescence intensities in the roots of Col-0, rbohD/F and noa1 seedlings exposed to 30 µM C10-HSL 1 h using the NO specific florescent probe DAF-2DA. (c) Quantification of ROS-specific fluorescence intensities in the roots of Col-0, rbohD/F and noa1 seedlings exposed to 30 µM C10-HSL 15 min using the ROS specific florescent probe DCF-DA. (d) Transcriptional regulation of NOA1 gene after exposed to 30 µM C10-HSL for up to 2 day in Col-0 and rbohD/F. (e) Relative root growth of Col-0 and mpk6 seedlings exposed to 30 µM C10-HSL with or without 30 µM SNP, 1.5 mM H2O2 and 500 µM CAT for 5 d compared with untreated seedlings, n = 40. (f) Quantification of NO-specific fluorescence intensities in the roots of Col-0 and mpk6 seedlings exposed to 30 µM C10-HSL 1 h using the NO specific florescent probe DAF-2DA. (g) Quantification of ROS-specific fluorescence intensities in the roots of Col-0 and mpk6 seedlings exposed to 30 µM C10-HSL 15 min using the ROS specific florescent probe DCF-DA. Control or Con refers to solvent control. All the error bars represent +/- SD. (Different letters indicate significantly different values, P < 0.05 by Tukey’s test)

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We further checked the NO and ROS production in roots of Arabidopsis mpk6 mutant seedlings. The induction of NO production by C10-HSL in wild-type seedlings was completely abolished in mpk6 seedlings (Fig. 5f). In contrast, ROS accumulation in response to C10-HSL treatment was comparable between mpk6 and wild-type plants (Fig. 5 g). Supplementation with CAT alleviated the C10-HSL-induced inhibition of primary root growth in wild-type roots, but did not further alleviate the C10-HSL-induced inhibition of primary root growth in the mpk6 mutant (Fig. 5e). In contrast, supplementation with exogenous H2O2 exacerbated the C10-HSL-mediated inhibition of primary root growth in wild-type seedlings, but did not further increase the inhibitory effects of C10-HSL on primary root growth in the mpk6 mutant (Fig. 5e). Supplementation with the NO donor sodium nitroprusside (SNP) increased the inhibitory effects of C10-HSL on the primary root growth of both mpk6 and wild-type seedlings (Fig. 5e). Considering all these results, we concluded that MPK6 mediates the C10-HSL-induced inhibition of primary root growth downstream of ROS and upstream of NO.

C10-HSL triggers an elevated concentration of intracellular calcium and controls ROS and NO accumulation

Cytosolic Ca2+ is a second messenger that plays an essential role in plant root growth [49, 50]. The effect of C10-HSL on [Ca2+]cyt was evaluated in root cells of transgenic Arabidopsis expressing the gene encoding intracellular apoaequorin. The results showed that the [Ca2+]cyt level in Arabidopsis root cells significantly increased within 30 s of adding C10-HSL at concentrations from 10 nM to 30 µM (Fig. 6a). Pretreatments with La3+, verapamil, or EGTA blocked the elevation in [Ca2+]cyt caused by 30 µM C10-HSL (Fig. 6b). To further monitor whether C10-HSL affects the level of [Ca2+]cyt in root cells, the fluorescence of the Ca2+-sensitive dye Fluo-4 AM was analyzed by confocal microscopy. The fluorescence intensity level significantly increased after exposure to C10-HSL at concentrations higher than 100 nM in a dose-dependent manner (Fig. 6c, d). These data suggested that C10-HSL immediately triggers an elevation of [Ca2+]cyt in Arabidopsis root cells. LiCl, a known inhibitor of the phosphatidylinositol cycle, inhibits the release of Ca2+ from intracellular calcium pools, while LaCl3 inhibits the import of Ca2+ across the plasma membrane. Addition of both these compounds to the medium containing C10-HSL buffered its inhibitory effect on primary root growth in wild-type seedlings (Fig. 7a and Fig S4), implying that Ca2+ plays a role in C10-HSL-induced inhibition of primary root growth in Arabidopsis. To investigate whether Ca2+ affects ROS and NO production induced by C10-HSL, we measured the fluorescence intensity of DCF-DA as well as DAF-2DA and found that the addition of Ca2+ inhibitors down-regulated both ROS and NO production induced by C10-HSL, consistent with the decrease in [Ca2+]cyt detected by Fluo-4 AM analyses (Fig. 7b–d). Taken together, these results indicate that C10-HSL increases the intracellular calcium concentration and controls the production of both ROS and NO.

Fig. 6
figure 6

C10-HSL improved cytosolic Ca2+ concentration in wild-type seedlings. (a) Changes in [Ca2+]cyt in apoaequorin-transformed Arabidopsis root cells with 10 nM, 100 nM, 1 µM, 10 µM, 30 µM C10-HSL. (b) Changes in [Ca2+]cyt in Arabidopsis root cells were measured upon addition of 30 µM C10-HSL after pretreatment with LaCl3, EGTA and Verapamil respectively. (c, d) Detection of cytosolic Ca2+ concentration in the roots of Col-0 seedlings exposed to 0 nM, 10 nM, 100 nM, 1µM, 10 µM and 30 µM C10-HSL using the Ca2+ specific florescent probe Fluo4-am, bar = 100 μm. Control or Con refers to solvent control. All the error bars represent +/- SD. (Different letters indicate significantly different values, P < 0.05 by Tukey’s test)

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Fig. 7
figure 7

Up-regulated cytosolic Ca2+ induced ROS and NO reaction which is necessary for C10-HSL induced root growth inhibition. (a) Relative primary root growth of Col-0 seedlings exposed to 30 µM C10-HSL with or without 100 µM LiCl plus 300 µM LaCl3 compared with control for 5 d, bar = 1 cm, n = 40. (b) Detection of cytosolic Ca2+ concentration in the roots of Col-0 seedlings exposed to 30 µM C10-HSL with or without 100 µM LiCl plus LaCl3 compared with untreated seedlings using the Ca2+ specific florescent probe Fluo4-am and quantification of Ca2+-specific fluorescence intensities, bar = 100 μm, n = 30. (c) Detection of ROS production in the roots of Col-0 seedlings exposed to 30 µM C10-HSL with or without 100 µM LiCl plus LaCl3 for 15 min using the specific florescent probe DCF-DA and quantification of specific fluorescence intensities, bar = 100 μm, n = 30. (d) Detection of NO production in the roots of Col-0 seedlings exposed to 30 µM C10-HSL with or without 100 µM LiCl plus LaCl3 for 15 min using the NO specific florescent probe DAF-2DA and quantification of NO-specific fluorescence intensities, bar = 100 μm, n = 30. Control or Con refers to solvent control. All the error bars represent +/- SD. (Different letters indicate significantly different values, P < 0.05 by Tukey’s test)

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Discussion

Many Gram-negative bacteria can use AHLs as intracellular signals to monitor their population density by QS [1,2,3]. AHLs not only play an essential role in QS-mediated physiological processes, but also modulate plant growth, defense responses, and development [11, 17, 18, 51,52,53]. C10-HSL or 3OC10-HSL can be produced by several bacteria including the rhizosphere-colonizing bacteria Pseudomonas fluorescens and Pseudomonas putida [54], the nitrogen-fixing bacterial symbiont Sinerhizobium meliloti [55], and the pathogenic bacteria Burkholderia pseudomallei and Burkholderia mallei [2]. The concentration of AHLs in natural niches is estimated to range from 1 nM to 10 µM in the bulk rhizosphere, although the concentrations can be higher (> 50 µM) in bacterial biofilms [56, 57]. Ortíz-Castro et al. (2008) tested the effects of seven AHLs with concentrations ranging from 12 to 192 µM on root growth, and found that C10-HSL showed the strongest effect to inhibit primary root growth and stimulate lateral root formation and root hair development [18]. A derivative of C10-HSL, 3OC10-HSL, was found to stimulate adventitious root formation in mung bean [11]. In this study, we confirmed the effect of C10-HSL and showed that treatment with 10 nM to 1 µM C10-HSL significantly reduced primary root growth and promoted lateral root formation. The effect of C10-HSL on root system architecture was concentration-dependent, and 30 µM C10-HSL caused a 74% reduction in primary root length that was accompanied by 3.5-fold increase in lateral root formation. These results were consistent with those reported by Bai et al. (2012) [11] and Ortíz-Castro et al. (2008) [18], and confirm that AHLs can mediate cross-kingdom interactions by modulating root development during the plant-bacteria interaction. Here, we focused on primary root growth and chose 30 µM C10-HSL as the effective concentration for further analyses. This concentration is somewhat higher than that produced by bacteria in the bulk rhizosphere but comparable to that present in bacterial biofilms.

In plants, ROS are involved in the responses to various phytohormones and environmental cues. We found that treatment with 30 µM C10-HSL increased ROS accumulation in roots, and this led to inhibition of primary root growth in Arabidopsis. The inhibitory effect of C10-HSL on primary root growth was alleviated by supplementation with CAT, but exaggerated by exogenous H2O2. This indicated that H2O2, one component of ROS, contributes to C10-HSL-mediated primary root growth inhibition in Arabidopsis. Further evidence was obtained from genetic analyses of the ROS-related mutant rbohD/F. We found that the primary roots of rbohD/F seedlings were less sensitive to C10-HSL treatment. Zhang et al. (2017) reported that ROS generation is required for exogenous H2S-induced inhibition of primary root growth in Arabidopsis [27]. Similarly, ROS were found to accumulate in the root tips after treatment with the nitrification inhibitor methyl 3-(4-hydroxyphenyl) propionate (MHPP), which inhibited primary root elongation in Arabidopsis [58]. These data show that AHLs influence primary root growth by regulating ROS accumulation in plant roots.

Another important signaling molecule is NO, which regulates root growth in plants. As AHL homologs, plant alkamides were found to induce accumulation of NO and promote adventitious root development in Arabidopsis thaliana explants [59]. Pharmacological analyses indicated that blocking the 3OC8-HSL-induced accumulation of NO with the NO scavenger cPTIO significantly reduced the effect of 3OC10-HSL to stimulate adventitious root formation in mung bean, suggesting a role of NO in 3OC10-HSL-mediated root development [11]. In our experiments, we found that C10-HSL triggered NO accumulation in root tips. Moreover, supplementation with an NO scavenger abolished the production of NO induced by C10-HSL, and repressed the inhibitory effects of C10-HSL on primary root growth. Furthermore, we found that NOA1 and NIA were upregulated by C10-HSL, and that C10-HSL-mediated primary root growth inhibition was markedly impaired in the NOA1-defective mutant noa1 and NIA mutant nia1nia2. These results provide pharmacological and genetic evidence that NO is required for C10-HSL-inhibited primary root growth in Arabidopsis. Heavy metals such as zinc and cadmium and toxic chemicals including H2S and MHPP inhibit primary root growth by increasing the concentration of intracellular NO, suggesting that NO is also involved in inhibiting primary root growth in response to environmental factors [27, 58].

In plants, MAPKs are activated in response to a number of environmental cues [60,61,62]. In this study, the MAPK inhibitor U0126 neutralized the C10-HSL-induced inhibition of primary root growth, and C10-HSL treatment strongly activated MPK6 and weakly activated MPK3 and MPK4 within 30 min. These findings indicate that activation of MAPK is required for the inhibition of primary root growth by C10-HSL. Consistent with this result, an MPK6 mutant showed reduced inhibition of primary root growth after C10-HSL treatment, providing molecular evidence for the essential role of MPK6 in C10-HSL-mediated inhibition of primary root growth. Previously, Schikora et al. (2011) reported that application of 3OC14-HSL induces resistance against bacterial and fungal pathogens, and this effect depends on strong and prolonged activation of MPK6 in Arabidopsis [9]. Accordingly, these data show that MPK6 is required for AHL-mediated changes in primary root growth.

MPK6 modulates plant growth and the response to environmental stimuli by interacting with ROS and/or NO [36, 37, 63, 64]. In Arabidopsis, NO stimulates cadmium-induced programmed cell death by enhancing MPK6-mediated caspase-3-like activation [65]. Compared with wild-type, the MPK6 mutant formed more and longer lateral roots after application of SNP but H2O2, indicating that MPK6 modulates NO accumulation and responses to H2O2 during root development in Arabidopsis [63]. Liu et al. (2016) reported that NO/ROS accumulation contributes to MHPP-mediated primary root growth inhibition and NO acts upstream of ROS in the response to MHPP in Arabidopsis [58]. Using pharmacological approaches, Bai et al. (2012) showed that H2O2 may modulate the NO signal during the response to 3OC10-HSL treatment in mung bean [11]. The results of those studies suggest that MPK6, ROS, and NO interact, but the mechanism underlying this interaction may be context-dependent. In this study, we observed that reducing ROS accumulation with the H2O2 scavenger CAT significantly decreased the activation of MPK6. We also found that C10-HSL-induced NO production was abolished in the rbohD/F mutant, while C10-HSL-induced accumulation of ROS was unaffected in the noa1 mutant. In addition, C10-HSL treatment did not upregulate NOA1 in the rbohD/F mutant. The induction of NO production by C10-HSL was completely abolished in the mpk6 mutant, while ROS accumulation caused by C10-HSL treatment was unaffected. Addition of CAT did not further alleviate the C10-HSL-induced inhibition of primary root growth in roots of the mpk6 mutant. In contrast, the defect in the C10-HSL-induced inhibition of primary root growth in the mpk6 mutant was rescued by application of SNP, an NO donor, but not by addition of exogenous H2O2. Our data indicate that ROS, MPK6, and NO might work together to regulate plant root responses to C10-HSL. A similar signaling pathway has been reported to participate in the plant response to H2S toxicity [27].

In plants, Ca2+ functions as an intracellular second messenger, and is essential for signal transduction processes [40, 41]. In our previous study, we showed that C4-HSL triggered an increase in the concentration of intracellular Ca2+ [9]. In the present study, we found that treatment with C10-HSL resulted in a transient and immediate increase in [Ca2+]cyt in root cells of Arabidopsis. The effect of C10-HSL to increase the [Ca2+]cyt concentration was investigated using the Ca2+-sensitive dye Fluo-4 AM. These analyses showed that the inhibition of the increase in the intracellular Ca2+ concentration reduced the inhibitory effect of C10-HSL on primary root growth. Moreover, the addition of a Ca2+ inhibitor significantly reduced the production of ROS and NO induced by C10-HSL. Our data indicate that Ca2+ signaling may be an early event in the plant response to C10-HSL, and it may regulate the down-stream production of ROS. These mechanisms also occur during incompatible host-pathogen recognition, when the flux of Ca2+ across the plasma membrane is one of the earliest cellular events and results in a set of oxidative bursts that produce ROS [66, 67].

In conclusion, our results show that C10-HSL triggered a transient and immediate increase in the concentration of cytosolic free Ca2+ and induced ROS accumulation, MPK6 activation, and NO production in Arabidopsis primary roots. The generation of ROS and NO induced by C10-HSL was mediated by Ca2+. Activation of MPKs was necessary for the inhibition of primary root growth by C10-HSL. MPK6 was shown to act downstream of ROS and upstream of NO. Thus, our data suggest that Ca2+, ROS, MPK6, and NO are all involved and might play roles in cascade in the C10-HSL-mediated inhibition of primary root growth in Arabidopsis (Fig. 8). Auxin plays a fundamental role in root system architecture in plants [68, 69]. However, Ortiz-Castro et al. (2008) pointed out that C10-HSL is independent of auxin signaling in Arabidopsis, although the C10-HSL-mediated inhibition of primary root growth is similar to the typical auxin signaling phenotype [18]. However, 3OC12-HSL and 3OC16-HSL were found to induce the tissue-specific expression of auxin-responsive GH3 in legume [13]. Bai et al. (2012) reported that 3OC10-HSL, an analog of C10-HSL, was able to mediate auxin-dependent adventitious root formation via H2O2- and NO-dependent cGMP signaling in mung bean seedlings [11]. C6-HSL was found to promote root elongation and increase the ratio of auxin: cytokinin towards higher auxin levels in both leaves and roots of Arabidopsis [51]. Zhang et al. (2017) reported that exogenous H2S repressed the distribution of auxin and inhibited primary root growth in Arabidopsis [27]. The nitrification inhibitor MHPP was found to modulate root system architecture via ROS/NO-mediated-accumulation and redistribution of auxin in Arabidopsis roots [60]. Moreover, Hu et al. (2018) found that N-decanoyl-homoserine lactone (C10-HSL) activated plant systemic resistance against Botrytis cinerea in tomato and C10-HSL-induced resistance was largely dependent on the JA signaling pathway [19]. Recently, we demonstrated that 3OC8-HSL primes plant resistance against necrotrophic pathogen Pectobacterium carotovorum by coordinating JA and auxin signaling pathway [70]. Therefore, comprehensive studies on the roles of phytohormones such as auxin and JA and their interplay with C10-HSL will shed light on the mechanism by which plants respond to C10-HSL, and will provide insight into novel applications of these biological molecules to regulate crop growth and development.

Fig. 8
figure 8

The tentative model for C10-HSL-induced inhibition of primary root growth. C10-HSL triggered cytosolic free Ca2+ and reactive oxygen species (ROS), increased the activity of mitogen-activated protein kinase 6 (MPK6), and induced nitric oxide (NO) production in Arabidopsis, and finally inhibited primary root growth

Full size image

Materials and methods

Plant growth and chemical treatments

The Arabidopsis thaliana ecotype Columbia-0 (Col-0), the mutants Atmpk6 (obtained from the TAIR stock center), nia1, nia2, noa1 (provided by Professor Chunpeng Song in Henan University, China), rbohD/F (provided by Professor Zhonglin Shang in Hebei Normal University, China) were used in this study. Seeds were surface sterilized with 75% ethanol for 1 min and 25% bleach for 5 min. After five washes with sterile distilled water, seeds were germinated on agar plates containing half-strength Murashige & Skoog (MS) medium [71] adjusted to pH 5.8. The plates were stratified at 4 °C for 2 days and then placed in a plant growth chamber with a photoperiod of 16 h: 8 h light: dark with 100 µmol·m2·s− 1 and temperature of 22 ± 2 °C. Seeds were grown on the vertical position. 3-d-old Arabidopsis thaliana seedlings were transferred to half-strength MS medium containing supplemented with various chemicals, including 30 µM C10-HSL, sodium nitroprusside (SNP), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), hydrogen peroxide (H2O2), catalase (CAT), U0126, lithium chloride (LiCl) and lanthanum chloride (LaCl3) and grown for additional 5 d. All chemicals were purchased from Sigma-Aldrich and C10-HSL was dissolved in ethanol as 30 mM stock solutions.

Analysis of root growth

Seeds were grown on the vertical position. 3-d-old Arabidopsis thaliana seedlings were transferred to half-strength MS medium supplemented with the indicated treatments and grown for additional 5 d. Growth of primary roots was measured at the same time every day. After 5 d of treatment, the primary root length was measured using a ruler. Lateral root (LR) number and lateral root primordia (LRP) were determined by counting the lateral roots or LRP present in the primary root, from the tip to the root/stem transition. Lateral root and primordia densities were determined by dividing the lateral root number by the primary root length and expressed as LR cm− 1 or LRP cm− 1. The stage C-D of lateral root primordia according to Zhang et al. (1999) [72] were considered for the research. All experiments were performed with the solvent (ethanol) as a control at an end dilution same as AHL tested.

Immuno-detection of phosphorylated MAPKs

According to the method mentioned by Schikora et al. (2011) [9], total protein was extracted from 3-d-old seedlings pretreated with C10-HSL in half-strength MS medium for additional 5, 15, 30, 45 and 60 min. The total proteins were separated on 12% SDS polyacryl gel. Western blot was done using Phospho-p44/42 MAPK antibody purchased from Cell signaling (CAT. #4370) [9]. Licor-ODYSSEY(CLX) was used to photo blotting images. To obtain higher definition, we used a drawing area function supported by the machine.

Dye loading and laser scanning confocal microscopy

The generation of NO, ROS and Ca2+ in root tissue was examined using 4, 5-diaminofluorescein diacetate (DAF-2DA) (Sigma Aldrich), 5-(and-6)-chloromethyl-29, 79-dichlorodihydrofluorescein diacetate (DCF-DA) (Sigma Aldrich) and Fluo-4 AM (Thermo) probes, respectively. Before experiments, roots of seedlings were pre-incubated in the dark in loading buffer (10 mM Tris + 50 mM KCL, pH 7.18) containing 5 µM DAF-2DA or 10 µM H2DCF-DA for 30 min, or in buffer (50 mM KCL + 10 mM Tris + 5 mM CaCL2 + 0.5 mM eserine, pH 7.18) containing 10 µM Fluo-4 AM for 1 h, and then washed with distilled water to remove excess dye. Examinations of fluorescence intensity were performed using a laser scanning confocal microscope (excitation, 488 nm; emission, 500–550 nm; Leica SP8). For the fluorescence of dyes and GFP, we measured the fluorescence value of all meristematic zone in every plant, using internal Quantity software of Leica SP8 confocal microscope.

RT-qPCR

Total RNA was isolated from root tissues of 12-d-old seedlings treated with the indicated reagents for 0, 1, 3, 6, 12, 24 and 48 h, using Trizol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using Moloney murine leukaemia virus reverse transcriptase (Invitrogen). PCR amplifications were performed according to standard protocols using Taq DNA polymerase or Pyrobest DNA polymerase (TaKaRa). The mean value of three replicates was normalized against that of ACTIN2.

[Ca 2+]cyt measurements

Arabidopsis seedlings constitutively expressing the aequorin gene were germinated and grown on the half-strength MS agar plates at 23 °C and a 10 h dark/14 h light regime for 10 days. The [Ca2+]cyt rises either as a result of uptake from the extra-cellular space through plasma membrane channels or from the release of internal stores. Roots were incubated in 10 µM coelenterazine (0.1 mM KCL, 1 mM CaCL2,10 mM MES, pH 5.5) for 24 h and rinsed with buffer for 30 min. Each seedling was placed in a luminometer cuvette containing 50 µl buffer solution. C10-HSL stock solution was then injected (50 µl per injection) to achieve the indicated concentration. Final concentrations of LaCL3, verapamil chloride and EGTA were 10 mM, respectively. Thirty pre-washed seedlings were used for each luminometric experiment. Real-time aequorin luminescence was recorded every 1 s over the course of the experiment. At the end of the experiment, 2 M CaCl2 and 10% ethanol were added to discharge any remaining aequorin.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on request.

Abbreviations

AHLs:

N-acyl-homoserine lactones

CAT:

catalase C10-HSL:N-decanoyl-L-homoserine lactone

cPTIO:

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

C6-HSL:

N-hexanoyl-homoserine lactone

DAF-2DA:

4, 5-diaminofluorescein diacetate

DCF-DA:

5-(and-6)-chloromethyl-29, 79-dichlorodihydrofluorescein diacetate

GCR:

G-protein-coupled receptor

H2O2 :

hydrogen peroxide

LaCl3 :

lanthanum chloride

LiCl:

lithium chloride

LR:

Lateral root

LRP:

lateral root primordia

MAPK:

Mitogen-activated protein kinase

MPK6:

Mitogen-Activated Protein Kinase 6

NO:

nitric oxide

QS:

quorum sensing

ROS:

reactive oxygen species

SA:

salicylic acid

SNP:

sodium nitroprusside

3OC8-HSL:

3-oxo-octanoyl-homoserine lactone

3OC10-HSL:

N-3-oxo-decanoyl-homoserine lactone

3OC14-HSL:

N-3-oxo-tetradecanoyl-homoserine-lactone

3OC16-HSL:

3-oxo-N-(tetrahydro-2-oxo-3-furaryl)-hexadecenamide

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Acknowledgements

We thank Professor Chunpeng Song (Zhengzhou University in China) for providing nia1, nia2, noa1 seeds, Professor Zhonglin Shang (Hebei Normal University in China) for providing rbohD/F seeds, and the TAIR stock center for the mutants Atmpk6 seeds. We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant Number 31601144) and the Central Guidance on Local Science and Technology Development Fund of Hebei Province(226Z6501G).

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Authors and Affiliations

  1. Biology Institute, Hebei Academy of Sciences, 46th, South Street of Friendship, 050051, Shijiazhuang, Hebei, China

    Xiang-yu Cao, Qian Zhao, Ming-Xiang Yu, Fang Liu, Zhe Zhang, Zhen-hua Jia & Shui-shan Song

  2. Hebei Engineering and Technology Center of Microbiological Control on Main Crop Disease, 46th South Street of Friendship, Shijiazhuang, China

    Qian Zhao, Fang Liu, Zhen-hua Jia & Shui-shan Song

  3. College of Life Science, Hebei University, 180th East Road of Wusi, Baoding, China

    Ya-na Sun

  4. Hebei Collaboration Innovation Center for Cell Signaling Environmental Adaptation, 20 East NanErhuan Road, Shijiazhuang, China

    Shui-shan Song

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  1. Xiang-yu Cao
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Contributions

S.S and C.X. design the experiment and conceived the study. Z.Q. and C.X. is responsible for Figs. 1 and 2; S.Y. is responsible for Fig. 3; C.X. is responsible for Figs. 4, 5 and 7; Z.Z. is responsible for Fig. 6; L.F. and J.Z. manipulate software and participated in data analyzation. S.S.is responsible for all the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shui-shan Song.

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We declare that we have no competing interests.

Author details

1 Biology Institute, Hebei Academey of Sciences, 46th South Street of Friendship, Shijiazhuang 050051, Hebei, China. 2 Hebei Engineering and Technology Center of Microbiological Control on Main Crop Disease, 46th South Street of Friendship, Shijiazhuang, China.3 College of Life Science, Hebei University, 180th East Road of Wusi, Baoding, China. 4 Hebei Collaboration Innovation Center for Cell Signaling Environmental Adaptation, 20 East NanErhuan Road, Shijiazhuang, China.

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12870_2022_3865_MOESM1_ESM.docx

Supplementary Material 1: Figure S1. Primary root length and lateral root density of wild-type seedlings exposed to C10-HSL. Figure S2. NIA1, NIA2 and NOA1 participate in C10-HSL induced short primary root. Figure S3. U0126 could rescue C10-HSL induced primary root inhibition. Figure S4. Relative primary root growth of Col-0 seedlings exposed to C10-HSL with or without LiCl plus LaCl3. Table 1. List of the primers for qRT-PCR analysis of the genes. Bloting images (Figure S5 and S6): Original blotting images.

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Cao, Xy., Zhao, Q., Sun, Yn. et al. Cellular messengers involved in the inhibition of the Arabidopsis primary root growth by bacterial quorum-sensing signal N-decanoyl-L-homoserine lactone. BMC Plant Biol 22, 488 (2022). https://doi.org/10.1186/s12870-022-03865-6

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BMC Plant Biology

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