Many Gram-negative bacteria can use AHLs as intracellular signals to monitor their population density, a process called quorum sensing (QS) (Steindler, 2007; Williams, 2007; Holm & Vikstrom, 2014). Accumulating evidence has shown that AHLs not only play an essential role in QS-mediated physiological processes but also modulate plant growth, defense response and development ( Ortíz-Castro et al., 2008; von Rad et al., 2008; Ortíz-Castro et al., 2009; Morquecho-Contreras et al., 2010; Bai et al., 2012; Jin et al., 2012). C10-HSL or 3OC10-HSL can be produced by several bacteria including the rhizosphere-colonized bacteria Pseudomonas fluorescens and Pseudomonas putida (Venturi, 2006), the nitrogen-fixing bacterial symbiont Sinerhizobium meliloti (Marketon et al., 2002) and the pathogenic bacteria Burkholderia pseudomallei and B. mallei (William, 2007). In natural niches, the concentration of AHLs have been estimated in the range from 1 nM to 10 µM in the bulk rhizosphere and higher concentration (> 50 µM) may be present in bacteria biofilms (Palmer et al. 2014; Campbell & Greaves, 1990). Ortíz-Castro et al. (2008) tested the effect of seven AHLs with concentration from 12 to 192 µM on root growth and found C10-HSL with the greatest activity in inhibiting primary root growth and stimulating lateral root formation and root hair development. The derivation of C10-HSL, 3OC10-HSL, was found to stimulate adventitious root formation in mung bean (Bai et al. 2012). In this study, we confirmed the effect of C10-HSL and revealed that 10 nM to 1 µM C10-HSL treatment already caused a significant reduction in primary root growth and promotion in lateral root formation (Fig. S1). 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 which was accompanied by 3.5-fold increase in lateral root formation (Fig. 1). These results were consistent to those reported by Ortíz-Castro et al. (2008) and Bai et al. (2012) and implicate that AHLs can mediate cross-kingdom interactions by modulating root development during the course of plant-bacteria interaction.
It has been shown that ROS are involved in the response of plant to variety of phytohormones or environmental cues. In this study, we found that treatment with 30 µM C10-HSL significantly increased the accumulation of ROS in roots, while this treatment caused a significant inhibition on primary root growth in Arabidopsis (Fig. 3). Supplement with CAT or exogenous H2O2 alleviated or exaggerated the inhibitory effect of C10-HSL on primary root growth, indicating that H2O2, one type of ROS, contributes to C10-HSL-mediated primary root growth inhibition in Arabidopsis (Fig. 3). Further evidence was obtained from the genetic analysis using ROS-related mutants rbohD/F. We found that the primary roots of rbohD/F seedlings were less sensitive to C10-HSL treatment (Fig. 3). Zhang et al. (2017) reported that ROS generation is required for the exogenous H2S-inhibited primary root growth in Arabidopsis. Similarly, ROS accumulation was induced by treatment with a nitrification inhibitor Methyl 3-(4-hydroxyphenyl) propionate (MHPP) in root tips of Arabidopsis while MHPP treatment inhibited primary root elongation in Arabidopsis (Liu et al., 2016). These data implicate that AHLs influences primary root growth by regulating the accumulation of ROS in plant roots.
NO is another important signal molecule to regulate root growth in plants. Plant alkamide as an AHL homologue can induce accumulation of NO and adventitious root development in Arabidopsis thaliana explants (Campos-Cuevas et al., 2008). Pharmacological analysis indicated that blocking the 3OC8-HSL-induced accumulation of NO with NO scavenger cPTIO significantly reduced the stimulatory effect of 3OC10-HSL on the formation of adventitious roots in mung bean, suggesting a role of NO in 3OC10-HSL-mediated root development (Bai et al., 2012). In our experiments, we found that C10-HSL triggered NO accumulation in root tips. Moreover, supplement with NO scavenger abolished the production of NO induced by C10-HSL while significantly repress the inhibitory effects of C10-HSL on primary root growth (Fig. 2). Furthermore, noa1 encoding NO synthesis-associated enzyme was found to be C10-HSL inducible and C10-HSL-mediated primary root growth inhibition was markedly impaired in the NOA1-defective mutant noa1(Fig. 2). These results provide the pharmacological and genetic evidence that NO is required for C10-HSL-inhibited primary root growth in Arabidopsis. Some heavy metals such as zinc and cadmium and some toxic chemicals including H2S and MHPP were found to inhibit primary root growth by increasing intracellular NO accumulation, suggesting the role of NO in the inhibition of primary root growth by the environmental factors (Liu et al., 2016; Zhang et al., 2017).
In plants, MAPK are activated in response to a number of environmental cues (Clarke; & Davies, 2000; Kumar, 2000; Takahashi et al., 2007). In this study, we demonstrated that the MAPK inhibitor U0126 neutralized the C10-HSL-induced inhibition of primary root growth and the treatment with C10-HSL caused a stronger activation of MPK6 compared to the untreated control plants, whereas a rather weak activation of MAPK3 and MAPK4 were observed 30 min after C10-HSL treatment (Fig. 4), indicating that the activation of MAPK is required for this inhibition. This notion was supported by the genetic evidence that the MPK6 mutant showed a reduced inhibition of primary root growth after C10-HSL exposure (Fig. 4), providing the 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 effects depend on a stronger and prolonged activation of MPK6 in Arabidopsis. Accordingly, these data implicate that MPK6 is required for AHL-mediated primary root growth alteration.
It has been reported that MPK6 modulates plant growth and the response to environmental stimuli by interacting with ROS and/or NO (Wang et al., 2010; Takahashi et al., 2011; Han et al., 2015; Jalmi & Sinha, 2015). NO stimulates Cd-induced programmed cell death (PCD) by enhancing MPK6-mediated caspase-3-like activation in Arabidopsis (Ye et al., 2013). The MPK6 mutant shows more and longer lateral roots than do wild-type plants after application of SNP or H2O2, indicating that MPK6 modulates NO accumulation and the response to H2O2 during Arabidopsis root development (Wang et al., 2010). 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 of roots to MHPP treatment in Arabidopsis. By using pharmacological approaches, Bai et al. (2012) found that H2O2 may modulate the NO signal during mung bean responses to 3OC10-HSL treatment. These studies suggest that the interaction between MPK6, ROS and NO exists and the mechanism underlying this interaction may be context-dependent. In this study, we observed that reducing ROS accumulation with H2O2 scavenger CAT significantly decrease the activation of MPK6 (Fig. 5). 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 noa1 mutants. In addition, C10-HSL treatment could not induce the transcript level of noa1 in rbohD/F mutants (Fig. 5). The induction of NO production by C10-HSL was completely abolished in mpk6 mutants, while ROS accumulation caused by C10-HSL treatment in mpk6 mutant seedlings was unaffected. Addition of catalase could not further alleviate the C10-HSL-induced inhibition of primary root growth in roots of mpk6 mutant (Fig. 5). On the other hand, application of SNP, a NO donor, could rescue the defect in the C10-HSL-induced inhibition of primary root growth in mpk6 mutants, while addition of exogenous H2O2 could not rescue the inhibition of primary root growth caused by C10-HSL treatment in mpk6 mutants (Fig. 5). Our data indicate that ROS, MPK6 and NO might act in line to regulates plant root responses to C10-HSL. A similar signaling pathway was reported in the responses of plant to H2S toxicity (Zhang et al., 2017).
Ca2+, as an intracellular second messenger, is essential for signal transduction processes in plants (Virdi et al, 2015; Tanejia et al, 2016). In our previous study, we showed that C4-HSL was able to trigger an increase in the concentration of intracellular Ca2+ (Song et al., 2011). In the present study, we found that treatment with C10-HSL resulted in a transient and immediate elevation of [Ca2+]cyt in root cells of Arabidopsis (Fig. 6). The effect of C10-HSL on induction of [Ca2+]cyt concentration increase was also observed by using the Ca2+-sensitive dye Fluo-4 AM and inhibition of intracellular Ca2+ elevation alleviated the inhibitory effect of C10-HSL on primary root growth. Moreover, we noticed that addition of Ca2+ inhibitor significantly reduced the production of ROS and NO induced by treatment with C10-HSL (Fig. 7). Our data indicate that Ca2+ signal may be the early event in plant response to C10-HSL and play its role by regulating the down-stream production of ROS. These acting mechanisms are often observed in the incompatible host-pathogen recognition in which flux of Ca2+ across the plasma membrane as an earliest cellular event results in a set of oxidative bursts that produce ROS (Harding, 1997; Reddy, 2003).
In conclusion, our present study revealed that C10-HSL triggered a transient and immediate elevation in concentration of cytosolic free Ca2+ and induced ROS accumulation, MPK6 activation, NO production in Arabidopsis primary roots. Ca2+ mediated the generation of ROS and NO induced by C10-HSL. Activation of MPKs were required for inhibition of primary root growth by C10-HSL. MPK6 was shown to act downstream of ROS and upstream of NO. Thus, our data suggested 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). It is well known that auxin plays a fundamental role in root system architecture in plants (Vanneste, 2009; Liu, 2015). 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. On the other hand, 3OC12-HSL and 3OC16-HSL were found to induce the tissue-specific expression of auxin-responsive GH3 in legume (Mathesius et al., 2003). Bai et al. (2012) reported that an analogue of C10-HSL, N-3-oxo-decanoyl-homoserine lactone (3OC10-HSL), mediated auxin-dependent adventitious root formation via H2O2- and NO-dependent cGMP signal in mung bean seedlings. C6-HSL was found to promote root elongation and increase the ratio of auxin:cytokinin toward higher auxin levels in both leaves and roots in Arabidopsis (Von Rad et al., 2008). Zhang et al. (2017) reported that exogenous H2S repressed the distribution of auxin and inhibited primary root growth in Arabidopsis. MHPP as a nitrification inhibitor can modulate root system architecture via ROS/NO-mediated-accumulation and maximum distribution concentration of auxin in Arabidopsis roots (Liu et al., 2016). Moreover, Hu et al. (2018) reported that N-decanoyl-homoserine lactone (C10-HSL) activates plant systemic resistance against Botrytis cinerea in tomato and C10-HSL-induced resistance is largely dependent on the JA signaling pathway. Therefore, the comprehensive investigation for the involvement of phytohormones such as auxin and JA and the interplay of C10-HSL with phytohormones will expand our understanding of the mechanism by which plants respond to C10-HSL and will provide insight into novel applications of this biological molecules for regulating crop growth and development.