Melatonin (N-acetyl-5-methoxytryptamine), a widely distributed endogenous bio-molecule in mammals, was first discovered in the bovine pineal gland in 1958 (Lerner et al., 1958). Melatonin regulates many important physiological processes in mammals, including sleep, body temperature regulation, circadian rhythms, mood, immune processes, etc. (Jan et al., 2009; Hardeland et al., 2012; Carrillo-Vico et al., 2013; Reiter et al., 2014).

The existence and discovery of melatonin in other species, especially in higher plants, indicates its extensive functions (Poeggeler et al., 1991; Dubbels et al., 1995; Hattori et al., 1995). Numerous studies have shown that melatonin is widely involved in regulating both the biotic and abiotic stress tolerance, biological rhythms, plant growth and development (seed germination, root architecture, shoot development, plant flowering, fruit ripening, and yield; Kang et al., 2010; Okazaki et al., 2010; Wang et al., 2012; Byeon et al., 2013, 2014b; Yin et al., 2013; Zhang et al., 2013, 2014; Zhao et al., 2013; Byeon and Back, 2014a; Shi and Chan, 2014; Weeda et al., 2014; Shi et al., 2015a,b,c,d,e, 2016).

In higher plants, melatonin is synthesized from tryptophan as substrate by four key enzymes [tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), and N-acetylserotonin O-methyltransferase (ASMT)] (Kang et al., 2007a,b, 2011, 2013; Okazaki et al., 2009; Fujiwara et al., 2010; Byeon and Back, 2014b, 2015; Zuo et al., 2014; Arnao and Hernández-Ruiz, 2015). Recent studies found that N-acetylserotonin can also be synthesized via caffeic acid O-methyltransferase (COMT) in Arabidopsis thaliana, except ASMT (Byeon et al., 2014a; Lee et al., 2014). Interestingly, there are two different pathways for the synthesis of auxin (IAA) in plants, one is tryptophan-independent, and the other is tryptophan-dependent, sharing the same substrate with melatonin (Wang et al., 2015). Tryptophan-dependent pathway is dependent on precursor tryptophan, through tryptamine (TAM), indole-3-pyruvic acid (IPyA) and indole-3-acetaldoxime (IAOx) pathways (Benjamins and Scheres, 2008; Strader and Bartel, 2008; Chandler, 2009).

Melatonin also showed some similar functions as auxin in the higher plants, in addition to sharing the same substrate for their biosynthesis. Previous studies suggested that melatonin acts as a growth-stimulating molecule in lupin tissues and some monocots, including canary grass, wheat, barely, and oat; its IAA-like activity is 10–55% of that of auxin (Hernández-Ruiz et al., 2004, 2005). However, there are also reports indicating that melatonin regulates Arabidopsis root growth independent of auxin signaling (Pelagio-Flores et al., 2012; Koyama et al., 2013).

To date, the function of melatonin in regulating root growth and the underlying mechanisms are still unclear in higher plants. Moreover, the relationship between melatonin and auxin remains unknown. In the present work, different concentrations of melatonin were used to treat the wild-type (WT, Col-0) Arabidopsis. The results showed that melatonin significantly suppressed root growth by reducing the size of root meristem. Additionally, comprehensive analyses of auxin synthesis, PIN (PINFORMED) proteins and a auxin response marker line of Arabidopsis (DR5 promoter marker line) suggested that melatonin might regulate the root growth through auxin signaling, at least partially.

As an important plant hormone, auxin plays vital roles in root cell division, differentiation, elongation, and the overall growth of roots (Benjamins and Scheres, 2008). In recent decades, more attention has been paid to the role of melatonin as a growth regulator of plants (Arnao and Hernández-Ruiz, 2015). Both of auxin and melatonin have been suggested to regulate similar growth processes. Our data showed 10 μM melatonin had no effect on primary root growth (Figures 1 and 2), and our previously work suggested that 10–50 μM melatonin had litter effect on endogenous melatonin content (Shi et al., 2015c). Based on previous studies and our preliminary experiments, 10–50 μM melatonin had litter effect on endogenous melatonin content (Shi et al., 2015c), and lower melatonin concentration also had litter effect on plant root development (Bajwa et al., 2014). Moreover, 100–600 μM melatonin were also widely used in other studies (Pelagio-Flores et al., 2012; Bajwa et al., 2014). In our study, we first tested the effect of melatonin on root growth of Arabidopsis and found that 100 μM melatonin had already shown an inhibitory effect on root growth, in accordance with previous reports (Chen et al., 2009). However, Bajwa et al. (2014) showed that 100, 200, and 400 μM melatonin treatment had no significant effects on plant root growth. The difference might be attributed to the big values of SD of their results and solvent effect. To limit the effect of solvent, the same volume of solvent including ethanol was used as a control in this study. Moreover, the average values of more than 30 seedlings per treatment from three independent experiments. The higher the concentration of melatonin, the more suppression of the root length (Figure 1B), indicating that melatonin inhibit root length in a dose-dependent manner. Although moderate auxin promotes root growth of plants, overproduction of auxin levels can cause a decay of root growth (Teale et al., 2005; Strader et al., 2011). After digging deeper into the effects of melatonin, we found high concentrations of melatonin reduced root meristem size, consistent with its effects on primary root length (Figures 2B,C).

The effects of auxin on root growth, is largely dependent on its biosynthesis and polar transport, which cause optimal auxin accumulation and distribution in the root apex during the whole developmental process (Blilou et al., 2005; Kim et al., 2007; Li et al., 2011). Quantitative real-time PCR showed that the expression levels of YUC1, YUC2, YUC5, YUC6, and TAR2, key genes of auxin biosynthesis, were significantly down-regulated after 600 μM melatonin treatment consistent with lower IAA content in 600 μM melatonin-treated roots (Figure 3). If we set the transcript level of YUC1 in control material as 1, the relative expression level of YUC2, YUC3, YUC4, YUC5, YUC6, YUC7, YUC8, TAA1, TAR1, and TAR2 was 28.7, 406.7, 4.6, 0.37, 151.9, 20.6, 170.3, 43.5, 41, and 693 separately. In melatonin-treated material, the relative transcript level of YUC1 and other genes were 0.4, 1.6, 564.1, 11.9, 0.1, 1, 132.8, 230.8, 23.1, 29.6, and 234.3, respectively. After melatonin treatment, the total relative expression abundance was decreased significantly. The decrease in expression of YUC1, YUC2, YUC5, YUC6, and TAR2, together with the effects of melatonin on auxin transport may cause the decrease in IAA levels in roots, at least partially. As reported recently, application of 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) results in a decay in the transcript levels of YUC1, YUC2, YUC4, YUC6, and TAR2 in Arabidopsis seedlings (Suzuki et al., 2015). We noticed there is a difference in the endogenous IAA level between this study and previous results (Zhao et al., 2001; Lee et al., 2012), which could be resulted from two possible reasons. One reason could be the sample differences. In our work, only the root tips were harvested and used for analysis. Another possibility is that different methods were used. In this study, we used ELISA method. Based on the consistence between the transcript levels of auxin biosynthesis genes and IAA level, we concluded that melatonin negatively regulated auxin biosynthesis.

Polar auxin transport is essential for the distribution of auxin in Arabidopsis root tips, and the auxin efflux machinery PIN proteins play important roles in controlling the size of root meristem (Friml et al., 2003; Blilou et al., 2005). Expression levels of PINs were always found down-regulated in the shortened root meristem after stresses (Liu et al., 2015; Yuan and Huang, 2016). Even in PIN1::GFP line roots exogenously treated with IAA, a significant decrease in GFP fluorescence was detected under high concentrations (5 and 10 μM; Omelyanchuk et al., 2016). Coincidently, our results demonstrated a decrease in both of the transcript levels and the protein expression levels of PIN1, PIN3, and PIN7 in melatonin-treated roots (Figures 4A,C, revised Figure 4B). Auxin transport inhibitor TIBA could also decrease the root meristem size and root growth (Blilou et al., 2005). Our study showed auxin transport inhibitor TIBA did not enhance melatonin-mediated reduction of root meristem size, indicating that PAT might be necessary for the regulation of root meristem size by melatonin treatment (Figures 5A,B). Moreover, the triple mutant pin1pin3pin7 was more tolerant than WT in response to melatonin treatment, suggesting that PIN1/3/7-mediated PAT might contribute to melatonin-regulated root meristem.

Previous study showed application of low concentration of melatonin (0.1 μM), increased the endogenous levels of IAA in Arabidopsis roots (Chen et al., 2009), and our findings demonstrated that exogenous application of low concentration of IAA (0.5 nM), also raised the endogenous melatonin content in roots (Figure 5E). On the contrary, 100 nM IAA caused reduced root meristem size, as previously reported (Rahman et al., 2007; Strader et al., 2011). In this study, simultaneous presence of 100 nM IAA and 600 μM melatonin led to more serious decrease in root meristem size than that of 600 μM melatonin alone (Figures 5C,D).

The expression of DR5 promoter marker line in root tips represents the responses and distribution pattern of auxin. Unlike the results showed by Pelagio and Koyama (Pelagio-Flores et al., 2012; Koyama et al., 2013), here we found that melatonin was able to change the expression pattern in both of DR5::GUS and DR5::GFP line roots (Figure 5G), just like the effects of IAA (Ottenschläger et al., 2003). In combination with 600 μM melatonin, 100 nM IAA caused a more expansion pattern of fluorescence signals in the whole root caps, including columella cells and lateral root cells, indicating that exogenous application of melatonin intensified the effect of IAA on the auxin responses in root tips. Considering altered auxin synthesis in root tip of treated seedlings, decreased signals of PIN1 in provasculature, reduced PIN3 and PIN7 signals in both root cap and provasculature, the DR5::GFP signal should not expand that strongly toward the lateral cap and tip cells after melatonin treatment. Then we also observed the expression and localization of PIN2 in the root cells, however, no significant difference was shown about the expression of PIN2 in cell membrane without and with 600 μM melatonin treatment (revised Supplementary Figures S1A–C). What’s surprised is that PIN2 signals in cytoplasm in melatonin-treated roots were obviously increased, although the signals in cytoplasm were much lower than that in cell membrane. This may, at least partially explain that why DR5 signals spread into the whole root cap after melatonin treatment. These results indicated that melatonin may have dual and complex effects on auxin transport. Besides PIN1/2/3/7, there may be other issues contribute to melatonin-mediated auxin signaling, which need to be further investigated.

Based on our results, we proposed a working model for the mechanisms by which melatonin regulates root meristem (Figure 6). Melatonin and auxin share the same substrate tryptophan during biosynthetic pathways, and exogenous application of IAA at low concentration increases melatonin production, while high concentration of melatonin decreases the level of IAA and PIN1, 3, 7 in Arabidopsis roots. Thus, melatonin regulates root meristem by repressing auxin synthesis and polar auxin transport in Arabidopsis. In summary, this study provides a direct link between melatonin and root growth, and indicates the novel involvement of auxin responses in melatonin-mediated root growth in Arabidopsis. We highlight the relationship between of melatonin and auxin in Arabidopsis root growth.

HS conceived and directed this study, revised the manuscript; QW designed and performed the experiments, analyzed the data, wrote and revised the manuscript; BA performed the experiments, analyzed the data and revised the manuscript; YW provided help in the melatonin and IAA content analysis; RR provided suggestions and revised the manuscript; HL designed the experiments and revised the manuscript; CH designed the experiments and revised the manuscript. All authors approved the manuscript and the version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.