Methanol elicits the biosynthesis of 4-coumaroylserotonin and feruloylserotonin in rice seedlings

Induced Synthesis of Serotonin Derivatives upon Methanol Treatment

Ten-day-old rice seedlings were grown in sterile water containing various concentrations of methanol for 6 days at 25°C under 12-h light/12-h dark, and leaves were harvested for quantifying serotonin derivatives by HPLC (Fig. 1). When rice seedlings were challenged with methanol, serotonin derivatives such as CS and FS were detected, whereas tyramine derivatives such as feruloyltyramine and 4-coumaroyltyramine were not detected in rice plants. Upon methanol treatment, CS and FS were produced and their levels were increased as the concentration of methanol increased. Rice leaves grown in 0.1% methanol showed 4.1 µg g ^{fresh weight (fw) of CS+FS, while the control leaves produced only 1.1 µg g fw of CS+FS. In response to 0.25% and 0.5% methanol, CS+FS were 6- and 28-fold higher than that of the control leaves. Maximum production of CS+FS was observed in 1% methanol treatment showing around 40 µg g fw of CS+FS which was 38-fold higher than that of the control leaves. In contrast, no induced synthesis of serotonin derivatives was observed upon 2.5% methanol treatment. No preferential production of either FS or CS was observed even though FS levels were slightly higher than those of CS. No phenotypical difference was observed in 1% methanol treated rice seedlings compared to those of the control leaves whereas 2.5% methanol treatment caused rice seedlings to wither quickly. Taken together, this data suggest that the synthesis of serotonin derivatives is induced upon methanol treatment, and that these methanol responses are manifested by a certain ranges of methanol concentrations. In particular, the roots of rice seedling did not showed the induction of serotonin derivatives even in the presence of 1% methanol. Thus, the induced synthesis of serotonin derivatives was specific to leaf tissues. The treatment of either CuSO}₄ or chitin did not result in the increased synthesis of serotonin derivatives (data not shown).

Figure 1

Synthesis of serotonin derivatives in response to methanol treatments in rice seedlings. 10-day-old rice seedlings (cv. Dongjin) were grown in sterile water containing various concentrations of methanol for 6 days at 25°C under 12-h light/12-h dark, and leaves were harvested for quantifying serotonin derivatives by HPLC. CS, 4-coumaroylserotonin; FS, feruloylserotonin. The data represent the means of the results of duplicate experiments ±SD.

Methanol Induces SHT Enzyme

To confirm whether the production of serotonin derivatives is specific to methanol treatment or not, several solvents with each 1% concentration are employed to test their effects on the synthesis of serotonin derivatives at 6 days after treatments. As shown in Figure 2A, ethanol, acetaldehyde and isopropanol had no eilicitor effects on the induced synthesis of serotonin derivatives in rice plants. To scrutinize for the exclusive role of methanol, we have checked the role of methanol catabolites. It is known in plant cells that methanol is oxidized sequentially into formaldehyde, formic acid and CO₂. The catabolites of methanol, such as formaldehyde and formic acid did not induce the synthesis of serotonin derivatives. This suggests that the induction of secondary metabolite serotonin derivatives is highly specific to methanol. Next, we have measured the plant hormonal effects on the methanol induced secondary metabolite production. 10-day-old seedlings were grown in 1% methanol in the presence or absence of 5 µM plant hormones. The leaves of rice seedlings at 6 days after treatments were subjected to HPLC analysis. Abscisic acid (ABA) has a strong inhibitory role on the induction of serotonin derivatives induced by methanol (Fig. 2B). Indoleacetic acid (IAA) and zeatin treatments also inhibit the production of serotonin derivatives. The level of serotonin derivatives in response to methanol plus IAA or zeatin was only 20% that of methanol treatment only. In contrast, gibberellic acid (GA) has a minor inhibitory effect on the synthesis of serotonin derivatives which was produced up to 70% that of methanol treatment only. This indicates that methanol's action on the synthesis of serotonin derivatives are closely interacted with plant growth hormones. Finally, we measured the serotonin N-hydroxycinnamoyl transferase (SHT) enzyme activity in leaves of rice seedlings exposed to methanol. SHT enzyme catalyzes the synthesis of serotonin derivatives by condensing two substrates serotonin and phenolic-CoA.⁹ In control leaves of seedlings, SHT enzyme activity remained unaltered until 5 days, then began to increase slightly at 7 days with a peak at 10 days which showed 2 pkat mg ^{protein of SHT enzyme activity (}Fig. 2C). This peak activity was rapidly reduced to basal level at 15 days. However, methanol treated leaves exhibited a rapid increase of SHT enzyme activity. SHT enzyme activity was 2 pkat mg ^{protein at 3 days, and reached a maximum activity with 5 pkat mg protein, which was 10-fold higher than that of untreated leaves (d = 0). After 7 days in methanol treatment, SHT enzyme activity decreased rapidly and was almost equal to the basal level at 20 days. The patterns of SHT enzyme activity upon methanol treatment is closely in parallel with the induced synthesis of serotonin derivatives. Therefore, the increased synthesis of serotonin derivatives in rice leaves when challenged with methanol is attributable to the transient induction of SHT enzyme activity. To date, no sequence information on rice SHT gene is reported although SHT gene was first cloned from hot pepper.10} On the basis of the induction of SHT enzyme activity, it will be of interest to investigate whether rice SHT gene is transcriptionally upregulated in response to methanol, and which part of SHT gene promoter sequence is responsible for methanol inducibility. In sum, several lines of evidence have provided strong support for the elicitation role of methanol on the synthesis of serotonin derivatives preceded by the induction of SHT enzyme activity in rice seedlings.

Figure 2

Effects of other solvents and plant growth hormones on the synthesis of serotonin derivatives (A and B) and SHT enzyme activity measurement (C). 10-day-old rice seedlings (cv. Dongjin) were grown in sterile water containing various solvents (1%) or plant growth hormones (5 µM) as described in Figure 1. SHT enzyme activity was measured in extracts of leaves of rice seedlings. SHT activity is expressed as pkat mg ^{protein. MeOH, methanol; EtOH, ethanol; AcA, acetaldehyde; IP, isopropanol; FA, formaldehyde; F, formic acid; H}₂O, sterile water; IAA, indoleacetic acid; GA, gibberellic acid; ABA, abscisic acid. The data represent the means of the results of duplicate experiments ±SD.