Conjugates of 3-phenyllactic acid and tryptophan enhance root-promoting activity without adverse effects in Vigna angularis

Abstract

3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure–activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.

The development of the root system is one of the key factors for water and nutrition uptake. The optimized root system gives better yield for crop production (Fageria and Moreira 2011; Koevoets et al. 2016; Nishizawa and Saito 1998; Seo et al. 2020). Although a growth control of crops is one of the major concerns in agriculture, the use of root promoting substances (RPSs) is limited, e.g., cuttings for root induction (Song et al. 2019). A natural auxin, indole-3-butylic acid (IBA) is frequently used for rooting of cuttings (Braha and Rama 2016; Crane and Mallah 1952; Owais 2010). Such an auxin substance is widely used for weed control agent because of the herbicidal activity and limits the usage for RPS (Grossmann 2010; Kelley and Riechers 2007). Bokashi is a kind of organic fertilizer made of plant residues with soil microorganisms which traditionally developed by Japanese farmers and notified for less adjustment of dose required (Jaramillo-López et al. 2015; Nikitin et al. 2018). Lactic acid bacteria are one of the common microorganisms of Bokashi (Maki et al. 2021; Xu 2001). 3-Phenyllactic acid (PLA) is identified as RPS from a culture of lactic acid bacteria but shows less phytotoxic activity (Maki et al. 2021). PLA exhibited a synergistic effect of root promotion when co-treated with tryptophan (Trp) (Maki et al. 2021). In this study, various stereoisomers of PLA-Trp conjugates and their ester derivatives is designed (Figure 1A), chemically synthesized and examined for root promotion activity (Figure 1B, Table 1).

Figure 1. Structures and synthesis of PLA-Trp conjugate and derivatives. (A) Structure of PLA-Trp conjugate and derivative. (B) Procedure for the synthesis of the PLA-Trp conjugate and derivatives. To obtain PLA-Trp methyl ester as intermediate, equimolar PLA and tryptophan methyl ester (Trp-me) was condensed using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as peptide synthesis reagent and one tenth molar 1-hydroxybenzotriazole as a racemization suppressor in methylene chloride as described previous report (Inoue et al. 2003; Maki et al. 2021). Methyl esters of conjugate were hydrolyzed in 0.1 M NaOH solution (H₂O : EtOH=1 : 1) to obtain the free form of PLA-Trp (Maki et al. 2021). In addition, the conjugate was esterified with the alcohol and p-toluenesulfonic acid (Fischer and Speier 1895). All synthesized compounds were purified with solvent extraction and recrystallization as described (Maki et al. 2021). Chemical structure and purity were confirmed by NMR spectrum (AL-300 spectrometer, JOEL Ltd., Japan) in dimethyl sulfoxide-d6 as solvent.

Table 1. Compound number of PLA and Trp stereoisomer conjugates, ester derivatives.

Compound No.	PLA	Trp	R
1	D	L	H
2	L	D	H
3	D	D	H
4	L	L	H
5	D	L	methyl
6	L	D	methyl
7	D	D	methyl
8	L	L	methyl
9	D	L	ethyl
10	D	L	propyl
11	D	L	isopropyl
12	D	L	butyl

The structure–activity relationship of stereoisomers is one of the research targets for agrochemical development (Jeschke 2018; Ulrich et al. 2012) and considered in this study. Twelve combinations of PLA-Trp stereoisomer conjugates and four combinations of their mixtures were examined for rooting activity by Adzuki root-promoting assay (Itagaki et al. 2003; Figure 2A). The relative activity of D-PLA-L-Trp conjugate (Table 1, No. 1) to a mixture of D-PLA and L-Trp (Figure 2B, No. 13) showed 41 times higher activity and L-PLA-L-Trp conjugate (Table 1, No. 4) showed 61 times higher activity to No. 13 (Figure 2B, Nos. 1, 4, 13). However, conjugates with D-Trp stereoisomer (Table 1, Nos. 2, 3, 7) abolished rooting activity (Figure 2B, Nos. 2, 3, 7). It is noteworthy that a mixture of L- or D-PLA and D-Trp exhibited comparable activity to a mixture of D-PLA and L-Trp (Figure 2, Nos. 13, 14, 15), suggesting that the sole D-Trp does not suppress PLA activity but steric structure of conjugate require rooting activity and incorporation of D-Trp moiety is not allowed for rooting activity.

Figure 2. Rooting activity of synthesized compounds. Adzuki root-promoting assay was carried out for an evaluation of synthesized compounds. Shortly seed of Adzuki bean cv. Erimoshouzu (Vigna angularis (wild.) Ohowi and Ohashi) were germinated on vermiculite and grown under continuous white light (44 µmol m⁻² s⁻¹, Tokyorikakikai) at 25°C for 12 days. The resulting shoot was cut 3 cm above from root-shoot junction and an apical bud was emasculated to remaining internode number. The cuttings were dipped into a test solution at 7 cm depth and grown under continuous white light at 25°C for 3 further days. Cuttings of submerged parts were washed and grown in distilled water for 4 days. (A) Pictures of adventitious roots in Adzuki root-promoting assay. Plants were treated with water control or each compound at 150 µM concentration. Bar indicates 1 cm. (B) Activity of compounds. The number of emerging adventitious roots was counted and the EC₅₀ (Half maximal effective concentration) was calculated from a curve-fitting function of dose-activity data point. The equivalent 50% root-promoting concentration of the assayed compounds to that of a mixture of D-PLA and L-Trp was calculated as EC₅₀eq. No.13 is an equal mole mixture of D-PLA and L-Trp, No.14 is an equal mole mixture of L-PLA and D-Trp, No.15 is an equal mole mixture of D-PLA and D-Trp, and No.16 is an equal mole mixture of L-PLA and D-PLA.

Five derivatives were synthesized (Table 1, Nos. 5, 9, 10, 11, 12) from compound No. 1 which is an amide conjugate of No. 13 as a lead compound and examined rooting activity (Figure 2). Compound No. 9, ethyl ester of D-PLA-L-Trp exhibited 3,400 times higher than compound No. 13 activity and this is the highest activity in this study (Figure 2, Nos. 9, 13). Compound No. 12, butyl ester of D-PLA-L-Trp showed 1,800 times higher activity of compound No. 13 activity (Figure 2, Nos. 12, 13). It is known that the hydrophobic moiety facilitates cell membrane permeability and enhances the activity of pesticides (Akamatsu 2011). In fact, alkyl esters of D-PLA-L-Trp conjugate, No. 9 and No. 12 showed higher values of LogP compared to the free forms of D-PLA and L-Trp (Table 2). Increased hydrophobicity in compound No. 9 and 12 may lead to enhancement on rooting activity. Although the methyl ester modification (Nos. 5, 8) enhances hydrophobicity as well, no enhancement of rooting activity detected (Figure 2B, Nos. 5, 8) and isopropyl ester modification abolished rooting activity (Figure 2B, No. 11). These results indicate that the hydrophobicity of conjugate is not sufficient for an activity increment but suggests a requirement of steric structure for rooting activity. Interestingly, L-PLA-D-Trp methyl ester (compound No. 6) exhibited low but significant activity while L-PLA-D-Trp (compound No. 2) does not show any rooting activity (Figure 2B, Nos. 6, 2). It is imaginable that undetectable rooting-activity of L-PLA-D-Trp (compound No. 2) has raised by methyl ester modification (Figure 2B, No. 6) which leads to higher cell permeability. However, D-PLA-D-Trp methyl ester (compound No. 7) did not show rooting activity (Figure 2B, No. 7) which contrast to the case of L-PLA-D-Trp methyl ester. Although it remains to be elucidated that an amide hydrolysis of conjugate for PLA and Trp occur inside of the cell, the difference of rooting activity between D-PLA-D-Trp methyl ester and L-PLA-D-Trp methyl ester suggests a PLA-Trp conjugate is an active form inside of the cell rather than amide hydrolysis of conjugate. Hydrophobicity and or steric structure of conjugate is associated with rooting activity.

Table 2. Hydrophobicity of PLA-Trp conjugates and derivatives.

	LogP
	pH 5.5	pH 7.0
PLA	−0.69	−1.37
Trp	−0.96	−1.12
No. 1	0.51	0.06
No. 2	0.68	0.07
No. 3	0.59	0.09
No. 4	0.71	0.12
No. 5	2.50	2.55
No. 6	2.71	2.33
No. 7	2.75	3.15
No. 8	2.91	2.93
No. 9	2.98	2.80
No. 10	3.20	3.36
No. 11	3.34	3.39
No. 12	3.23	3.17

For the measurement of hydrophobicity, LogP (partition coefficient in 1-octanol/water) value (Akamatsu 2011) was measured. Shortly aquatic compounds were prepared to pH 5.5 or pH 7.0 and partitioned by 1-octanol. Aqueous or 1-octanol phases were separated by HPLC and UV absorption was measured simultaneously and the LogP (Log (OcOH/AQ)) value was calculated.

Compound Nos. 9, 10, and 12 show higher rooting activity in a dose-dependent manner up to 0.5 mM while PLA saturated rooting activity at 0.1 mM (Figure 3; Maki et al. 2021). This result suggests that compound Nos. 9, 10, and 12 are highly controllable for rooting activity by the administration of their concentration and potentiate for practical usage. Commercially and widely used RPS, IBA was compared to No. 9 in terms of herbicidal activity. The leakage of electrolyte is increased with a failure of cell-membrane integrity (Hatsugai and Katagiri 2018). Electrical conductivity of leaves treated with IBA or compound No. 9 was measured by conductivity meter (model CM-40V, HORIBA, Tokyo) (Figure 4B). Maximum effective concentration of IBA was 0.2 mM in Adzuki root-promoting assay and IBA did not showed phytotoxicity at this concentration (Figure 4B). Beyond of maximum effective concentration, IBA exhibited leaf wilting (Figure 4A) and electrolyte leakage (Figure 4B). Although maximum effective dose of compound No. 9 is higher than that of IBA (0.5 mM, Figure 3 top), No. 9 treated plants remained vigorous shape (Figure 4A) and modest electrolyte leakage (Figure 4B) beyond of maximum effective concentration. Together with dose-dependent activation of rooting in compound Nos. 9, 10 and 12 indicates a potent non-phytotoxic rooting agent, which is controllable with various concentrations.

Figure 3. Dose response of active conjugates of PLA-Trp ester, PLA and Trp. The number of inductive adventitious-root is calculated by the subtraction of the number of emerged adventitious-root in water control from the number of adventitious-root treated. The statistical analysis was performed using two-tailed Student’s t-tests. The plotted data is a means (SD) (n=5). The asterisks indicate significant differences as * p<0.05, ** p<0.01.

Figure 4. Phytotoxic activity of No. 9 and IBA. (A) 1 mM of IBA or compound No. 9 were examined according to Adzuki root-promoting assay and picture was taken at 5 days after treatment before adventitious root formation. Bar indicates 1 cm. (B) Electrical conductivity of leaves. The leaves of cuttings treated with chemical for 3 days were submerged in distilled water for 24 h and electrical conductivity was measured by conductivity meter (model CM-40V, HORIBA, Tokyo). The statistical analysis was performed using two-tailed Student’s t-tests. The plotted data is a means (SD) (n=5). The asterisk indicates significant differences as * p<0.05.

In this study, an amide conjugates, D-PLA-L-Trp (compound No. 1) as well as L-PLA-L-Trp (compound No. 4) showed enhanced activity compared to a mixture of D-PLA and L-Trp (compound No. 13). Such a rooting activity is dependent on Trp stereoisomers and D-Trp decreases or abolished rooting activity (Figure 2B, Nos. 2, 3, 6, 7). However, D-Trp itself does not inhibit L-PLA activity (Figure 2B, Nos. 14, 15). These results suggest that the L-Trp of conjugates are required for rooting activity. Further esterification of D-PLA-L-Trp with ethyl and butyl residues resulted in 3,400 times higher activity and 1,800 times higher activity than that of compound No. 13 respectively. These increment of activity correlates with a chemical hydrophobicity (Table 2) which increased permeability through the cell membrane. However, esterification by isopropyl reside abolished PRS activity like as D-Trp conjugate. This result also supports a structure–activity relationship. Although membrane permeability has not been measured directly in this study, such an increment of hydrophobicity of compound No. 1 and 4 may explain its higher rooting activity than PLA and Trp. The rigidity of stereoisomer and ester moiety of PLA-Trp conjugate suggests the function of conjugate sole, however, hydrolysis of amide conjugate can not be ruled out. Phytotoxicity of D-PLA-L-Trp and its active derivatives is less visible, which is similar property to PLA (Maki et al. 2021), which also indicates the similar signaling pathway as PLA. A compound with a high activity is, in other words, a compound with low environmental impact (Abet et al. 2017; Casida 2017; Jeschke 2018). Further research on the environmental toxicity of D-PLA-L-Trp and derivatives will be necessary for practical usage and signaling pathways for rooting activity.

Abbreviations

IBA

indole-3-butylic acid

LogP

partition coefficient in 1-octanol/water

PLA

3-phenyllactic acid

RPS

root promotion substance

Trp

tryptophan