Study on the induction of exogenous plant hormones to enhance the weed suppression ability of allelopathic and non-allelopathic rice accessions

Rice seedling cultivation and induction treatments

An allelopathic rice accession PI312777 and a non-allelopathic rice accession Lemont, which had been identified previously (Dilday, Lin & Yan, 1994) and obtained in our lab, were used as target plants for induction. The induction experiment with PI and LE was conducted following the manner detailed in our prior paper (Zhang et al., 2018). Briefly, the homogeneous rice seedlings of each accession at the 1-leaf stage were cultivated in a plastic cup containing 0.4 L of Hoagland’s solution, with one rice seedling per cup. At the four-leaf stage, the culture solution was switched new Hoagland’s solution (pH 6.0) containing different induction treatments as subsequent experiments. Hoagland’s solution without an inducing solution was used as a control. The experiments followed a completely randomized design, with twenty repetitions of each treatment. Rice was grown in a growing space with diurnal and nocturnal temperatures of 20−30 °C and relative humidity varying from 65% to 90%.

Selection of appropriate plant hormones with optimum induction conditions

Experiment 1 was run to evaluate the changes of the induced-allelopathy of PI and LE treated with the optimum induction phytohormones. In the preliminary experiments, a total of 10 plant hormones with three concentrations were used to investigate their effects on the root growth of two rice cultivars (‘PI’ and ‘LE’) in hydroponic solution. Based on the preliminary screening results (see results in Table S1), four exogenous plant hormones were selected for the induction dose experiments, including ethephon (ETH) (0, 0.17, 0.34, 0.68, 1.36 µmol/L), abscisic acid (ABA) (0, 1, 3, 5, 7 µmol/L), brassinolide (BR) (0, 0.01, 0.03, 0.05, 0.07 nmol/L), and 24-epibrassinolide (EBL) (0, 0.1, 0.3, 0.5, 0.7 nmol/L). All compounds were first dissolved in a small volume of ethanol to prepare a high-concentration solution according to the needs of the experiment, and then diluted with Hoagland’s solution into the above different final concentrations which were used for rice cultivation. Seven days post-treatment, the rice leaves were harvested, sectioned into two cm segments, and immersed in distilled water (8% w/v) for 24 h in an incubator set at 25 °C. The extracts were filtered with a sterile 0.22 µm membrane filter for the subsequent bioassay, which can estimate the induced- allelopathy of two rice cultivars.

Experiment 2 for the effect of treatment duration was similar to the first experiment described above. The dosages were set as ABA (three and five µmol/L) and EBL (0.3 and 0.5 nmol/L), which was based on the results of Experiment 1 above (see results in Figs. S1, S2). The treatment duration was set at 1, 3, 5, and 7 days. The preparation of the rice leaf extracts and their bioassays were conducted as previously described in Experiment 1.

The results of two experiments above determined which induction dosage (three µmol/L ABA, 0.5 nmol/L EBL) and treatment duration (3 days) of two appropriate phytohormones (ABA or EBL) were selected for further validation. Hoagland’s solution without inducing solution was used as control. The preparation of the rice leaf extracts for the subsequent bioassay were conducted as previously described in Experiment 1. In addition, rotary evaporation at 40 °C ± 1 °C was used to collect and concentrate the culture solution of each treatment to 200 mL. The concentrated culture solutions were refrigerated at 4 °C for 24 h, then filtered through a 0.22 µm filter and diluted to a volume of 200 mL, which served as the concentrated root fluids for subsequent laboratory bioassays.

Bioassay of the induced-allelopathy of two rice cultivars

The alteration in rice-induced allelopathy was assessed by quantifying the inhibiting effects of rice extracts from leaves or rice root exudates on the root length of lettuce (Lactuca sativa L.) in a laboratory bioassay, as previously detailed (Zhang et al., 2019). Briefly, five mL of the rice leaf extracts or concentrated root exudates obtained from the induction experiments above were introduced into tissue culture flasks that were lined with filter paper at the bottom. Hoagland’s solution was serving as a control in this experiment. Five sprouted lettuce seedlings were sowed on filter paper and subsequently positioned in a Petri dish, which was subsequently set in an incubator sustained at 25 ± 2 °C, with a daily light cycle of 12 h from 6:00 to 18:00. Each experimental trial was conducted five times, and the root length of the lettuce was assessed within three days.

The inhibiting ratio (%) was employed to evaluate the impact of the experimental solutions compared to the control, calculated using the following formula: inhibition rate (%) = (1−treatment/control) ×100%. The inhibition rate of rice accessions on lettuce with and without induction was defined as total allelopathy (TA) and genetic allelopathy (GA), respectively, and the increment of inhibition rate was defined as induced-allelopathy (IA), wherein IA = TA–GA (Zhang et al., 2018).

Fine-root trait calculation of two rice cultivars

To investigate the impact of exogenous plant-produced hormones on the root morphology of Rice PI with allelopathic effects and rice LE without allelopathic effects, the optimum induction conditions (that is, three µmol/L ABA or 0.5 nmol/L EBL, and 3 days of treatment) were used in a hydroponic experiment. Hoagland’s solution without inducing solution was used as a control. After the seedlings were harvested, the pristine and undamaged roots were scanned using an Epson Expression LA2400 scanner (Seiko Epson Co., Nagano-ken, Japan) to produce a grayscale image. The image was analyzed using WinRHIZO software (Regent Instruments Inc., Quebec, Canada) to extract morphological characteristics following the methodology of Li et al. (2019). Rice roots were investigated in three diameter ranges (0–0.2 mm, 0.2–1.0 mm, and 1.0–2.0 mm) for the evaluation of three fine-root morphology traits (root length, root surface area, and root volume). The number of root tips for the entire root was also obtained. The root biomass was obtained by oven-drying the roots at 105 °C for 30 min and 80 °C for 48 h.

Analysis of phenolic acids and terpenoid contents

The quantification of phenolic acids in rice leaves and root exudates was conducted utilizing a combination of liquid extraction and solid-phase extraction, subsequently analyzed through high-performance liquid chromatography (SPE-HPLC). After three days of treatment with three µmol/L ABA or 0.5 nmol/L EBL, the leaf extracts or concentrated root exudates were extractedusing Cleanert PEP solid phase extraction cartridges (Agela Technologies, Tianjin, China). Methanol was added after the cartridge was diluted with water, and then concentrated with N2 before being diluted in 500 µL of methanol for quantitative analysis by HPLC. The quantitative analysis of seven phenolic allelochemicals, namely, protocatechuic acid, 4-hydroxybenzoic acid, syringic acid, vanillic acid, salicylic acid, ferulic acid, and cinnamic acid, was undertaken as previously outlined (Li et al., 2022). The phenolic acid contents in rice leaves were quantified as the number of micrograms per gram of fresh leaves. The concentration of phenolic acids in the root exudates was quantified in micrograms per liter of the rice culture solution.

After three days of treatment with three µmol/L ABA or 0.5 nmol/L EBL, the rice leaf extracts were sampled. The contents of terpenoids in rice leaves were determined by microwave-assisted extraction and semi-quantitative analysis by GC-MS, depending on the method reported by Li et al. (2020). The concentrations of terpenoids were determined by multiplying the area of the measured terpenoid peaks by the quantity of the internal standard (n-dodecane), and then this product was divided by the product of the measured peak area of the internal standard and the weight of the soil sample.

Quantitative real-time PCR

Based on the RNA-seq results of Zhang et al. (2019), we selected ten key genes involved in the phenolic acid (PAL, C4H, F5H and COMT) and terpenoid (HMGR, MK, MTS, STS, PS and SQS) biosynthesis pathways for quantitative real-time PCR (qPCR) analysis. Rice roots and the second internode leaf were sampled at 3 d after treatment with three µmol/L ABA or 0.5 nmol/L EBL as described above. The relative expression of each target gene was assessed using the 2^−ΔΔCt method (Zhang et al., 2018) and a bar chart was drawn representing the expression level values of 10 key genes induced by ABA or EBL compared with the control of the same rice cultivar. All primers required to perform qRT-PCR are given in Table S2.

Statistical analysis

Prior to statistical analysis, homogeneity of variance was confirmed by residual plots, and normality was verified using Q–Q plots and Shapiro–Wilk’s normality test. The analysis of the significance of differences among various treatments applied to the same rice cultivars was conducted using one-way ANOVA utilizing SPSS (Version 20; SPSS Inc., Chicago, IL, USA), followed by Tukey’s honestly significant difference test at P < 0.05. The correlation analysis was carried out by the “Corrplot” package in R (version 0.93; https://github.com/taiyun/corrplot).

Induced-allelopathy of two rice cultivars treated with appropriate phytohormones under the optimum induction conditions

Preliminary screening results of 10 plant hormones with three concentrations were presented in Table S1. Compared with the other plant hormones, low concentrations of four exogenous plant hormones (ETH, ABA, BR and EBL) were selected more suitable for the growth induction of the roots of allelopathic rice PI and non-allelopathic rice LE. Further screening Experiment 1 and Experiment 2 were run to obtain the optimum induction phytohormones, evaluating the changes of the induced-allelopathy of PI and LE under induction treatment of ETH, ABA, BR and EBL at different concentrations. Compared with the inhibition rate of rice accessions without induction (defined as genetic allelopathy, GA), the inhibition rate of two rice accessions with induction of ETH, ABA, BR and EBL (defined as total allelopathy, TA) were increased with different concentrations in both PI and LE (Fig. S1). Based on the results of the induced-allelopathy (IA, IA = TA–GA) of four hormones between the two rice cultivars in Fig. S2, we will select three, five µmol/L ABA and 0.3, 0.5 nmol/L EBL for induction with different time gradients to analyze the time-effect of induced-allelopathy in the two rice cultivars.

As shown in Figs. 1A and 1B, regardless of ABA or EBL induction, the induced-allelopathy (IA) of non-allelopathic rice LE was greater than that of allelopathic rice. Although the peaks of IA of the two rice varieties after treatment with the same hormone appeared at different treatment times, it is more practical to improve the allelopathy of non-allelopathy rice. Therefore, we focused on the increment of IA in non-allelopathy rice. We considered that a treatment time of 3d to be the appropriate induction time for both rice cultivars, and the optimal plant hormones were three µmol/L ABA (Fig. 1A) and 0.5 nmol/L EBL (Fig. 1B). Under these induction conditions, the inhibitory impact of leaf extracts and exudates of roots from varieties of rice was markedly enhanced. After treatment with three µmol/L ABA, the total allelopathy (TA) values of rice root exudates were 39.82% in PI, and 32.17% in LE. The induced-allelopathy (IA) values (9.62%) accounted for 23.9% of TA values in PI, while IA values (13.76%) in LE accounted for 43.1% of TA values (Fig. 1C). As shown in Fig. 1D, treatment with 0.5 nmol/L EBL also substantially enhanced the inhibitory effect of the two rice cultivars. The TA values in PI and LE induced by 0.5 nmol/L EBL were 37.42% and 31.33%, respectively. The IA values induced by EBL were 7.83% in PI which accounted for 20.4% of TA values, whereas the IA values in LE were 11.51% that accounted for 36.7% of TA values (Fig. 1D). The trends in the IA and TA values of the two rice leaf extracts were in agreement with the results of their root exudates. Obviously, the extent of the increase in the induced-allelopathy (IA/TA) of non-allelopathic rice was markedly greater than that in allelopathic rice.

Fine-root traits in allelopathic and non-allelopathic rice cultivars treated with two appropriate phytohormones

As shown in Figs. 2A–2D, ABA treatment increased the root length, root surface area and root volume in the 0–0.2 mm diameter range of PI root by 1.49, 1.85 and 1.40-fold, respectively. LE treated with ABA showed a maximal enhancement in root surface area within the 0.2–1.0 mm diameter range. After EBL treatment, the root length, root surface area, and root volume of PI increased exhibited the most substantial increases at a diameter range of 0–0.2 mm, which were 1.95 times, 2.49 times, and 1.85 times that of the control, respectively. The root length, root volume, and root surface area of LE increased most in the diameter range of 1.0–2.0 mm, which were 1.56, 1.73 and 1.57 times greater than those of the control, respectively, after EBL treatment. Regardless of ABA or EBL induction, The quantity of tips of root and the dry weight of roots in two rice cultivars exhibited a considerable increase (Figs. 2E–2F).

Appropriate phytohormones induced the production of rice allelochemicals

The production of secondary metabolites by rice during the seedling stage is the main mechanism for inhibiting weed growth. Based on previous reports (Li et al., 2020), this study examined the impact of exogenous hormones on the levels of phenolic acids and terpenoids. Eight phenolic acids (protocatechuic, p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic, salicylic, and cinnamic acid) are identified as allelochemicals in rice (Li et al., 2019). When the seedlings of the two rice cultivars were exposed to ABA or EBL under the optimum induction conditions, the contents of some phenolic acids increased in both rice leaf- extracts and root-exudates, while some decreased compared with the untreated control (Figs. 3A–3B). However, upon aggregating the concentrations of eight phenolic acids, it was observed that the cumulative levels of the selected phenolic allelochemicals in the hormone-treated groups were significantly higher in both PI and LE compared to the control group, regardless of the levels of total phenolic acids in rice leaves and roots (Figs. 3A–3B). In particular, the total contents of eight phenolic acids present in the root-exudates of PI and LE induced by EBL were 1.16 and 2.33 times greater, respectively , relative to the untreated control (Fig. 3B). At the same time, ABA treatment also elevated the concentration of phenolic acids secreted by PI and LE roots by 1.48 and 1.87 times, respectively.

GC−MS analysis results identified approximately 30 terpenoids in rice leaves, mainly including monoterpenoids, sesquiterpenoids and diterpenoids. The contents of each group were summarized and analyzed. As shown in Fig. 3C, the terpenoids in the two rice leaves of the control and treatment groups were mainly monoterpenes and sesquiterpenoids, and The levels of these two categories in the hormone-treated group were significantly higher compared to the control group, regardless of PI and LE. In addition, the contents of oxygenated terpenoids in the rice leaves were further compared. Two hormones increased the ratio of oxygenated terpenoids, especially the ratio of the contents of oxygenated sesquiterpenes in total sesquiterpenes. ABA treatment increased the ratio of the contents of oxygenated sesquiterpenes in total sesquiterpenes from 7.41% to 21.92% in PI, and from 6.67% to 10.7% in LE; After EBL induction, the ratio of oxygen-containing sesquiterpenes in PI was increased to 15.23%, while the ratio in LE was increased to 16.24%.

Expression patterns of key genes involved in the biosynthesis of rice allelochemicals

When two rice cultivars were incubated with ABA or EBL, ten genes involved in the biosynthesis of phenolic acids (PAL, C4H, F5H and COMT) and terpenoids (HMGR, MK, MTS, STS, PS and SQS) were expressed in variety-dependent manners (Figs. 3D–3E). For four genes from the phenolic acid pathway, ABA treatment significantly induced the expression of F5H in the roots and PAL in the leaves of both PI and LE, while expression of phenolic acid biosynthesis genes PAL, C4H, and COMT in rice leaves of LE was significantly up-regulated by EBL, and PAL induction also occurred in rice roots of PI. In particular, the expression of these four genes was much stronger in both tissues of non-allelopathic rice LE than allelopathic rice PI, regardless of ABA or EBL application. Furthermore, ABA and EBL also regulated gene expression levels of terpenoids biosynthesis genes in rice tissues of both PI and LE. EBL strongly induced (more than twofold changes) HMGR and SQS in LE leaves, the expression of MK and SQS in both tissues of PI were also increased after EBL treatment. However, ABA decreased the all of the biosynthesis genes in the leaves of both the allelopathic and the non-allelopathic variety. Compared with the untreated control group, the expression of PS in rice roots of PI was strongly increased (more than twofold changes) and were less strongly induced in LE roots.

Correlation between root morphological traits and induced-allelopathic potential in rice after hormone induction

According to the experimental results in Fig. 2, root length and root volume with diameters ranging from 0–0.2 mm and 0.2–1.0 mm were selected to represent root morphological indexes. At the same time, the induced-allelopathy (IA) of rice root exudates (Figs. 1B and 3C), the contents of allelochemicals (phenolic acids and terpenoids) (Figs. 3B and 3C) and the expression level of genes related to allelochemical synthesis (Figs. 3D and 3E) represented the allelopathy potential of rice, and the correlation between two hormones’ induced allelopathic potential and root morphological features, was investigated.

The correlation results showed that the induced-allelopathy (IA) after EBL application (Fig. 4A) was significantly positively correlated with the root volume with a diameter of 0.2–1.0 mm, phenolic acid contents in the root-exudates, the expression levels of phenolic acid synthesis genes (C4H,COMT) and terpenoid synthesis genes (MK, STS, PS and SQS). Under ABA treatment, IA was also positively correlated with root length with a diameter of 0.2–1.0 mm, phenolic acid contents in the root-exudates and terpenoid contents in rice leaves (Fig. 4B). However, IA was significantly negatively correlated with the expression levels of the phenolic acid synthesis genes (C4H, F5H, COMT), the terpenoid synthesis genes (MK, MTS, PS, STS) (P  <  0.01), and the MK and expression levels (P  <  0.05).