Identification, characterization, and expression of Oryza sativa tryptophan decarboxylase genes associated with fluroxypyr‐meptyl metabolism

Abstract

Tryptophan decarboxylase (TDC) belongs to a family of aromatic amino acid decarboxylases and catalyzes the conversion of tryptophan to tryptamine. It is the enzyme involved in the first step of melatonin (MT) biosynthesis and mediates several key functions in abiotic stress tolerance. In Oryza sativa under pesticide‐induced stress, TDC function is unclear. Three TDC differentially expressed genes (DEGs) and six TDC‐coding genes were found to be expressed in fluroxypyr‐meptyl (FLUME)‐treated rice transcriptome datasets, which allowed researchers to explore the properties and roles of rice TDC family genes under pesticide‐induced stress. By applying sequence alignment and phylogenetic analysis, two subfamilies of the TDC gene family—DUF674 and AAT_I—were found in rice, Glycine max, Zea mays, Hordeum vulgare, and Solanum lycopersicum. According to chromosomal location studies, segmental duplication aided in the expansion of the OsTDC gene family, and the three TDC DEGs in rice were irregularly distributed on two of its 12 chromosomes. In addition, nine rice TDC genes displayed a collinear relationship with those of soybean, maize, barley, and tomato. Rice TDC genes can encode a variety of biotic and abiotic stress responses because of their diverse gene architectures, cis‐elements, motif compositions, and conserved domains. Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analysis confirmed that a proportion of TDC genes (Os08g0140300, Os08g0140500, and Os10g0380800) were preferably expressed under 0.08 mg L⁻¹ FLUME stress, with a 5.2‐, 3.2‐, and 3.9‐fold increase in roots and a 2.1‐, 2.4‐, and 2.6‐fold increase in shoots, respectively. MT treatment further increased the expression of these genes, with a 2.1‐fold, 3.1‐fold, and fivefold increase in roots and a 1.5‐, 1.1‐, and 1.1‐fold increase in shoots than that treated with 0.08 mg L⁻¹ FLUME only, respectively. When rice seedling roots and shoots were subjected to 0.08 mg L⁻¹ FLUME stress, TDC activity was increased by 2.7 and 1.6 times higher than in the control, respectively. MT application also further promoted TDC activity in rice tissues; TDC activity in rice roots and shoots was twofold and 1.4‐fold higher, respectively, than that under 0.08 mg L⁻¹ FLUME alone. These findings indicate that TDC genes respond effectively to FLUME stress, and the application of MT could enhance the expression of these TDC genes, which comprise a set of candidate genes that regulate pesticide metabolism and degradation with the application of MT.

Abbreviations

• ABRE
• ABA-responsive cis-acting elements
• DEG
• differentially expressed gene
• GST
• glutathione S-transferase
• HPLC
• high-performance liquid chromatography
• MeJA
• methyl jasmonate
• MW
• molecular weight

BACKGROUND

Pesticides are crucial to agricultural development because they can increase the quantity and quality of food yield and lower the loss of agricultural products (Yu et al., 2023). However, the acceleration of biodiversity loss, largely caused by the excessive use of chemicals, remains one of the biggest challenges to the functions and services of natural and agricultural ecosystems worldwide (Ruuskanen et al., 2023). The extensive use of agrochemicals, such as herbicides, is a major contributor to contamination, exposing humans, animals, and nontarget plants.

Among living organisms, plants are most frequently exposed to the ever-changing environment due to their sessile growth. Upon harsh environmental conditions such as extreme drought, salinity, heavy metals, and pesticides, plants implement various mechanisms to cope with environmental changes and adversities (Ahmad et al., 2013). Mechanisms to avoid pesticide toxicity include the synthesis of a series of secondary metabolites (amino acids, phenols, and polyamines) and plant hormones (salicylic acid, brassinolide, and jasmonic acid). Their synthesis often enhances pesticide stress resistance (Chen et al., 2022a; Godoy et al., 2021; Waadt et al., 2022). For example, the increase in salicylic acid content in crops can enhance the activity of antioxidant and detoxifying enzymes such as that of CAT, POD, P450, and glutathione S-transferase (GST), thereby promoting the degradation and metabolism of isoproturon and propazine (Lu et al., 2020; Zhang et al., 2018). The increase in jasmonic acid content in crops also promotes CAT, POD, APX, and GST activity and thus reduces isoproturon toxicity (Ma et al., 2018).

Previously, it was believed that the pineal glands of animals are the primary source of melatonin (MT) (N-acetyl-5-methoxytryptamine) (Zhou et al., 2020). Numerous recent researches have shown that MT is produced by all living beings, including plants. Parts of plants that contain MT include leaves, stems, roots, fruits, and seeds. The structural characteristics of MT are similar to those of indoleacetic acid (Arnao & Hernandez-Ruiz, 2015). Additionally, many studies on the physiological advantages and functions of MT in plants, which may serve as a defensive mechanism against a range of abiotic stressors, have been conducted (Kaur et al., 2015; Liu et al., 2021; Zhou et al., 2020). An increase in MT in transgenic Oryza sativa or through exogenous MT application could regulate seed germination and root elongation under abiotic stress factors such as cold and osmotic stress (Zhou et al., 2020). Furthermore, some studies have reported that an increase in MT content can also alleviate crop pesticide toxicity by increasing monodehydroascorbate reductase, dehydroascorbic acid reductase, glutathione reductase, and GST activity (Liu et al., 2021). However, the effects of exogenous MT application on the alleviation of pesticide phytotoxicity and residues in plants remain largely unknown. In plants, key genes involved in MT biosynthesis include tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), and caffeic acid O-methyltransferase (COMT) (Zhou et al., 2020). Yan et al. (2019) revealed that MT synthase gene (COMT1) overexpression could alleviate toxicity and reduce the accumulation of carbendazim in Solanum lycopersicum. However, the involvement of TDC in pesticide metabolism and detoxification in plants must be elucidated in future studies.

TDC is an aromatic L-amino-acid decarboxylase (AADC), evolutionarily classified as a type II pyridoxal-5′-phosphate-dependent decarboxylase (PLP_DC), which catalyzes tryptophan to tryptamine conversion and is the first enzyme in MT biosynthesis (Zhou et al., 2020). Many TDC genes from a range of plant species have been isolated, and their expression patterns demonstrate that they are closely related to MT biosynthesis (Zhao et al., 2019). TDC gene overexpression in plants leads to increased tryptamine and serotonin levels, which ameliorate H₂O₂ and O₂⁻ accumulation, decrease cell membrane damage under multiple abiotic stresses (Li et al., 2016; Zhao et al., 2019; Zhou et al., 2020). Therefore, we hypothesized that TDC can also alleviate pesticide stress in plants by increasing MT synthesis.

The organic heterocyclic herbicide fluroxypyr-meptyl (FLUME) exhibits internal absorption and transmission properties. In wheat, Hordeum vulgare, Zea mays, and rice fields, this pesticide is frequently used to suppress broad-leaved weeds because it is inexpensive and effective (Hu et al., 2014). However, serious environmental issues arise as a result of the widespread use of FLUME in agricultural output. It is common to find FLUME, especially its hydrolysate fluroxypyr, in soil, lakes, and groundwater (Hu et al., 2014; Wu et al., 2009). Crop growth can be impeded by FLUME and fluroxypyr residues in the soil, particularly in root systems, which can be absorbed by crops and eventually reach edible crop sections (Wu et al., 2009). Furthermore, a number of studies have shown that FLUME and its hydrolysate fluroxypyr can significantly influence earthworm activity (Velki et al., 2019) and damage rat, mouse, and dog renal function (Wang et al., 2011; Zhong et al., 2016). Furthermore, a fatal instance of poisoning occurred in humans who consumed a combination of FLUME and chlorpromazine (Jang et al., 2021). Therefore, consumption of crops or water sources polluted with FLUME may pose a risk to human health. To reduce the risk of pesticide accumulation to human health, it is necessary to delve deeper into how the MT synthase genes respond to and participate in the MT and degradation of pesticides in plants.

Currently, the response of rice TDC genes to FLUME stress and the involvement of TDC genes in pesticide metabolism in rice plants require further investigation. Thus, we screened genome-wide TDC gene transcripts responsive to FLUME in the presence or absence of MT to examine the potential molecular and biochemical roles of TDCs. The chromosomal placements, collinearity, structures, cis-elements, motif compositions, and conserved domains of these genes were also thoroughly examined. The responses of these TDC genes to FLUME with or without MT were verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We further analyzed the overall TDC activity levels in rice plants exposed to FLUME with or without MT. Together, we have created a practical screening method for rice TDC genes that are susceptible to FLUME + MT, which may alter toxicological responses and FLUME resistance in settings and crops. In addition, we provided data supporting the regulation of residual pesticide degradation and metabolism by MT synthesis genes in crops.

Core Ideas

• Identified nine tryptophan decarboxylase (TDC) expression genes in rice under fluroxypyr-meptyl (FLUME) stress with melatonin (MT) treatment.
• The nine rice TDC genes display a collinearity relationship with those of soybeans, maize, barley, and tomato.
• The cis-elements function of nine rice TDC genes is mainly involved in responding to abiotic stress.
• The expression level of rice TDC genes and protein activity were obviously enhanced under FLUME stress with MT t.
• Molecular docking analysis revealed that TDC proteins had good binding ability with FLUME.

METHODS

Growth condition and treatment of plants

Wild-type rice (O. sativa, Japonica) seeds were sterilized with 3% H₂O₂, rinsed thoroughly with distilled water, and germinated in an incubator in the dark at 30°C for 48 h. Next, germinated seeds were cultured in 50% strength Hoagland nutrient solution (pH = 5.8) for 8 days in growth chambers (14 h photoperiod at 200 µmol m⁻² s⁻¹ light intensity, 75% relative humidity, and 30°C/25°C during the day/night) (Chen et al., 2024a). After 2 days, uniform seedlings (20 seedlings for each pot) were transferred to the fresh nutrient solution containing FLUME at 0.04, 0.06, 0.08, 0.1, and 0.12 mg L⁻¹ with or without MT (200 mg L⁻¹) and allowed to grow for 6 days for total TDC activity determination and qRT-PCR. The nutrient solution was changed every 2 days. Each treatment was repeated three times.

Building transcriptome libraries and using high-throughput RNA sequencing

Samples of rice shoots and roots were collected at 2, 4, and 6 days after treatment with 0.08 mg L⁻¹ FLUME with or without 200 mg L⁻¹ MT. Root and shoot samples from the three time points were pooled for quantitative analysis. Total RNA contents from each sample were isolated using Trizol (Invitrogen) for RNA sequencing (RNA-seq), and DNA was removed using DNaseI (Takara). Six libraries were constructed, including Shoot-FLUME (control), Root-FLUME (control), Shoot + FLUME (with FLUME), Root + FLUME (with FLUME), Root + FLUME + MT (with FLUME and MT), and Shoot + FLUME + MT (with FLUME and MT), and sequenced using Illumina HiSeq 2500 with three biological replicates for each treatment. After deleting low-quality bases, the clean bases and reads were mapped to the rice genome () (Chen et al., 2024b).

TDC activity assay

The activity of TDC was determined according to Kang et al. (2007) with some minor modifications. Following a 6-day exposure period to 0–0.12 mg L⁻¹ FLUME with or without 200 mg L⁻¹ MT, 0.5 g of fresh rice samples were frozen and ground with liquid nitrogen and mixed with 2 mL of extraction solution (100 mM Tris-HCl buffer [pH 8.0, containing 1 mM EDTA, 1 mM DTT, 2% PVPP]). Samples were then centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was used to determine the total TDC activity in 1 mol L⁻¹ Tris-HCl buffer (pH 8.0) solution with 10 mmol L⁻¹ L-tryptophan, 4 mmol L⁻¹ PLP, and 30 mmol L⁻¹ DTT. Finally, after finishing the reaction, the solution was filtered through a 0.22 µm syringe membrane. Tryptamine contents in rice tissues were detected by high-performance liquid chromatography (HPLC) using an XDB-C18 column (250 × 4.6 mm, 5-Micron, Agilent) and an HPLC UV detector (Waters) at a wavelength of 280 nm. The flow rate was 1 mL min⁻¹ with the mobile phase containing acetonitrile and 0.05% phosphoric acid water (70:30 v:v), and the injection volume was 20 µL. Enzyme activity was characterized according to the amount of tryptamine produced per unit time, expressed in µmol g (fresh weight [FW]) h. Standard curves were used to determine the tryptamine concentration in rice tissues. The linear equation of the calibration curve was y = 19,366x – 20,991 (R² = 0.9998).

Quantitative RT-PCR analysis of TDC genes in rice tissues

After applying 0, 0.04, 0.06, 0.08, 0.1, and 0.12 mg L⁻¹ FLUME with or without 200 mg L⁻¹ MT to 10-day-old rice seedlings, rice root and shoot samples were obtained at 2, 4, and 6 days, and samples from different time points were combined for quantitative analysis. The methodology outlined by Chen et al. (2024b) was used to extract comprehensive RNA from rice tissue. After crushing rice shoots and roots into a powder in liquid nitrogen and extracting the material with 1 mL of Trizol, the mixture was incubated for 5 min and centrifuged at 12,000 × g and 4°C for 10 min. Note that 800 µL of the supernatant was then centrifuged for 10 min in a 1.5 mL centrifuge tube containing 400 µL of chloroform. After mixing and incubating for more than 30 min at −20°C, 400 µL of the supernatant was mixed with an equivalent volume of isopropyl alcohol.

Excess supernatant was discarded, samples were centrifuged again for 10 min, and the precipitate was repeatedly washed with a 75% ethanol solution before being dried almost completely under sterile airflow. After autoclaving treatment, a suitable volume of 0.1% DEPC water was dissolved and precipitated. An ultra-microspectrophotometer (NanoPhotometer N60) was used to detect the quality of the sample. A 2 × Q3 SYBR qPCR Master Mix (Universal) kit (Tolo Biotechnological) was used to process the collected RNA. The reaction was started with the primers listed in Table S1, and it was run for 40 cycles with one round of denaturation at 95°C for 30 s and 40 cycles of annealing (95°C for 5 s) and extension (60°C for 30 s). To determine the expression of the three TDC differentially expressed genes (DEGs) in rice, a Light Cycler 96 Real-Time PCR System (Roche) was used. The method of 2^−ΔΔCt was employed to measure relative gene expression.

Chromosome distribution and interspecific collinearity of rice TDC genes

We downloaded the genome annotation files for rice, Glycine max, maize, barley, and tomato from EnsemblPlants () (Bolser et al., 2016). The China Rice Data Center () provided nine rice TDC protein and nucleic acid sequences for download. The distribution of the nine rice TDC genes on the chromosomes was visualized using the TBtools-TM program () (Chen et al., 2020). The collinearity of TDC genes between rice and soybean, maize, barley, and tomato was also analyzed and visualized using TBtools-II software (Chen, et al., 2024b).

Phylogenetic tree analysis of rice TDC genes

Nine rice TDC gene sequences were obtained () and used as query sequences using the Rice Genome Annotation Project. A sequence similarity threshold of no less than 40% was employed to search for protein sequences in soybean, maize, barley, and tomato that were comparable to rice TDC gene sequences in the database using the BLAST website (). To create phylogenetic trees and understand evolutionary links among species, the maximum likelihood approach was employed using ultrafast bootstrap approximation with 1000 and SH-aLRT test set to 1000 random addition replicates in IQ-TREE software (Cao et al., 2023). The phylogenetic tree was visualized with online iTol () and FigTree software.

Analysis of rice TDC gene domain structure and cis-acting promoter elements

TBtools-II was used to extract the first 2000-bp nucleic acid sequence of the three rice TDC genes, and PlantCARE () was used to predict cis-acting elements (Lescot et al., 2002). Conserved motifs were analyzed using multiple expectation maximizations for motif elicitation (), setting the number of motifs to six (Bailey et al., 2006). To determine the domain structure of each of the nine rice TDC genes, we used the NCBI CD-search Tool () (Lu et al., 2020). Results were exported using TBtools-II.

Protein–protein interactions between rice TDC family genes analyzed by network analysis

In order to examine protein–protein interactions using STRING (), we searched for O. sativa TDC (OsTDC) gene analogs in Arabidopsis () based on the rice genome (). The protein–protein interaction network was visualized using Cytoscape () (Chen et al., 2024b).

Protein structure and molecular docking analysis

We obtained secondary structure predictions using P, including the number of amino acids, molecular weight (MW), theoretical isoelectric point (PI), instability index, and grand average of hydropathicity value () (Duvaud et al., 2021). SWISS-MODEL () was used to predict the tertiary structure of nine TDC proteins (Waterhouse et al., 2018). For molecular docking, we selected three FLUME-induced rice TDC genes and constructed their corresponding proteins by eliminating H₂O molecules and heteroatoms. The 3D structure file of FLUME was obtained from PubChem (), and the format was converted using Open Babel (O'Boyle et al., 2011). The docking model cutoff was a binding affinity of −7 kcal mol⁻¹, and molecular docking was conducted using AutoDock Vina 1.2.5, setting the exhaustiveness to 24. In the docking results, we selected a low binding energy and good conformation. The interaction between residues and ligands was visualized using Pymol 2.6.0 Open-Source, and 2D images of the interaction were generated using Discovery Studio 2019 (Chen et al., 2024b).

Statistical analysis

For every experiment, three independent replicates (n = 3) were used to compile all of the study's results. Statistical significance of the data was assessed using standard deviation and analysis of variance (ANOVA, p < 0.05). All analyses were performed using SPSS 19.0 software (IBM).

RESULTS

Effect of exogenous MT on TDC gene expression in rice tissues under FLUME stress

To identify the FLUME + MT-responsive TDC genes in rice, we performed RNA-seq using the following criteria: false discovery rate (FDR) < 0.001 and log₂fold change (FC) > 1. Based on a two-by-two comparison of Root + FLUME versus Root + FLUME + MT and Shoot + FLUME versus Shoot + FLUME + MT, nine TDC genes that responded to FLUME + MT treatment were identified (Table S2). Genes tend to perform a specific function based on their expression in an organism, and their expression patterns often reflect their functions (Zhou et al., 2018). To explore the function of OsTDC genes, we analyzed their expression levels in different tissues under different treatment conditions (different exposure concentrations [0–0.12 mg L⁻¹]) with or without 200 mg L⁻¹ MT (Figure 1). OsTDC gene expression in all tissues under different treatment conditions was different to that in control, and expression levels varied across tissues and treatment conditions. Compared with control, there were three TDC genes with significantly upregulated expression (Os08g0140300, Os08g0140500, and Os10g0380800) with a 5.2-, 3.2-, and 3.9-fold increase in roots and a 2.1-, 2.4-, and 2.6-fold increase in shoots after 6 days of 0.08 mg L⁻¹ FLUME stress, respectively. MT treatment further increased the expression of these genes. In comparison with the addition of 0.08 mg L⁻¹ FLUME alone, the expression of Os08g0140300 in shoots increased by 1.5-fold under MT + 0.08 mg L⁻¹ FLUME treatment, whereas there was no significant difference in the expression of Os08g0140500 and Os10g0380800. In roots, Os08g0140300, Os08g0140500, and Os10g0380800 expression under MT + 0.08 mg L⁻¹ FLUME was significantly higher (by 2.1-, 3.1-, and 5-fold, respectively) than that treated with 0.08 mg L⁻¹ FLUME only. These results suggest that MT treatment is associated with increased TDC gene expression, which may be related to secondary metabolism and xenobiotic detoxification (Samanta et al., 2021).

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Exogenous MT application enhances TDC protein activity in rice tissues under FLUME stress

TDCs serve a variety of purposes and are essential for plants’ ability to resist xenobiotic stress (Li et al., 2016; Yan et al., 2019; Zhou et al., 2020). To analyze the role of TDCs under FLUME stress, total TDC activity was measured in different rice tissues subjected to different treatment conditions (different exposure concentrations of 0–0.12 mg L⁻¹ FLUME and with or without 200 mg L⁻¹ MT) for 6 days (Figure 2). OsTDC activity was enhanced in all tissues under treatment compared to control, varying across tissues and treatment conditions. Compared to control, TDC activity in rice roots and shoots treated with FLUME for 6 days was highest for the 0.08 mg L⁻¹ treatment, with values increasing by 1.4- and 2.7-fold, respectively (Figure 2). Similar to OsTDC gene expression, MT application also further promoted TDC activity in rice tissues. TDC activity in rice roots and shoots reached a maximum value after 6 days of MT + 0.08 mg L⁻¹ FLUME treatment, and it was twofold and 1.4-fold higher, respectively, than that under 0.08 mg L⁻¹ FLUME alone. These results revealed that TDC activity in rice tissues can be further enhanced by exogenous MT, particularly in roots, which supports the involvement of TDC in xenobiotic stress responses.

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Analysis of the synteny and chromosomal distribution of rice TDC genes

We used TBtools to construct chromosome location maps and determine the overall distribution of OsTDCs in the rice genome. The nine TDC genes were unevenly distributed among the 12 chromosomes of the rice genome (Figure S1). The number of genes on each chromosome ranged from 0 to 2, whereas three genes were located on individual chromosomes (Chr 1, 4, and 7) as solitary genes. Several other genes were clustered together on the same chromosomes (Chr 5, 8, and 10), indicating variations in the functions of different rice TDC genes. The primary mechanism for the synthesis of new genes is gene duplication, which is also essential for the development and growth of plant gene families (Hou & Cao, 2016). To further understand the evolutionary mechanisms and homologous genes of these nine OsTDCs, an intraspecific and interspecies collinearity map was constructed in rice and among rice, dicotyledons (soybean and tomato), and monocotyledons (barley and maize). In this study, we identified one duplicate gene pair on two chromosomes (Figure 3A; Table S3). A total of one of nine OsTDC genes showed collinearity with soybean (Figure 3B; Table S4), two with tomato (Figure 3C; Table S4), four with barley (Figure 3D; Table S4), and four with maize (Figure 3E; Table S4), suggesting that rice TDCs are phylogenetically more closely related to monocotyledonous plants.

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Phylogenetic investigation of the TDC gene family

To assess the evolutionary relationships among the nine rice TDC genes, we constructed an intraspecific phylogenetic tree using their amino acid sequences. To further investigate the relationships among rice, soybean, maize, barley, and tomato genes with >40% similarity, we created an interspecific phylogenetic tree based on the amino acid sequences of nine TDC genes using MEGA 5.0 and the NJ technique. In both intra- and interspecies relationships, two branches were found in the phylogeny based on the degree of kinship. TDC domain counts showed that the AAT_I and DUF674 subfamilies were represented by branches I and II of the intraspecific rice phylogenetic tree, respectively (Figure 4A). Interspecific phylogenetic analyses revealed that group I included seven rice, four soybean, five tomato, 18 barley, and eight maize genes and Group II included two rice, one soybean, nine barley, and seven maize genes (Figure 4B; Table S5). The genes in the same group were homologous. Phylogenetic tree analysis showed that rice, soybean, tomato, barley, and maize all exhibited intra- and interspecific similarity, despite the variation in gene count along the branches. This suggested that there is a preserved evolutionary link among the TDC families of maize, barley, tomato, rice, and soybean.

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Cis-acting element analysis of OsTDC promoters

Cis-acting promoter elements, which are important molecular switches involved in the transcriptional regulation of genes under biotic and abiotic stress, can be induced through independent signal transduction pathways (Chen et al., 2024b). To understand the underlying function of TDC genes, the 2000-bp upstream promoter regions of OsTDCs were submitted to PlantCARE for cis-element identification. These nine TDC genes contained light-responsive cis-acting elements (MRE, GATA-motif, ACE, AE-box, chs-CMA1a, G-Box, GT1-motif, I-box, Sp1, TCCC-motif, and TCT-motif); stress-responsive cis-acting elements (ARE, GC-motif, MBS, and TC-rich repeats); hormone-responsive cis-elements, including ABA-responsive cis-acting elements (ABRE), salicylic acid (TCA-element), methyl jasmonate (MeJA)-related elements (CGTCA-motif, TGACG-motif), and gibberellin-responsive elements (GARE-motif, P-box, TATC-box); cis-acting elements associated with plant development (CAT-box, AT-rich element, O₂-site); and other cis-elements (Figure 5; Table S6). These indicated that OsTDC genes may be induced by exogenous factors (such as hormones, light, and stress) and involved in environmental stress responses.

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Gene structure, conserved domain, and motif analysis of TDC genes in rice

To investigate the functional relationship among TDC genes, conserved motifs, gene structures (exon-intron), and conserved domains were further studied. Differences in protein structure were compared using MEME software to identify the number and distribution of motifs among rice TDC genes. A total of 11 distinct conserved motifs were predicted, namely motifs 1–11 (Figure S2; Tables S7 and S8). The conserved motifs in the nine OsTDC proteins were analyzed using MEME, and 11 different conserved motifs were searched, among which the Os05g0115600 and Os04g0594500 genes did not have conserved motifs, and the remaining seven OsTDC genes contained 11 motifs with the same order of motif alignment. This suggests that there were significant variations in the motif composition among subfamilies. On the other hand, motifs within the same subfamily displayed commonalities, and the general distribution pattern of motifs within each subfamily remained roughly the same (Lai et al., 2024). Generally, motifs can be exclusive to a family, clade, or group, and genes with similar pattern compositions typically share similar biological roles; however, further research is needed to validate this (Khan, 2022). Intron quantity and plant evolution are associated, and genes with fewer introns may be efficiently transcribed in response to environmental changes (Schmitz-Linneweber et al., 2015). In this study, gene structure results revealed that, except for Os07g0437500, which has 12 introns, the other eight remaining TDC genes contain fewer or no introns (Figure 6; Tables S7 and S8), suggesting that they could provide a prompt solution to outside problems. Os07g0437500’s high intron density made alternative splicing more likely. Differential splicing gives genes different functions (Rühl et al., 2012). Hence, multiple functions of TDC subfamily genes may exist in rice. We also searched the conserved domains of the nine rice TDC genes using the NCBI Conserved Domains Database. The nine rice TDC genes exhibited typical aldehyde dehydrogenase domains (DUF674 and AAT_I) (Figure 6; Tables S7 and S8). Collective findings indicate that distinct architectures are represented by rice TDC genes located in the same gene evolutionary tree branch.

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Analysis of the interaction network, physicochemical properties, and secondary and tertiary structures of rice TDC proteins

Based on the above results, these three TDC DEGs (Os08g0140500, Os10g0380800, and Os08g0140300) may play key roles in alleviating abiotic stress and mediating the detoxification and metabolism of xenobiotics (pesticides). Thus, we predicted the physicochemical properties and secondary and tertiary structures of these three TDC DEGs (Figure S3; Table S9). The web-based ExPASy technology was utilized to forecast the physicochemical characteristics of the trio of TDC proteins. The number of amino acids varied from 514 to 526, the MW varied from 55.70211 to 56.50976 kDa, the PI varied from 5.49 to 6.79, and the instability index varied from 37.89 to 42.91, which identified the proteins as stable (Table S8). The grand average of hydropathicity of Os08g0140500 and Os10g0380800 was 0.007–0.055, respectively, which indicated that they may be hydrophobic, whereas that of Os08g0140300 was −0.073, classifying it as a possibly hydrophilic protein. To further understand the secondary structure of these proteins, we used the SOPMA server to predict their structural composition. Their alpha helix (Hh), extended strand (Ee), beta turn (Tt), and random coil (Cc) percentages were in the ranges of 33.66%–41.83%, 15.97%–18.36%, 0%, and 42.21%–48.64%, respectively (Table S9). Additionally, the tertiary structures of all TDC proteins were predicted using SWISS-MODEL. More than 90% structural coverage between the proteins and the matching model sequence suggested that the predicted TDC protein structure was fairly reliable (Table S10).

To evaluate possible protein–protein interactions between TDC proteins and other proteins in rice, we used the online database STRING to obtain functional protein association networks. Three TDC proteins and their target proteins were shown to be related in the interaction map, including tryptophan synthase (Q6ZL61_ORYSJ and Q67VM1_ORYSJ), extradiol ring-cleavage dioxygenase (Q0JH82_ORYSJ), serotonin N-acetyltransferase (SNAT2 and SNAT1), prephenate dehydratase (Q8H0A1_ORYSJ), and phenylalanine ammonia-lyase (Q7 × 720_ORYSJ and Q6K6Q1_ORYSJ). (Figure 7; Table S11). Os08g0140300, Os08g0140500, and Os10g0380800 interacted with Q8H0A1_ORYSJ, SNAT2, SNAT1, PAL, Q0JH82_ORYSJ, Q67VM1_ORYSJ, Q7 × 720_ORYSJ, Q6K6Q1_ORYSJ, Q0JA59_ORYSJ, and Q6ZL61_ORYSJ, whereas there was also an interaction between Os08g0140500 and Os08g0140300 (Figure 7). Some of these proteins are related to the synthesis of betaine and melatonin, which can regulate plant growth, metabolic processes, tissue development, and biosynthetic processes and play an important role in biotic and abiotic stress acclimatization in rice (Ahmad et al., 2013; Chen et al., 2022a; Iqbal et al., 2011; Liu et al., 2021; Zhou et al., 2020). Our investigation on the protein–protein interaction network indicated that TDC proteins are involved in abiotic stress responses in plants, based on their correlation with target proteins.

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Docking analysis

Molecular docking is a widely applied virtual screening method used to predict the interaction between receptors and ligands, given that protein activity can be affected by structural modifications upon ligand binding (Chen et al., 2024b). We conducted a molecular docking study to examine the interactions of FLUME with the binding pocket of three OsTDC proteins. The models with the lowest binding energy were selected for subsequent analysis, which identified strong interactions between FLUME and the active site of OsTDC proteins (Tables S12 and S13; Figure 8). The FLUME complex interacts with Os08g0140300 through eight interacting amino acids, with a binding energy of −6.5 Kcal mol⁻¹ (Table S12 and S13; Figure 8A). Each of these amino acids has a unique binding energy and bond length. Interactions between FLUME and amino acid residues included conventional hydrogen bonds (LYS-330, THR-175, SER-327, and PHE-104), a pi-sigma interaction (HIS-214), and pi-alkyl interactions (HIS-329, ALA-300, and PHE-103). There was an important contribution of hydrogen bonds in protein docking. The FLUME complex interacts with Os08g0140500 through nine interacting amino acids, with a binding energy of −6.5 Kcal mol⁻¹ (Tables S12 and S13; Figure 8B). The interactions between amino acid residues were formed by conventional hydrogen bonds (THR-176, HIS-330, LYS-331, and PHE-104) and pi-alkyl interactions (ALA-106, ALA-301, HIS-215, LEU-337, and TRP-95). Hydrogen bond and pi-alkyl were the dominant interactions between Os08g0140500 and FLUME. The FLUME complex interacted with Os10g0380800 through eight interacting amino acids, with a binding energy of −5.7 Kcal mol⁻¹ (Tables S12 and S13; Figure 8C). Interactions between FLUME and amino acid residues included conventional hydrogen bonds (LYS-334, THR-181, and ASN-331), a pi-sigma interaction (TRP-106), pi-alkyl interactions (HIS-114, HIS-333, and PHE-115), and a pi-cation bond (HIS-217). The binding score was attributed to the high number of pi-alkyl interactions and halogen bonds at the enzyme active site. The three protein docking results suggested that OsTDC proteins may play a crucial role in pesticide metabolism, and Os08g0140300 showed a better docking efficiency with FLUME, indicating that it may be more closely related to FLUME metabolism.

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DISCUSSION

MT plays important roles in the regulation of many physiological processes in plants, including an antioxidant to protect plants from various biotic and abiotic stresses and functioning as a regulator of plant growth and development (Khanna et al., 2023; Kolodziejczyk & Kazmierczak, 2024). Many studies have reported that the exogenous application of MT or its increased accumulation could enhance plant tolerance to adversity by increasing the activity of some antioxidant and detoxification enzymes (Cherono et al., 2021; Karumannil et al., 2023). Investigating the genes involved in MT production under pesticide stress may yield novel targets for genetic engineering and the discovery of new genes that reduce the hazards associated with pesticide residues in the environment. TDC, as one of the MT synthases, enhances abiotic stress tolerance in rice (Zeng et al., 2022). In this study, we identified nine TDC-coding genes in FLUME-treated rice transcriptome datasets, which may be crucial for FLUME phytotoxicity mitigation and metabolism promotion in rice. The structures, cis-elements, motif compositions, conserved domains, and chromosomal locations of these nine TDC genes were then identified, and an analysis was performed to ascertain their phylogeny and collinearity with those found in maize, tomato, soybean, and barley. Moreover, the expression levels of the three TDC DEGs, along with the activity and protein–protein interaction network of TDC proteins, were also analyzed under FLUME or MT + FLUME treatment. In addition, the ability of three TDC proteins to bind FLUME was determined by molecular docking. Numerous studies have demonstrated that gene duplication is essential for the evolution of genetic systems and genomes. Thus, gene families may be generated as a result of tandem and segmental duplications, and plants can use these mechanisms to quickly adapt to environmental stress (Moustafa-Farag et al., 2020). To fully understand their expansion pattern, we examined the duplication events of OsTDC genes. As shown in Figure S1, Os08g0140300, Os04g0594500, Os05g0115600, Os05g0510600, Os07g0437500, Os08g0140500, Os10g0380800, Os10g0400500, and Os01g0770200 are located on chromosomes 1, 4, 5, 7, 8, and 10, respectively, and display segmental duplication events. This indicates that segmental duplication has a major impact on the evolution of rice TDC genes and their capacity to adapt to stress. The expression patterns of these nine previously described genes under MT + FLUME therapy suggest that TDC genes may be implicated in the MT + FLUME response. These findings highlight the close relationship between duplication events and gene function.

To explore homology relationships among these OsTDC genes with those of other plant species, we further analyzed their intraspecific and interspecies collinearity with TDC genes in soybean, maize, barley, and tomato. The results revealed genomic collinearity between OsTDC genes and significant collinearity of these OsTDC genes with those of soybeans, maize, barley, and tomato. Amplification and replication led us to conclude that TDC gene evolution involved several species. Several plant species possess distinct TDC genes, consistent with our evolutionary investigation. With TDC genes from rice, soybeans, maize, barley, and tomato, a phylogenetic tree was constructed to determine the evolutionary links between rice TDC genes and other plants (Figure 4; Table S5). In accordance with their evolutionary relationships, the nine rice TDC genes were divided into two categories. Among them, Os05g0510600 and Os04g0594500 belonged to DUF674, and the other seven TDC genes belonged to the AAT_I subfamily. Moreover, we found close evolutionary relationships between TDC genes in rice and barley (Os05g0115600 and HORVU.MOREX.r3.1HG0008660, respectively). An interspecies collinearity map of barley and rice was used to confirm these tight ties (Figure 3D). Furthermore, these results were consistent with rice, soybean, maize, barley, and tomato phylogenetic studies, which found that TDC genes belonging to the same evolutionary subgroup shared conserved motifs, functional domains, and gene structures of OsTDC genes. Together with previous studies on TDC in a variety of species, such as grapevine, rice, Arabidopsis, barley, bermudagrass, apple, and grapevine, we discovered that monocot-dicot plants—which are crucial for adapting to a range of stresses—share relatively conserved gene family structures, roles, and evolutionary histories (Kolodziejczyk & Kazmierczak, 2024; Moustafa-Farag et al., 2020; Zeng et al., 2022). Therefore, we postulated that the FLUME stress response involves these TDC genes. On the other hand, the significant variance that has been identified among the groups implies that functional differences among rice TDC proteins were gradually formed throughout the course of development. This explanation is consistent with that of Zhou et al. (2020) findings, which demonstrated that the origins and beginnings of TDC gene differentiation can be traced back to the evolutionary history of monocotyledons and dicotyledons.

A motif is a brief sequence of generally conserved proteins that is recognized by certain functional protein sequences and contributes to various biological activities (Jiao et al., 2022; Theune et al., 2019). A crucial foundation for protein functional studies can be provided by motif prediction (Schmitz et al., 2022). The majority of TDC family genes in the same group share conserved domains, proteins, and motifs, according to this phylogenetic tree analysis in this study and conserved motif studies. Their phylogenetic clustering was further supported by this. Additionally, all TDC proteins, except Os05g0115600 and Os04g0594500, had conserved motifs 1–11, demonstrating the conserved domains of the TDC family (Figure S2), which might point to a closer evolutionary kinship between OsTDC proteins and different plant species. The unique role of TDC proteins in the MT signaling pathway may also be influenced by the variations in group I and II motif composition patterns. Os08g0140300 and Os08g0140500 were found to be the closest and they were composed of motifs 1–11. Thus, we can reasonably speculate that rice, soybean, maize, barley, and tomato TDC proteins located in the same phylogenetic tree group (Figure 4B) have similar motif structures. Similar conserved motifs and protein sequences among genes in the same group suggest that they may perform related tasks (Tan et al., 2023; Zhou et al., 2023). Based on the above results, we inferred that rice Os08g0140300, Os05g0510600, Os07g0437500, Os08g0140500, Os10g0380800, Os10g0400500, and Os01g0770200 proteins may participate in FLUME stress resistance. However, certain variations in sequence and motif between species may have resulted from genomic loss in a common ancestor or from environmental alterations, which led to the expansion or limiting of genomic mutations (Anwar et al., 2023).

In addition, according to a plethora of research, transcription factors react to cis-elements to control gene expression and improve plant stress tolerance (Schmitz et al., 2022). The data for nine TDC genes revealed that MeJA-related elements have the greatest distribution in the Os04g0594500 gene, with four MeJA-related elements identified upstream of it. ABA and MeJA were widely distributed upstream of the Os08g0140300 and Os08g0140500 genes (Figure 5). Plants may be able to synthesize MeJA and ABA in response to abiotic stimuli, such as salinity, low temperature, drought, and pesticides, because of the high frequency and wide range of MeJA, ABA, and ABRE components (Chen et al., 2024b; Zhang & Yang, 2021). The resistance of rice to abiotic stress is efficiently increased by ABA (Dashevskaya et al., 2013), whereas MeJA is essential for the metabolism and detoxification of pesticides (Zhang & Yang, 2021). For example, MeJA may enhance isoproturon detoxification or degradation and increase isoproturon resistance in wheat (Ma et al., 2018). Following up on our earlier findings, which showed that the cis-acting region of acetyltransferase reacts to gibberellin and ABA under fomesafen stress (Chen et al., 2022a), we finally demonstrated that this gene stimulates the breakdown and metabolism of fomesafen in rice plants (Chen et al., 2023). Thus, OsTDC may participate in multiple biotic and abiotic stress responses, including possibly being involved in FLUME degradation and metabolism. Additionally, numerous plant hormone-related cis-elements, including abscisic acid- and MeJA-responsive elements, are present in the OsTDC family genes and may interact or be implicated in MT signaling pathways. This may explain why the expression of nine OsTDC genes was significantly increased upon FLUME or FLUME + MT treatments (Figure 1). An earlier study found that via triggering hormone signaling pathways and stress-responsive gene expression, exogenous MT administration promotes stress tolerance (Khanna et al., 2023; Kolodziejczyk & Kazmierczak, 2024; Zeng et al., 2022). These findings also suggest that during plant growth and FLUME tolerance, the OsTDC genes may interact with plant hormones.

According to earlier research, the promoter's binding locations, cis-elements, and gene expression patterns may all be crucial for environmental influence adaptability and for creating a stable genetic framework that improves resistance to abiotic stress (Schmitz et al., 2022; Tan et al., 2023; Zhou et al., 2023). To investigate the role of OsTDC genes and proteins in FLUME stress with or without MT, we analyzed OsTDC gene expression and protein activity in different tissues at different FLUME concentrations with or without MT treatment (Figure 1; Figure 2). OsTDC gene expression and protein activity were considerably enhanced under FLUME stress compared with their respective control levels in all tissues, especially in roots. Regarding high gene expression and protein activity of TDC in rice tissues, predicting TDC may play an important regulatory role in FLUME metabolism in rice seedlings. These findings further imply that the OsTDC DEGs might activate stress-responsive cis-elements, which would interact with downstream genes (Figure 5) involved in abiotic stress tolerance, based on the identification of cis-elements in these genes, which consisted of components that might adapt to general stress, anaerobic environments, light, drought, and defense. The rise in OsTDC gene expression could be explained by this (Figure 1).

In the promoter sequences of eukaryotic genes, transcription factors have the ability to specifically bind cis-acting regions. Stress circumstances such as low temperature, drought, and salinity can promote the expression of plant transcription factor genes, which can improve plant resistance to such conditions (Chen et al., 2024b). Myeloblastosis (MYB) transcription factors play a widespread role in the regulation of development, primary and secondary metabolism, and abiotic and biotic stress throughout the kingdom of plants (Wang et al., 2021). In this study, we found that a MYB binding site associated with abiotic stress, such as drought stress, was distributed upstream of TDC genes (Figure 5). Thus, OsTDC gene expression may be triggered by stress-responsive cis-element activation to enhance FLUME metabolism. Likewise, MT treatment significantly induced OsTDC gene expression and enhanced OsTDC protein activity (Figure 2). TDC genes are involved in MT biosynthesis, whereas the cis-elements (MBS, LTR, TC-rich repeats) in OsTDC gene promoters (Figure 5) may bind during FLUME stress with MT treatments. These findings support earlier research suggesting that MT biosynthesis genes may play a role in abiotic stress responses (Khanna et al., 2023; Kolodziejczyk & Kazmierczak, 2024; Zeng et al., 2022).

For the functional validation of genes, protein–protein interactions are essential (Anwar et al., 2023; Theune et al., 2019). In this work, OsTDC proteins interacted with key proteins (Q7 × 720_ORYSJ and Q6K6Q1_ORYSJ) involved in the synthesis and metabolism of phenylalanine ammonia-lyase (Figure 7), whose accumulation in large amounts in plants was identified to play an important role in plant development and resistance to abiotic stress (Timofeeva et al., 2024). Previous studies supported these findings, showing that the OsTDC promoter had important cis-elements linked to development, metabolism, and stress (Figure 5). Consequently, we determined that OsTDC genes might be essential for FLUME stress resistance.

To gain more insight into the molecular functions of OsTDC domains in pesticide metabolism and degradation, the interactions between pesticides and their receptor ligands require further classification. The strength of the reported association between a pesticide molecule and a target protein enhances the probability that a detoxifying enzyme will be involved in the detoxification metabolism of the pesticide (Qiao et al., 2022). The results of this study showed that the proteins produced by the three TDC genes, particularly Os08g0140300, had significant hydrogen bonding with FLUME molecules (Figure 8). This discovery is essential for understanding the functions of the TDC gene family in the metabolism of plant pesticides.

CONCLUSIONS

This study provided a thorough analysis of the OsTDC gene family, and nine OsTDC genes were found in the rice genome. The nine OsTDC genes that were expressed following MT + FLUME therapy comprised two distinct subfamilies (DUF674 and AAT_I) based on their structural and functional properties. These upstream promoter regions of OsTDC genes included multispecific cis-elements, indicating a connection between the transcriptional and translational activation of TDC family genes and the stress response to FLUME in plants. With the ability to interact with different substrates for environmental adaptation, most TDC proteins feature unique domains or motifs. According to investigations of classification, chromosomal distribution, and collinearity, OsTDC genes are distributed throughout six chromosomes in two groups. One, five, four, and two of these genes have a collinearity link with tomato, soybean, maize, barley, and other crops, respectively. Further evidence that one or more large-scale replication events played a role in the evolution of the OsTDC gene family was obtained from the discovery of segmental duplication in the genome-wide replication of OsTDC. Numerous amino acid residues are involved in the binding of FLUME to TDC proteins, as shown in docking studies. Furthermore, OsTDC may be essential for the prediction of the FLUME stressor, according to qRT-PCR and TDC activity measurements. The physiological and molecular mechanisms underpinning the TDC family's activity in rice can be studied with the help of these findings, which also opens up new avenues for research on the role of OsTDC in the regulation of FLUME metabolism and detoxification in rice with the application of MT.

AUTHOR CONTRIBUTIONS

Hao Wen Wang: Data curation; formal analysis; investigation; methodology; software; validation; visualization; writing—original draft. Xu Zhen Shi: Data curation; formal analysis; methodology; software; validation; visualization. Xiao Yu Zhong: Software; validation; visualization. Gan Ai: Resources; software; supervision; validation; visualization. Yan Hui Wang: Resources; software; supervision; validation. Zhi Zhong Zhou: Methodology; software; validation; visualization. Dan Lu: Data curation; software; validation. Xiao Liang Liu: Funding acquisition; project administration; resources; supervision; writing—review and editing. Zhao Jie Chen: Conceptualization; funding acquisition; project administration; resources; supervision; writing—original draft; writing—review and editing.

ACKNOWLEDGMENTS

The authors acknowledge the Guangxi University doctoral initiation projects (202201264) and the Natural Science Foundation of Guangxi Zhuang Autonomous Region (2024GXNSFBA010236).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

Data will be made available on request.

References

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