3,4-Dichlorophenylacetic acid acts as an auxin analog and induces beneficial effects in various crops

Auxins, primarily indole-3-acetic acid (IAA), are crucial plant hormones regulating growth, development, and responses to environmental cues. Their asymmetric accumulation in tissues directs organogenesis, pattern formation, and tropic growth. This precise distribution depends on membrane-bound transporters like AUX1/LAX influx carriers, PIN-FORMED (PIN) efflux carriers, and ABCB transporters. The polar localization of PIN proteins, maintained by endocytosis and transcytosis, is crucial for directed auxin flow. Auxin's nuclear action involves binding to TIR1/AFB receptors, triggering the degradation of AUX/IAA repressors and activating AUXIN RESPONSE FACTOR (ARF) transcription factors, ultimately regulating the expression of auxin-responsive genes. Besides IAA, other endogenous auxins exist, such as indole-3-butyric acid (IBA), phenylacetic acid (PAA), and 4-chloroindole-3-acetic acid (4-Cl-IAA), each with unique characteristics. Synthetic auxin analogs, like NAA and 2,4-D, are widely used in research and agriculture, but limitations exist regarding efficacy, application scope, and potential side effects. This study aimed to screen new auxin analogs to overcome these limitations. The researchers designed over 2000 compounds based on known auxin structures; 82 were chosen for physiological activity testing, ultimately identifying 3,4-dichlorophenylacetic acid (Dcaa) as a promising candidate.

The literature extensively documents the role of auxins in plant growth and development, highlighting the importance of their polar transport and interaction with specific receptors. Studies have characterized various endogenous auxins and their distinct properties. The use of synthetic auxin analogs in agriculture and research has been widely explored, but their drawbacks motivated the search for new, more effective, and safer alternatives. Previous research provides a basis for understanding the mechanism of auxin action and the design of new analogs. The existing auxin-like PGRs still suffer from drawbacks and limitations.

The research involved a multi-stage process. Initially, more than 2000 chemical compounds were designed using 'me too' and 'active substructure splicing' methods based on the chemical structures of known auxins (indole, naphthalene, and benzene rings). 82 compounds were selected for physiological activity tests based on factors such as material availability, synthesis difficulty, stability, toxicity, and cost. The oat coleoptile elongation assay was used to assess auxin activity, comparing Dcaa to NAA, IBA, and other controls. Adventitious root generation in mung bean seedlings was also examined to evaluate Dcaa's root-promoting effects. The root growth of various crops (cucumber, cabbage, tomato, maize) was assessed after treatment with Dcaa via root irrigation or foliar spraying, comparing Dcaa's effects to a potassium indole butyrate/sodium naphthylacetate control. Maize nitrogen use efficiency was examined after Dcaa treatment. The effect of Dcaa on auxin-responsive reporters (DR5:GUS and DR5rev:GFP) was assessed via histochemical staining and enzyme activity assays. RT-qPCR was used to analyze the expression of auxin-responsive genes (ARF7, ARF19, IAA19, LBD16, SAUR22, and SAUR24). Molecular docking studies investigated Dcaa's binding to TIR1 and AFB1-5 auxin receptors. The sensitivity of auxin transport mutants (*aux1-T* and *pin2-T*) to Dcaa was assessed using DR5:GUS reporter expression and root growth assays. Finally, the effect of Dcaa on PIN2 protein endocytosis was investigated by co-treating PIN2-GFP seedlings with Dcaa and BFA (brefeldin A). Statistical analyses (Student's t-test) were performed to evaluate the significance of the results.

Dcaa demonstrated auxin-like activity, promoting oat coleoptile elongation and mung bean adventitious root formation, although less potently than IBA and NAA. In various crops (cucumber, cabbage, tomato, maize), Dcaa application significantly enhanced root growth parameters (fresh/dry weight, total length, surface area, and volume), often surpassing the control treatment (potassium indole butyrate/sodium naphthylacetate). Dcaa treatment improved maize nitrogen use efficiency. Dcaa enhanced the expression of auxin-responsive reporters (DR5:GUS, DR5rev:GFP) and modulated the expression of auxin-responsive genes in a pattern similar to NAA. Molecular docking studies showed that Dcaa binds to auxin receptors, with the highest binding affinity to TIR1. *pin2-T* mutants exhibited hypersensitivity to Dcaa, indicating a role for PIN2 efflux carriers in Dcaa transport. In contrast, *aux1-T* mutants showed resistance to 2,4-D but responded similarly to Dcaa as the wild type. Dcaa, like other auxins, inhibited PIN2 protein endocytosis.

This research demonstrates that Dcaa functions as a novel auxin analog, effectively promoting root growth across diverse crop species. While exhibiting lower activity than established auxins (like IBA and NAA) in some assays, Dcaa's superior root-promoting effects in intact plants compared to a standard PGR mixture highlight its potential for agricultural applications. Dcaa's mechanism involves interactions with the auxin signaling pathway, including binding to auxin receptors (especially TIR1) and modulating the expression of auxin-responsive genes. Its transport properties appear similar to NAA, relying on PIN2 efflux carriers and possibly also AUX1 influx carriers. The ability of Dcaa to inhibit PIN protein endocytosis further supports its auxin-like activity. The differential responses of *aux1-T* and *pin2-T* mutants confirm the involvement of auxin transport mechanisms in Dcaa's action. These findings provide strong evidence for Dcaa as a promising plant growth regulator.

This study successfully identified and characterized 3,4-dichlorophenylacetic acid (Dcaa) as a novel auxin analog with significant root growth-promoting effects across multiple crop species. Dcaa's efficacy and mechanism, involving auxin receptor binding, gene expression modulation, and PIN protein regulation, make it a promising candidate for agricultural applications. Future research could focus on optimizing Dcaa application methods, evaluating its effects on yield and other agronomic traits, and exploring potential applications in improving nutrient use efficiency and stress tolerance.

While this study provides compelling evidence for Dcaa's auxin-like activity and beneficial effects on plant growth, some limitations exist. The study primarily focused on root growth; further investigations into the effects of Dcaa on shoot growth and overall plant development are warranted. The molecular mechanisms underlying Dcaa's interaction with PIN proteins warrant further exploration. Long-term field trials are needed to fully assess the efficacy and safety of Dcaa under various environmental conditions. Finally, comparisons with commercial auxin analogs under identical experimental conditions would strengthen the conclusions.