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

The study addresses the need for new auxin-like plant growth regulators (PGRs) that are effective across diverse crops while minimizing side effects associated with existing auxins. Auxin regulates plant growth and development via directional transport (AUX1/LAX influx, PIN efflux, ABCB transporters) and nuclear signaling through TIR1/AFB receptors that target AUX/IAA repressors. Endogenous auxins (IAA, IBA, PAA, 4-Cl-IAA) differ in biosynthesis, transport, and activity. Widely used synthetic auxins (e.g., NAA, 2,4-D) have distinct application scopes and limitations, including phytotoxicity in sensitive species and environmental/health concerns. The authors designed and screened new analogs, identifying 3,4-dichlorophenylacetic acid (Dcaa) as a candidate auxin analog, and set out to characterize its physiological activity and signaling mechanism.

Background covers auxin biology: polar transport mediated by AUX1/LAX influx, PIN efflux, and ABCB transporters; PIN polarity maintained by endocytosis/transcytosis and inhibited by auxin. Nuclear perception involves TIR1/AFB receptors that promote AUX/IAA degradation, freeing ARFs to drive auxin-responsive gene expression. Endogenous auxins include IAA (Trp-derived via TAA/YUC), IBA (IAA precursor/storage), PAA (widely present, lower activity, non-polar transport), and 4-Cl-IAA (legumes, higher activity, carrier substrates). Synthetic auxins (NAA, 2,4-D, dicamba) are used as herbicides and PGRs with differing properties; NAA is potent for rooting/fruiting but can be phytotoxic in cucurbits; 2,4-D is effective in fruit set and postharvest but has environmental/health concerns. Prior structural insights show TIR1 accommodates diverse auxin ring systems via a conserved carboxyl-binding site (Arg403, Ser438) and a hydrophobic cavity, enabling binding of IAA, NAA, and 2,4-D. Transport properties of analogs differ: IAA uses influx and efflux; NAA diffuses in and requires efflux; 2,4-D requires influx and is poorly effluxed.

- Compound design and screening: >2000 auxin-like compounds designed using 'me too' and active substructure splicing approaches based on indole, naphthalene, and benzene scaffolds; 82 shortlisted considering raw material availability, synthesis difficulty, stability/toxicity, and cost. Pot and field trials used for efficacy/safety, identifying Dcaa (3,4-dichlorophenylacetic acid) as promising. - Plant materials: Arabidopsis Col-0; mutants/reporters aux1-T, pin2-T, DR5:GUS, DR5rev:GFP, ProPIN2:PIN2-GFP, tir1-1, tir1-1 afb1,2,3. Crop cultivars: cucumber (Jinpei 98F1), cabbage (Xiaguan F1), tomato (Kaideyali 1832), maize (Yudan 9953), mung bean (Jizaolvzhenzhu 2), oat (Avena sativa). Standard growth conditions on MS medium or soil/greenhouse. - Chemicals: NAA, IAA, 2,4-D, IBA, naphthalene acetamide, naphthoxyacetic acid, vitamin B1, potassium indole butyrate, sodium naphthalene acetate (commercial sources). - Oat coleoptile elongation assay: 30 segments per treatment; Dcaa at 1, 10, 100 μM; controls at 10 μM (NAA, IBA, naphthalene acetamide, naphthoxyacetic acid, VB1); dark; length after 40 h quantified by ImageJ. - Mung bean adventitious rooting: Roots excised; bases soaked 24 h in Dcaa (3–120 ppm) or 25 ppm NAA; then water-cultured 7 days; measured rooting zone length, number and length of adventitious roots. - Crop root growth assays: • Cucumber/cabbage: Root irrigation with 5 mL Dcaa at 1.5, 3, 6, 12 ppm; control PGR mix (1 ppm potassium indole butyrate + 1 ppm sodium naphthalene acetate). Assessed after 7 days. • Tomato: Foliar spray Dcaa at 3, 6, 12 ppm; control PGR mix at 10, 20, 40 ppm; assessed after 7 days. • Maize: Root irrigation with 5 mL Dcaa at 3, 6, 12 ppm; same control mix; assessed after 7 days. Root traits quantified by WinRHIZO: fresh/dry weight, total length, projected area, surface area, average diameter, volume, branch number. - Maize nitrogen use efficiency: Plants grown with 8.75 g/L urea; Dcaa applied by foliar spray or root irrigation at 3, 6, 12 ppm; repeated after 1 week; shoots analyzed 2 days later for total nitrogen; nitrogen use efficiency calculated. - Reporter assays: • DR5:GUS: 7-day seedlings treated 2 h with 10 μM NAA or 250/500 μM Dcaa; histochemical GUS staining and quantitative GUS enzymatic activity measured (4-MUG to 4-MU fluorescence, normalized to protein). • DR5rev:GFP: 7-day seedlings treated 2 h with 10 μM NAA, 10 μM 2,4-D, or 500 μM Dcaa; fluorescence imaged and quantified. - RT-qPCR: 7-day Col seedlings treated with NAA (1 or 10 μM) or Dcaa (100 μM, 250 μM, 500 μM, 1 mM) for 0–6 h; RNA extracted (TRIzol), cDNA synthesized; qPCR for ARF7, ARF19, IAA19, LBD16, SAUR22, SAUR24; TIP41 as internal control; 2^-ΔΔCt method. - Auxin receptor dependence: Root growth assays on MS with 100 nM 2,4-D, 2.5–5 μM Dcaa using Col, tir1-1, tir1-1 afb1,2,3; root length measured at 7 days. - Molecular docking: Dcaa 3D structure minimized; receptor TIR1 crystal (PDB 2P1Q) and AFB1-5 homology models (SWISS-MODEL); ligand/receptor preparation (Mgltools 1.5.6); docking with AutoDock Vina v1.2.3; best binding modes by binding energy; interactions visualized. - Auxin application at root tip: 5-day seedlings placed with root tips on plastic barrier; 1% agar strips containing IAA (100 μM), 2,4-D (50 μM), NAA (100 μM), or Dcaa (1.5 mM) applied overlapping root tips; incubated 13 h; DR5:GUS staining or DR5rev:GFP imaging; performed in Col and auxin transport mutants (aux1-T, pin2-T). - PIN endocytosis assay: PIN2-GFP seedlings pretreated 30 min with DMSO, 5 μM NAA, 5 μM 2,4-D, or 75 μM Dcaa; then co-treated 30 min with 25 μM BFA; confocal imaging; BFA bodies per cell quantified. - Statistics: Student’s t-test; mean ± SD; biological repeat numbers per figure; significance thresholds P<0.05, P<0.01, P<0.001.

- Discovery and activity: From 82 shortlisted analogs, 3,4-dichlorophenylacetic acid (Dcaa) emerged as a promising auxin-like PGR from pot and field screening. - Oat coleoptile elongation: Dcaa increased elongation by ~27%, 33%, and 83% at 1, 10, 100 μM vs untreated; IBA at 1, 10, 100 μM increased ~89%, 165%, 156%; 10 μM NAA increased ~202%. VB1, naphthoxyacetic acid, and naphthylacetamide inhibited elongation. - Adventitious rooting (mung bean): Dcaa increased rooting zone length and number of adventitious roots dose-dependently; root length increased at low Dcaa but decreased at high; 120 ppm Dcaa comparable to 25 ppm NAA. - Cucumber roots (root irrigation): Dcaa reduced shoot height but increased root fresh/dry weight with dose; 12 ppm Dcaa comparable to control PGR mix for fresh/dry weight. Max total root length and surface area at 3 ppm (252.83 cm and 36.64 cm²) vs untreated (194.01 cm; 27.95 cm²) and control (194.94 cm; 30.37 cm²). Diameter and volume increased with dose, highest at 12 ppm; branch number unchanged. Overall, 3 ppm most effective across indices. - Cabbage roots: Root fresh/dry weights increased with Dcaa concentration and exceeded control PGR; diameter and volume significantly larger at 12 ppm vs untreated; Dcaa outperformed control. - Maize roots (root irrigation): Dcaa significantly increased root fresh/dry weight, total length, and surface area vs untreated; branch number, diameter, and volume not significantly changed. Control PGR increased length/surface/volume but had smaller effects on fresh/dry weight than Dcaa. - Tomato roots (foliar spray): 6 ppm Dcaa significantly increased projected area, surface area, and volume; total length difference (61.2 vs 44.7 cm) was not statistically significant; diameter not increased. 6 ppm identified as reasonable dose. - Nitrogen use efficiency (maize): Dcaa increased shoot nitrogen content at all tested concentrations. Leaf-spray 3 ppm Dcaa achieved 34.7% nitrogen use efficiency (declined at higher doses). Root irrigation showed highest promotion at 6 ppm. - Auxin-responsive reporters: Dcaa (250, 500 μM) enhanced DR5:GUS signals in QC, columella, and root tip stele similar to 10 μM NAA; GUS enzymatic activity significantly increased and was dose-dependent. DR5rev:GFP fluorescence intensified after 500 μM Dcaa (and 10 μM NAA/2,4-D). - Gene expression: Dcaa elicited time- and dose-dependent transcriptional responses similar to NAA in ARF7, ARF19, IAA19, LBD16, SAUR22, and SAUR24. - Receptor dependence: tir1-1 and tir1-1 afb1,2,3 mutants showed weaker root growth inhibition by Dcaa and 2,4-D than wild type, indicating action via TIR1/AFBs. - Molecular docking: Dcaa bound in silico to TIR1 and AFBs with calculated energies (TIR1 and AFB1-5: −6.037, −5.503, −5.412, −5.149, −5.620, and 5.610, respectively), with strongest predicted binding to TIR1. Dcaa carboxyl formed H-bonds with Arg403 and Ser438, and additionally Arg436 in TIR1. - Transport behavior: Root-tip application induced DR5 signal propagation consistent with basipetal movement. In pin2-T DR5:GUS roots, IAA, NAA, and Dcaa did not elevate DR5 in root hair zone (2,4-D did), suggesting Dcaa requires efflux (PIN2). In aux1-T DR5:GUS roots, NAA and Dcaa (but not IAA, 2,4-D) increased DR5 in root hair zone, suggesting influx carriers are not required. Sensitivity assays showed aux1-T resistant to 2,4-D but not to NAA/Dcaa; pin2-T hypersensitive to NAA/Dcaa but not 2,4-D, indicating Dcaa is a PIN2 substrate and likely enters via diffusion. - PIN endocytosis: Dcaa co-treatment with BFA reduced PIN2-GFP BFA body formation, similar to NAA and 2,4-D, indicating inhibition of PIN2 endocytosis.

The work demonstrates that Dcaa functions as an auxin analog that promotes root system development across multiple crops and engages canonical auxin pathways. Physiologically, Dcaa stimulates coleoptile elongation, adventitious root formation, and root growth; molecularly, it enhances DR5 reporters, induces auxin-responsive gene expression, requires TIR1/AFB perception, and exhibits transport dependencies similar to NAA (passive entry, efflux carrier-mediated export). Docking suggests Dcaa fits the TIR1 pocket via conserved carboxyl interactions (Arg403, Ser438) with an additional Arg436 interaction, consistent with receptor engagement. Transport assays and mutant sensitivities indicate Dcaa is exported by PIN2 and can enter cells without AUX1. Dcaa also inhibits PIN2 endocytosis like other auxins, supporting its auxin-like cellular effects. Notably, while Dcaa’s auxin activity is lower than IBA/NAA in isolated tissue assays, in intact crops Dcaa outperformed a standard PGR mix for some root traits, implying advantageous stability, transport, or signaling properties that translate to improved root architecture and potentially enhanced nitrogen use efficiency.

Dcaa (3,4-dichlorophenylacetic acid) is identified as a new auxin-like compound that effectively promotes root growth in cucumber, cabbage, tomato, and maize. It activates auxin signaling (DR5 reporters, auxin-responsive genes), is perceived by TIR1/AFB receptors (supported by mutant analyses and docking), likely undergoes polar transport requiring PIN-mediated efflux, and inhibits PIN2 endocytosis. Dcaa improved root architecture and increased nitrogen use efficiency in maize at specific doses, providing a basis for its application as a plant growth regulator in agriculture.

- Reporter transport assay cannot exclude that increased DR5 activity reflects persistent local signaling rather than true long-distance transport. - Molecular docking provides in silico evidence for receptor binding without biophysical validation. - Dcaa concentrations required to elicit reporter responses were higher than those of NAA, indicating lower potency in some assays. - Direct transport measurements (e.g., radiolabeled uptake/efflux) were not performed; conclusions on transport rely on reporter localization and mutant phenotypes. - Field performance and safety are referenced from screening/field trials but detailed agronomic and environmental impact assessments are not presented.