Proteomic and ultrastructural characterization compatible with programmed cell death in Conyza sumatrensis resistant to the herbicide 2,4-D.
Sumatran fleabane, Auxin mimetics, Oxidative stress.
The herbicide 2,4-D is among the most widely used in Brazil for controlling broadleaf weeds in various cropping systems. Among the main broadleaf weeds in the country, Conyza sumatrensis (buva) stands out for its resistance to 2,4-D. This resistance is atypical and characterized by symptoms of rapid physiological response, with leaves necrotizing within 4 hours after application (HAA), preventing herbicide translocation. The objective of this study is to confirm whether the rapid physiological response to the herbicide 2,4-D is a process of programmed cell death of the hypersensitivity type (PCD-HR), induced by specific proteins in the cell degradation process. The first experiment was conducted at DIC, with treatments for biotypes susceptible and resistant to the herbicide 2,4-D, with and without herbicide application, containing four replicates each. The herbicide was applied to plants at the 8-10 leaf stage, using a CO₂-pressurized backpack sprayer at 150 L ha⁻¹ at a dose of 1005 g a.i. ha⁻¹. Phytotoxicity and chlorophyll α fluorescence were obtained at 20, 30, 40, 60, and 120 min after application (MAA). Plant gas exchange was evaluated using an infrared gas analyzer at 30 and 120 MAA. Twenty-eight days after application, the aerial part was collected and kept in a drying oven until it reached a constant mass. The values were submitted to ANOVA (p≤0.05) and, when significant, to Tukey's test (p≤0.05). For proteomics, the material was collected at 0, 20, and 30 MAA and frozen at -80°C. It was then subjected to label-free analysis, and the identified proteins were analyzed by the Student's t-test (two-tailed), with proteins with a p-value < 0.05 considered “up-accumulated” if the Log2 of the fold change (FC) > 0.6 and “down-accumulated” if the Log2 of the FC was less than < -0.6. The transmission electron microscopy (TEM) experiment was performed in DIC, with three treatments: functional leaves from resistant plants without herbicide application and leaves collected 1h and 2h HAA from 2,4D, with three replicates. The plant material was processed and the images analyzed descriptively. Proteomic analysis confirmed a differentiation of proteins in the 2,4-D-resistant biotype compared to the susceptible one. Proteomics identified 387 differentially accumulated proteins, grouped into six dynamic clusters. In the resistant biotype, early induction of proteins related to oxidative stress, cell signaling, and structural reorganization was observed, unlike in the susceptible biotype. Proteins involved in defense signaling pathways, such as serine/threonine phosphatases, were found to be accumulated, suggesting activation of MCP-HR defense routes. MET analyses show that in the resistant biotype, mesophyll cells present structural damage between 1 and 2 HAA with membrane disorganization, tonoplast rupture, and mitochondrial swelling, characteristics of MCP-HR. In vascular bundle cells, the structure remained preserved in resistant biotypes, suggesting no herbicide translocation. It is concluded that proteomic and ultrastructural analyses confirm that resistance to 2,4-D in Conyza sumatrensis is associated with the activation of MCP-HR, with localized damage in the mesophyll and limited herbicide translocation.