Insights into profiling of resistance mechanism of alfalfa to atrazine

Abstract

Medicago sativa L. is a type of forage grass belonging to the legume family, known as the "king of forage" due to its high nutritional value, excellent quality, and good palatability. Atrazine is а triazine herbicide and is the primary agent used to control corn fields in China. However, the long-term and frequent use of atrazine in the corn-to-alfalfa planting model will have apparent toxic effects on alfalfa in the soil residues, affecting the average growth and development. It needs further solutions in alfalfa production. This experiment aims to screen out alfalfa varieties with different resistances by identifying atrazine resistance in 60 varieties and taking resistant and sensitive varieties as research objects. Based on the perspective of non-target resistance mechanisms, alfalfa has different resistance to atrazine. The non-target resistance mechanism of atrazine was studied, and the differences in atrazine absorption and metabolism of alfalfa were further analyzed. A comparative analysis of the transcriptomes of resistant and sensitive alfalfa varieties exposed to atrazine was conducted to understand the mechanisms of resistance. The development of a novel and effective method for determining atrazine will allow for the rapid analysis of a significant number of samples for the determination of herbicide residues. Field experiments on the study of atrazine residues in soil and plants helped to establish the patterns of their accumulation. These studies provided a theoretical basis and practical solutions for breeding varieties in different regions agricultural production. The resistance of 60 varieties of alfalfa to atrazine was analyzed with the detection of resistant (SF8001 (SF)) and sensitive (Juneng 2 (J2)) by the soil toxicity method. The experimental results showed that residual atrazine had a significant toxic effect on alfalfa, but different alfalfa varieties had different resistance to atrazine. The most resistant variety SF with an IC50 of 14.8750 mg/kg and the most sensitive J2 with an IC50 of 0.2424 mg/kg were screened. The use of a hydroponic method to determine the inhibitory effect of atrazine on alfalfa varieties with different resistance showed that with increasing atrazine, the fresh weight inhibition rates of SF and J2 gradually increased, and the differences between the fresh weight inhibition rates were significant. The results of the herbicide toxicity study showed that the IC50 of the resistant variety (SF) was 8.28, and the IC50 of the sensitive variety (J2) was 0.29. The effects of P450s (inhibitor malathion) and GSTs (inhibitor NBD-Cl) on the growth of SF and J2 were determined using hydroponic method. The results showed that the highest safe dose of malathion for SF and J2 was 25.00 mg/kg, and the highest safe dose of NBD-Cl for SF and J2 - 0.25 mg/kg, with no significant difference compared to control. The effects of malathion and NBD-Cl on the resistance levels of SF and J2 were determined using the hydroponic method. Compared with the use of atrazine alone, the IC50 values for a resistant and favorable variety were significantly reduced after combined treatment with malathion, with IC50 ratios of 3.02 and 2.21 for SF and J2 alfalfa, respectively. Therefore, it was speculated that the resistance of SF and J2 to atrazine may be related to P450s. Similarly, compared with the use of atrazine alone, the IC50 of SF and J2 decreased significantly after the combined treatment with NBD-Cl. The IC50 values of SF and J2 were 1.08 and 1.21, respectively. Therefore, it was speculated that the resistance of SF and J2 to atrazine may not be related to GSTs. When studying the effect of atrazine stress on plant height (PH), root length (RL), shoot dry weight (SDW), and root dry weight (RDW) of varieties with different resistance, the following patterns were established: with increasing atrazine application concentration, these indicators decreased, the difference in the suppression of SF and J2 was significant, and the atrazine-sensitive variety was suppressed more strongly than the resistant one. When the concentration of atrazine was 2.0 mg/kg, SF decreased PH, RL, SDW, and RDW by 50.84%, 55.03%, 44.42%, and 54.13%, respectively, under atrazine stress. J2 reduced these indicators by 81.84%, 81.73%, 92.31%, and 85.00%, respectively. The results showed that the resistance of SF and J2 to atrazine was related to photosynthesis. The study of the effect of atrazine stress on the photosynthetic characteristics of SF and J2 showed that with increasing atrazine concentration in the soil, the net photosynthesis rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) gradually decreased, while the intercellular carbon dioxide concentration (Ci) gradually increased. At a concentration of 2.00 mg/kg of atrazine, the Pn, Gs, Tr, and Ci of J2 decreased by 83.42%, 84.15%, 91.57%, and 46.69%, respectively. At 0.5 mg/kg, the Pn, Gs, and Tr of SF significantly decreased by 34.03%, 17.90%, and 27.59%, respectively, while Ci increased by 16.38% and showed significant differences compared to J2. Determination of the effect of atrazine stress on the fluorescence parameters of chloroplasts of varieties with different resistance showed that with an increase in the residual amount of atrazine maximum quantum yield (Fv/Fm), maximum photochemical efficiency (Fv/Fo), actual photosynthetic efficiency (Y(II)), photosynthetic electron transport rate (ETR), and coefficient of photochemical fluorescence quenching (qP) all decreased, while non-photochemical quenching coefficient (NPQ) was the opposite. At a concentration of 2.00 mg/kg of atrazine, Fv/Fm, Fv/Fo, Y (II), ETR, and qP decreased by 11.76%, 76.90%, 80.00%, 63.12%, and 46.44%, respectively, while NPQ significantly increased by 37.64% compared to the control at this concentration. When the concentration of atrazine was 0.50 mg/kg, the Fv/Fm, Fv/Fo, Y (II), ETR, and qP of SF decreased by 10.11%, 8.00%, 24.56%, 26.62%, and 11.11%, respectively, while NPQ increased by 19.72%, which was significantly different from J2. The chlorophyll and MDA (Malondialdehyde) contents of SF and J2 were both affected after atrazine stress. The results showed that with the increase of application days, the content chlorophyll a and b in SF remained higher than that in J2. Compared with the control, on the 10th day, the content of chlorophyll a in SF and J2 was 1.35 times and 4.53 times that of the control, respectively, and the content of chlorophyll b was 3.4 times and 1.06 times that of the control, with significant differences between the two levels. The MDA content of SF remained lower than J2 with increasing application days. On the third day, the MDA content of J2 was 4.19 times that of the control and 1.91 times that of SF, with a slightly slower growth rate after 3 days. The resistance of SF and J2 to atrazine was related to the activity of defense enzyme systems. The conditions of the UPLC-MS/MS method for the determination of atrazine in alfalfa plants were optimized. Differences in atrazine absorption and metabolism between SF and J2 were evaluated using this method. The results showed that the residual amount of atrazine in J2 was always higher than that in SF, and there was a significant difference. SF had a significantly higher metabolic rate of atrazine than J2. The emergence of resistance to herbicide in alfalfa may be related to the increased level of resistance absorption and metabolic capacity. Comparative analysis of SF and J2 genes altered under atrazine stress using transcriptome sequencing technology showed that under the treatment of atrazine, there were 4032 differentially expressed genes in the aboveground part between J2-T and SF-T, of which 2233 genes were up-regulated and 1799 genes were down regulated. The GO and KEGG annotation analysis results indicated that differentially expressed genes were mainly involved in photosynthetic carbon utilization and metabolism, amino acid synthesis and UDP galactosyltransferase. Determination of atrazine residues in soil and plants during two alfalfa growing seasons (2022-2024) showed a threat to plant growth. The amount of atrazine in the soil gradually decreased and then stabilized. The residual amount in SF and J2 varieties first increased and then decreased.