Leaf proteome analysis provides insights into the molecular mechanisms of bentazon detoxification in rice
Graphical abstract
Introduction
Herbicides are the dominant method of crop weed control in most broad-area grain cropping systems, and their use has contributed significantly to improved crop productivity and sustainability of agricultural systems in many parts of the world. However, herbicide-resistant weeds present an increasing challenge to this practice, and have become a major threat to the sustainability and profitability of cropping systems. The two primary mechanisms of herbicide resistance in weeds are resistance conferred by mutations in genes that encode proteins containing target sites of the herbicide (target-site resistance), and resistance as a result of mutations outside of target sites (non-target-site resistance) [1]. Most herbicides are designed to target specific enzymes or proteins, thus target-site resistance develops from either genetic diversity or mutations in target sites. Mutations that produce structural differences at the protein level can prevent binding of a single herbicide or a group of related herbicides, such as ACCase (acetyl-CoA carboxylase), the target of DM (diclofop methyl), in rice [2]. DM selectively inhibits the activity of ACCase in monocotyledonous plants, but lacks efficacy in dicotyledonous plants, due to structural changes in ACCase not present in monocotyledonous plants.
In contrast to target-site resistance, non-target-site herbicide resistance involves multi-herbicide resistance proteins, including but not limited to P450s (cytochrome P450 monooxygenase), glutathione S-transferase, glycosyltransferase, and ABC transporters [3]. Non-target-site resistance depends on sophisticated detoxification mechanisms against toxic chemicals, in which the oxidation phase reaction is carried out by P450s. Reactions mediated by P450s produce oxygenated compounds with increased reactivity or solubility, thus setting the stage for subsequent detoxification reactions [4]. The function of P450s in non-target herbicide resistance has been established through the correlation of P450 enzyme activity with herbicide resistance [5], [6].
Bentazon [3(1-isopropyl)-(1H)-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide] is a widely used herbicide that selectively removes broad-leaf weeds by competing with plastoquinone (QB) for the binding site in the D1 protein and interrupting the PET (photosynthetic electron transfer) chain [7]. Rice develops resistance to this herbicide due to the ability of bentazon to induce expression of the P450 gene CYP81A6. Sensitivity of CYP81A6 mutants to bentazon further confirmed that CYP81A6 is a key mediator of bentazon resistance [8], [9]. It has also been shown that unknown protein factor(s) can rapidly induce CYP81A6 transcription within 2 h of bentazon exposure [10]. CYP81A6 is therefore used as a selective mrker in modern crop breeding [[11], [12], [13]].
The majority of previous studies related to herbicide–plant interactions on their specific metabolism and detoxification pathways in plants focused on effects at the physiological and biochemical levels. However, little is known about the impact of herbicides on transcription or translation, particularly on transcriptomic or proteomic scales. In this study, we employed a 2-D DIGE proteomic approach to evaluate changes in expression patterns after bentazon treatment. DIGE is a powerful tool for separating complex mixtures of proteins by charge and size (electrophoresis), and different sample types are detected by different fluorescent. To the best of our knowledge, this is the first study to identify protein that may play important roles in upregulation of CYP81A6 transcription, as well as to clarify the molecular mechanisms by which these pathways participate in CYP81A6 induction and bentazon detoxification in rice.
Section snippets
Plant materials
Indica rice seeds (Jiazhe B) were germinated on filter paper at 30 °C in the dark for 2 d. The germinated seeds were transferred to a net floating on liquid culture medium for 3 weeks. The seedlings were then incubated in a controlled-environment growth chamber at 28 ± 0.5 °C with a light:dark cycle of 12 h:12 h, with light provided by cool-white fluorescent bulbs (≈ 54 μEm− 2 s− 1). After one month, 5-leaf rice seedlings approximately 20 cm in height were treated with 500 mg/l bentazon. At this
Photosynthesis efficiency was affected after bentazon exposure
To investigate the time course of bentazon effects on the photosynthesis system, four parameters were measured after bentazon treatment using a portable chlorophyll fluorometer. The values of four chlorophyll fluorescence parameters remained at high levels at every time point in control rice samples. However, photosynthesis efficiency was significantly suppressed after treatment of rice for 1 h with 500 mg/l bentazon, and inhibitory effects increased in a time-dependent manner. ETR and ФII
Discussion
Bentazon is a commonly used herbicide for control of dicotyledon weeds, and acts by binding to photosynthetic system II components, and inhibiting electron transfer and carbon fixation [7]. However, monocotyledons, including rice, exhibit strong resistance to bentazon. The concentration of bentazon used in our study (500 mg/l) did not induce phenotypic changes in the rice [10]. However, chlorophyll fluorescence analysis demonstrated that bentazon affected the PET chain of rice during the initial
Conclusion
The results of our study indicate that rice was sensitive to bentazon treatment at the initial time of exposure. Using a DIGE proteomic approach, we demonstrate that bentazon treatment rapidly suppressed photosynthesis and carbohydrate metabolism pathways, among other pathways. The physiological results from chlorophyll fluorescence analysis also confirmed that photosynthesis was inhibited and that electron transfer was blocked. However, numerous antioxidant and biotic stress response proteins
Acknowledgments
This work was financially supported by the Natural Science Foundation of China (21277127), and Zhejiang Provincial Natural Science Foundation of China (LR14B070001).
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These authors contributed equally to this work.