Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application

Abstract

Abstract:The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuronmethyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides.

Leaf proteome analysis provides insights into the molecular mechanisms of bentazon detoxification in rice

“…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 exposure time (Fig.…”

“…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].…”

“…After one month, 5-leaf rice seedlings approximately 20 cm in height were treated with 500 mg/l bentazon. At this concentration, the rice grew normally without morphologic changes and effectively induced CYP81A6 transcription to an extent that changes in protein expression could be detected following bentazon exposure [10].…”

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing

“…The following program was used for all qPCRs: 10 min at 95 °C; and 40 cycles of 10 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C. Relative gene copy number was calculated using the 2 −ΔΔC T method, with the rice actin gene as the internal standard (Lu et al, 2015).…”

Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa1 in rice

“…Transgene positive T 0 plants were identified by PCR analysis of the hygromycin resistant gene using primer Hyg R (Table 1) and T 1 seeds were harvested and grown into plant lines according to Lu et al (2015). Root, stem, leaf, flower tissues and seeds (developing and mature) were histochemically stained for 24 h at 37 °C with GUS dye solution containing 100 mmol/L phosphate buffer (pH 7.0), 20% (v/v) methanol, 0.5% (v/v) Triton X-100, and 0.5 mg/ml X-Gluc, according to Sieburth and Meyerowitz (1997).…”