Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion

Abstract

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice.

Keywords: AHAS activity; multi-herbicide tolerance; rice; the W548 deletion.

Figure 1

Herbicides bind and block the channel leading to the active site. The molecular surfaces of the monomers were depicted as pink and cyan, respectively. The residues were labeled on the surfaces. ’ indicated residues from different monomers. W548 was shown as a red stick-ball model with the red surface. The herbicides were shown as color stick-ball models with white carbon atoms, blue nitrogen, red oxygen, cyan fluorine, yellow sulfur, and green chloride.

Figure 2

Gel image of SDS-PAGE for the purified AHAS. Lane 1: GST-AHAS-WT; Lane 2: GST-AHAS-ΔW548; Lane 3: mature AHAS-WT; Lane 4: mature AHAS-ΔW548. M: protein marker. The molecular weights of bands in the marker were indicated.

Figure 3

Gel filtration chromatography analysis of purified AHAS. AHAS-WT and AHAS-ΔW548 are shown as red and blue traces, respectively. The black trace is the molecular weight standard. From left to right, the molecular weights of the standards were 670, 158, 44 and 1.35 kD. The dimer peak (128 kDa) and tetramer peak (256 kDa) are indicated separately by the black arrow and red arrow.

Figure 4

Bioassay curves of AHAS activities in the presence of six herbicides. AHAS-WT was inactive when an herbicide was more than 10 μM. AHAS-ΔW548 remained active at high concentrations of herbicides. Panels of (a–f) displayed the AHAS remaining activities in solutions of different herbicides: (a) sulfometuron-methyl (SM), (b) rimsulfuron (RS), (c) chlorimuron-methyl (CM), (d) flucarbazone-sodium (FC), (e) bispyribac-sodium (BS), and (f) imazethapyr (IT). Error bars represented the errors from triplicate measurements. Triangles are AHAS-WT, and circles are AHAS-ΔW548.

Figure 5

Diagram of T-DNA cassette for rice transformation. The ahas-ΔW548 gene replaced the hygromycin resistant gene and served as a selection marker. This gene was driven by a 35S promoter. RB and LB: right and left border of T-DNA; 35S: 35S promoter of the CaMV; ter: terminator of the CaMV.

Figure 6

PAGE analysis of PCR products for ahas fragments. Lane 1-10: PCR products from Line 1-10 of transgenic rice. P: positive control (plasmid DNA of 1300-AHAS-ΔW548). N: negative control (genomic DNA of Xiushui134). M: Low MW DNA marker-A. The molecular weights were indicated. Two arrows showed two amplified fragments: upper arrows indicated wildtype ahas fragments, lower arrows indicated ahas-ΔW548 fragments.

Figure 7

Image showing rice plants with herbicide treatment. The plants were treated with 0.25 g/L SM, 0.8 g/L BS or 2.2 g/L IT at the seedling stage. After two weeks, the Xiushui134 plants withered, while the Line9 plants survived with the herbicide treatment.

Figure 8

Plant relative height of Xiushui134 and Line9 at the seedling stage. The Xiushui134 plants were killed with herbicide treatments, and the relative heights were around 20%. The Line9 plants grew well with SU herbicide treatments. The growth of Line9 was inhibited by a PYB (BS) and IMI (IT) herbicides. Error bars represent the errors from multiple measurements.

Figure 9

The W548 location in the AHAS-WT tetramer. The interfaces were labeled by dash Lines. The backbone of the monomer was colored as pink and cyan, respectively. W548 was shown as a red stick-ball model, S627 was shown as a blue stick-ball model.