In silico analysis and identification of P197S tolerant mutation in rice AHAS

A multiple sequence alignment involving the AHAS enzyme of different plants resulted in a consecutive 35 amino acid long conserved stretch from 190–224 amino acid positions in all AHAS enzymes (Fig. 1A). The amino acid proline was highly conserved in all the tested plant species at 197 with reference to Arabidopsis; this residue was located at position 171 in the rice AHAS protein. There have been many AHAS-inhibiting herbicide-resistant weeds with the P197S substitution mutation reported to exist in nature10 and the multiple sequence alignment revealed the conserved position of this amino acid in the protein sequence, confirming the crucial role of proline in AHAS enzyme function. Thus, a naturally selected tolerance mutation in the AHAS gene could be a valuable resource for the development of herbicide-tolerant transgenic plants.

Figure 1 In silico analysis of AHAS proteins and the schematic representation and PCR confirmation of the expression cassette. (A) Multiple sequence alignment of AHAS proteins in various plant species. The rectangle shows the presence of a 35 amino acid long conserved stretch, and the arrow shows the position of the conserved proline amino acid at position 171. (B) Schematic representation of expression cassette containing mutated rice AHAS and bar genes. (C) PCR confirmation of the expression cassettes (Full images are available in Supplementary information as Figs SI3 and 4). Full size image

Development and molecular confirmation of transgenic plants

The in silico analysis revealed that the amino acid at position 171 in the rice AHAS protein is the conserved mutation point for herbicide tolerance; this amino acid was subsequently replaced by serine to develop herbicide-tolerant plants (Fig. 1A). While creating the P171S mutation in rice AHAS, the XbaI restriction site was introduced to help to distinguish transgenic plants from non-transgenic plant (Fig. 2E). We used the Agrobacterium-mediated transformation method for the generation of transgenic plants. The putative transgenic plants were initially screened for confirmation of the presence of transgene construct with the help of bar primers by PCR (Fig. SI1A and B). Since AHAS is a native gene, it was not possible to screen putative transgenic plants with AHAS primers because there would be no difference between native and transgenic PCR bands.

Figure 2 Molecular analysis of transgenic plants. (A) and (B) Transgene integration analysis of PCR positive transgenic plants by southern blotting. First, the membrane was hybridized with AHAS probe showing the presence of common native and unique transgene bands (A). The membrane was rehybridized with bar probe which shows only a single unique transgene integration (B). (C) Expression analysis of transgenes by northern blotting. To see the expression of the AHAS and bar genes, the same membrane was hybridized at first with AHAS and then with bar probes. The membrane was rehybridized with Act1 probe used as internal standard. (D) Analysis of the relative expression of both transgenes using semi-quantitative RT-PCR. The expression of the native rice Act1 gene was used as internal standard. (E) Restriction digestion analysis of cDNA-amplified PCR products showing the difference between the expression of native and trans-AHAS genes and simultaneous confirmation of the introduced P171S mutation. The digestion of PCR products from transgenic plants yielded three bands of 401, 294 and 107 bp molecular weight while no digestion occurred in the wt plants. wt: wild type, L: Line number of transgenic plants (Full images of all the blots and gels are available in supplementary information from Figures SI5–SI8). Full size image

The PCR positive T1 transgenic plants were analyzed to confirm the presence of the transgene integration by southern blotting. The hybridization of nylon membrane with AHAS DIG labelled probe resulted in a common band for all the transgenic lines (including wild type (wt) control plant) (Fig. 2A); this band represents the native AHAS gene. In addition to native copy, there was an additional band found for all 5 transgenic lines (line 1, 2, 4, 5, 6), which showed the single copy AHAS transgene integration. Subsequently, the same nylon membrane was re-hybridized with bar DIG probe. The bar probe resulted in the same pattern as observed in AHAS hybridization except the common AHAS band due to the heterologous origin of bar gene (Fig. 2B). Among the transgenic plants (lines 1, 2, 4, 5 and 6), lines 4 and 6 showed similar band patterns for both the AHAS and bar genes; thus considered as the same transgenic events. Further, transgenic line 3 and the wt control did not show signals for any of the genes; this line was considered non-transgenic. Thus, the southern blot analysis confirmed a total of 4 different positive AHAS and bar single copy integrated independent transgenic events.

The expression levels of the AHAS and bar transgenes in all 4 southern-positive transgenic lines were further validated by northern blotting and semi-quantitative RT-PCR. Northern hybridization was performed consecutively with AHAS and bar DIG probes. Initially, the membrane was hybridized with AHAS DIG probe which resulted in an ~2 kb band for all the plant samples; however, the hybridization signal was found to be higher in all the transgenic lines compared to the wt controls (Fig. 2C). Furthermore, the membrane was reprobed with bar DIG probe which resulted in an ~500 bp hybridization signal for all transgenic lines; this band was absent in the wt control. The experiment confirmed the expression of both transgenes. Further, the expression of the ActI constitutive gene was used as an internal standard which showed equal expression in all plant samples (Fig. 2C). Similarly, the semi-quantitative RT-PCR experiment showed a higher expression (almost 2-fold) of the AHAS gene in the transgenic lines compared to the wt control plants due to the active transcription of the AHAS transgene copy (Fig. 2D). All four transgenic lines showed expression of the bar gene in semi quantitative RT-PCR which was absent in wt control plants. The expression of the ActI gene was used as the internal standard to confirm equal quantities of cDNA used in all the experiments which showed its uniform expression in all plant samples (Fig. 2D).

The restriction digestion analysis of semi quantitative RT-PCR product of the AHAS gene from all the transgenic and wt control plants further helped to distinguish between the expression of OsmAHAS and OsAHAS. The internal AHAS gene primers were designed to amplify a region flanking the P171S (XbaI) mutation site. A 401 bp PCR band was amplified from all the transgenic and wt plants using cDNA and was digested with XbaI restriction enzyme. The PCR product of wt plant could not digested with XbaI restriction enzyme while all the other transgenic lines showed three distinct bands with 107, 294 and 401 bp DNA products. The PCR-based strategy was used as a molecular marker to estimate the relative expression of the native AHAS versus trans-AHAS genes in all transgenic lines (Fig. 2E).

Transgenic plants showed an increased tolerance to BM

The tolerance mutations of AHAS exhibit different degree of resistance to various classes of AHAS-inhibiting herbicides. Among all the tolerance mutations, the amino acid substitution at position P171 in the AHAS protein is frequently observed in many tolerant weed biotypes imparting a higher degree of tolerance to sulfonylureas (SU) and relatively lower level of tolerance to pyrimidinylcarboxylates (PC) herbicides22. To validate the level of tolerance offered by the introduced P171S mutation in rice AHAS, the transgenic rice seeds were screened against two popular AHAS-inhibiting herbicides BM (belongs to SU) and bispyribac sodium (BS; belongs to PC) at the germination stage. Initially we checked the natural tolerance of control rice seeds against various concentrations of BM and BS. The results showed a complete inhibition of root growth at 25 μM concentration for BM and 20 μM for BS. There was no significant difference in shoot growth observed (data not shown). The surface sterilized T3 homozygous seeds were inoculated on half-strength MS media containing 100 μM concentration of BS or BM herbicides (Figs 3 and 4). The wt seeds treated with and without herbicides were used as positive (+) and negative (−) controls respectively, and handled alike. In BM supplemented growth media, all the transgenic lines showed vigorous root growth which was comparable to that of the wt (−) control, as recorded after 7, 14 and 21 days of seed germination (Fig. 3). Further, the wt (+) control showed poor root growth even after 14 days; however, no significant difference in shoot lengths was observed which was similar to the wt (−) control. The root growth of the wt (+) control plants was completely inhibited after the 7th day, and no subsequent growth was observed (Fig. 3A, 3B).

Figure 3 Physiological analysis of the tolerance of transgenic plants to BM herbicide at the seed germination stage. (A) Transgenic lines (L1, L2, L4 and L5) grew normally in the 100 μM BM supplemented media with root and shoot growth similar to those of wt (−) plants. However, the root growth of wt (+) plants was severely arrested at this concentration, while no shoot length was affected, which was comparable to wt (−) plants. (B) Comparison of the effects of BM on root growth in wt and transgenic line 1. (C) Graphical representation of the root-shoot length ratio. wt (−): wild type without herbicide treatment, wt (+): wild type treated with herbicide, L: Line number of transgenic plants, DASG: Days after seed germination. Full size image

Figure 4 Comparative analysis of herbicide tolerance of transgenic plants to BM and BS at seed germination stage. (A) Effects of 100 μM BM and BS herbicides on seed germination and plant growth. Transgenic lines 1 and 2 efficiently tolerated to BM herbicide and showed normal phenotype similar to wt (−) plants. On the other hand, the root growth of transgenic lines 1 and 2 were significantly inhibited in BS supplemented media similar to wt (+) plants. (B) Comparison of root lengths between wt and transgenic line 1. (C) Graphical representation of the root-shoot length ratios of wt and transgenic lines. Wt (−): wild type without herbicide treatment, wt (+): wild type treated with herbicide, L: Line number of transgenic plants. Full size image

To compare the degree of tolerance offered by the transgenic lines to sulfonylureas (SU) and pyrimidinylcarboxylates (PC) class of herbicides, the T3 transgenic lines (1 and 2) were grown in half strength MS media supplemented with 100 μM BM and BS separately. The root growth of these transgenic lines in the BM supplemented media showed normal morphology, which was comparable to that of wt (−) controls (Fig. 4). However, the root growth was significantly arrested in both the transgenic lines in the BS media which was comparable to that of wt (+) plants (supplemented with 100 μM BS). Although, the transgenic plants showed a moderate level of tolerance to 50 μM BS at germination stage (data not shown). Furthermore, we did not observe any significant shoot growth differences among the transgenic lines, including the wt (−) and wt (+) controls, even at 21 days after seed germination (Fig. 4A–C).

The tolerant transgenic lines (1 and 2), which were confirmed by seed germination tests, were further examined to determine the effect of BM herbicide on the overexpressed mutant OsmAHAS gene in transgenic plants via color tests. Transgenic lines 1 and 2 developed a light red color due to the accumulation of acetoin resulting from the enzymatic action of mutant OsmAHAS proteins in the presence of 0.1 µM BM. The color of the wt (−) control reaction was dark red because of the uninhibited accumulation of high acetoin due to the lack of BM. However, the color of the wt (+) control reaction remained yellowish due to the presence of the inhibitor BM and the absence of mutant OsmAHAS. The native OsAHAS protein is unable to produce acetolactate due to the inhibitory action of BM, thus leading to no acetoin formation and the color of the reaction remained yellow (Fig. SI2A). The results showed active expression of the mutant OsmAHAS gene.

Confirmation of transgenic lines tolerance to phosphinothricin herbicide

Chlorophenol red is a color indicator dye that appears red at pH 6 and turns yellow with a decrease in pH. To validate the activity of the bar gene in transgenic plants, seeds from the wt control and transgenic groups were germinated on half strength MS media supplemented with 30 mg/L phosphinothricin herbicide. We observed a gradual change in media color from red to yellow after 10 days of seed inoculation in all transgenic lines, indicating active expression and function of the bar gene. The bar protein detoxified phosphinothricin rapidly which resulted in healthy growth of all transgenic plants (Fig. 5A–D); this result was comparable to that of the wt (−) controls. On the other hand, the wt (+) control plants were unable to detoxify phosphinothricin due to the absence of the bar, and consequently the media color remained unchanged (Fig. 5A,B). Unlike BM and BS herbicide, phosphinothricin has equal effects on both root and shoot growth (Fig. 5E). The root and shoot development was severely arrested in wt (+) plants while the transgenic plants showed normal growth.

Figure 5 Effects of phosphinothricin on transgenic plants at the seed germination stage. (A) Effect of 30 mg/L of phosphinothricin herbicide on the growth of transgenic and wt plants. All the transgenic plants survived in phosphinothricin and changed the color of the media from deep red to yellow; however, the wt (+) plants could not grow much and the color of media remained the same. (B) Stunted growth of wild-type plants in the presence of phosphinothricin. (C) Effects of phosphinothricin on the shoot growth of wt and transgenic plants. (D) Figure showing variation in the media color by wt and transgenic line. (E) Graphical representation of root-shoot length ratios of transgenic and wt plants in the presence of phosphinothricin herbicide. Full size image

We tested the effective tolerance of transgenic plants against commercially available phosphinothricin (marketed as ‘Basta’) during the vegetative stage. Basta (in which phosphinothricin is the active ingredient) is a contact herbicide that has a localized toxic effect on plant tissue. Phosphinothricin irreversibly binds to glutamine synthetase (GS), an important enzyme in the ammonia detoxification mechanism. The transgenic lines as well as control plants were painted (on leaf tip region) with 3% (v/v) basta herbicide at the 8–12 leaf stage. The localized toxic effect of basta killed the control plant leaves within 10 days of application due to the accumulation of ammonia, however, the transgenic lines (except line 4) did not show any leaf necrosis due to the detoxification of phosphinothricin by bar (Fig. SI2B). Phosphinothricin irreversibly binds to glutamine synthetase (GS), an important enzyme in the ammonia detoxification mechanism, and results in plant death.

Characterization of transgenic plants under the foliar application of BM and basta herbicides

The T3 homozygous transgenic lines as well as two wt control plants (one month old) were analyzed for their field level tolerance against the herbicides BM and basta. The first set of transgenic lines and wt controls were foliar sprayed with 300 μM BM (Fig. 6A, 6B). Necrotic symptoms started on wt (+) control plants after 4–5 days of BM application, and the leaves turned yellow and completely died after 15–20 days. However, all four transgenic lines showed healthy growth without any necrotic symptoms in response to the same BM application; their leaves stayed green which was comparable to the wt (−) control plants (without BM application) (Fig. 6A, 6B). There was no significant difference between the transgenic and wt (−) plants in terms of growth and morphology (Fig. 6C). The measurements of the total fresh weight of the herbicide treated transgenic lines did not show any growth penalty which suggests that the introduced P171S mutation added an advantage to the transgenic plants in providing tolerance to BM herbicide.

Figure 6 Effects of foliar spraying of BM herbicide on transgenic plants. (A) The transgenic plants efficiently tolerated 300 μM BM herbicide application without any visible necrotic symptoms while the wt (+) plants died shortly thereafter. (B) Effects of BM herbicide application on leaves. (C) Graphical representation of gram fresh weight of herbicide treated transgenic and wt plants. Full size image

Similarly, a second set of plants (transgenic lines and wt control) were sprayed with 2% (v/v) commercially available basta solution. The transgenic lines showed normal morphology with healthy leaves without any leaf necrosis similar to wt (−) plants. Furthermore, the total fresh biomass was analyzed which did not show any noticeable differences between the transgenic and wt (−) control plants, indicating no morphological penalty on the transgenic plants due to the active expression of bar (Fig. 7C). However, the wt (+) plants started showing necrotic symptoms after 6–7 days and completely died within 10–15 days (Fig. 7A, 7B).

Figure 7 Effects of foliar application of basta herbicide on transgenic plants. (A) Transgenic plants showed significant tolerance to 2% basta and were healthy even after 20 days of application, while the wt (+) plants died shortly thereafter. (B) Effects of basta on the leaf of wt and transgenic lines. (C) Graphical representation of total biomass of basta treated plants at 20 days after application. The graph shows no significant difference between the transgenic and wt (−) plants in terms of phenological attributes. Full size image

We also examined the effects of dual applications of the herbicides basta and BM on the transgenic plants. A set of transgenic lines and wt control plants were initially sprayed with a 2% (v/v) basta solution. At 7 days of basta application, the same set of plants was similarly sprayed with 300 μM BM. There was no phenological injury observed in the transgenic lines which remained green and healthy even at 20 days post-application (Fig. 8A). Further, we allowed the transgenic plants to grown until the maturity stage to determine the effects of dual applications of herbicides on yield. The transgenic plants did not show any yield penalty having normal seed setting and seed number per panicle which was similar to the wt (−) control plants (Fig. 8B, 8C).