A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops

Roles of stomata in gramineous crops growth and biomass production

Stomata, microscopic pores surrounded by two guard cells, play essential roles in the most important plant physiological processes: photosynthesis and transpiration. Unlike dicotyledons, grasses, including major gramineous crops, have distinctive dumbbell-shaped guard cells and specialized subsidiary cells, forming a more efficient stomatal complex. Stomata are capable of governing growth, development, and biomass production by means of regulating the transpiration and gas exchange process in a plant; that is, the main functions of stomata are to permit CO2 entry and control H2O movement and supply nutrients for biomass accumulation via photosynthesis. However, little is known about the roles of stomata in gramineous crops growth and biomass production. Stomatal conductance (gs) proves to be a vital aspect for high-yield potential in crops by influencing all the key traits of a crop's life cycle, particularly its biomass accumulation. Furthermore, transpiration enables stomata to stimulate biomass allocation in the phloem tissue, facilitating the translocation of assimilates and signals from the designated source to the sink, further endorsing floral transition and biomass allocation to the reproductive organs including the seed yield characteristic. This review focuses on stomatal function of gramineous crops, like rice, wheat, maize, barley, and so on. While stomata enforce majority of the essential processes in crops, their performance remains highly prone to the effects of unfavorable environmental conditions. Thus, manipulation of stomatal regulation is useful for the promotion of crop growth and biomass production

Phylogenetic analysis of the five EPSPS and related proteins.

<p>The phylogenetic tree was based on homologous sequences of the EPSPS proteins and the neighbor-joining methods (MEGA4.0). The percentage of the tree from 1000 bootstrap resamples supporting the topology is indicated when above 50. Accession numbers or international patent publication numbers are shown in parentheses. The scale bar represents 0.1 substitutions per position.</p

Glyphosate tolerance of transgenic tobacco plants in a greenhouse when sprayed with 6 L ha⁻¹ Roundup®.

<p>T1 tobacco seeds were germinated on the MS medium containing 10 mg L⁻¹ PPT and grown for 7 days at 100 µmol s⁻¹ m⁻² with a 16 h light/8 h dark period. The live seedlings were transferred to soil in pots and grown for another month. Six-to eight-leaf stage transgenic plants were sprayed with 6 L ha⁻¹ Roundup®. Two weeks after treatment, injury was observed, and survival rate was measured. (A) Photograph of tobacco plants two weeks after glyphosate treatment. (B) Survival rate of tobacco plants. Data are shown as the average ± S.E. of seven to ten independent transgenic lines. Experimental data was tested by ANOVA analysis and different letter in each column means significant difference at <i>P</i><0.05 level.</p

Southern blot analysis of transgenic tobacco plants.

<p>(A) HTG7; (B) AM79; (C) A1501; (D) RD; (E) G2. Five transgenic lines each construct were analyzed. 100 µg genomic DNA was digested with <i>Hin</i>dIII which has only one site in the plasmid, so the band numbers are equal to the copy number of transgene.</p

Glyphosate tolerance of transgenic tobacco under different glyphosate concentrations.

<p>Tobacco seeds of generation T1 were germinated on the MS medium containing 10 mg L⁻¹ PPT and grown for 7 days at 100 µmol s⁻¹ m⁻² with a 16 h light/8 h dark period. The live seedlings were transferred to MS medium in plates containing different amounts of glyphosate and grown vertically for another two weeks.</p

Glyphosate tolerance of <i>E. coli</i> containing five <i>aroA</i> genes.

<p>The plasmids pACYC184, pACYC-HTG7, pACYC-AM79, pACYC-A1501, pACYC-RD and pACYC-G2 were transformed into <i>E. coli</i> ER2799 competent cells for growth curve measurement. M9 liquid medium was supplemented with different concentrations of glyphosate. OD₆₀₀ was recorded every two hours starting 6 h after treatment. Data are shown as the average ± S.E. of three independent experiments. Experimental data was tested by ANOVA analysis and different letter means significant difference at <i>P</i><0.05 level. (A) Growth curve of ER2799 and the strain harboring different plasmids under 0 mM glyphosate. (B) Growth curve of ER2799 and the strain harboring different plasmids under 20 mM glyphosate. (C) Growth curve of ER2799 and the strain harboring different plasmids under 100 mM glyphosate. (D) Growth curve of ER2799 and the strain harboring different plasmids under 150 mM glyphosate. (E) Growth curve of ER2799 and the strain harboring different plasmids under 200 mM glyphosate.</p

Glyphosate tolerance of transgenic tobacco seedlings on plates containing 1 mM glyphosate.

<p>T1 tobacco seeds were germinated on the MS medium containing 10 mg L⁻¹ PPT and grown for 7 days. The live seedlings were transferred to MS medium on plates containing 1 mM glyphosate. Photographs were taken two weeks later, and the fresh weight was measured at the same time. (A) Photograph of tobacco harboring <i>HTG7 aroA</i> and WT grown on the medium containing 1 mM glyphosate. (B) Photograph of tobacco harboring <i>AM79 aroA</i> and WT grown on the medium containing 1 mM glyphosate. (C) Photograph of tobacco harboring <i>A1501 aroA</i> and WT grown on the medium containing 1 mM glyphosate. (D) Photograph of tobacco harboring <i>RD aroA</i> and WT grown on the medium containing 1 mM glyphosate. (E) Photograph of tobacco harboring <i>G2 aroA</i> and WT grown on the medium containing 1 mM glyphosate. (F) Photograph of tobacco plants grown on the medium without glyphosate. (G) Fresh weight of the tobacco plants. Data are shown as the average ± S.E. of seven to ten independent transgenic lines. Experimental data was tested by ANOVA analysis and different letter in each column means significant difference at <i>P</i><0.05 level.</p

T-DNA cassette containing <i>aroA</i> gene for plant transformation.

<p>LB and RB, left and right border of T-DNA; bar, phosphinothricin acetyltransferase gene; nos, <i>NOS</i> terminator; CaMV 35S, cauliflower mosaic virus 35S promoter; SP, coding sequence of signal peptide of pea rib-1,5-bisphospate carboxylase (<i>rbcS</i>) small subunit; EPSPS gene, five microorganism <i>aroA</i> gene.</p