A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

Abstract

Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.

Keywords: Arabidopsis thaliana; circadian rhythm; glyphosate; herbicide resistance; phyB; shikimate pathway.

Figure 1

Gre1 plants show glyphosate resistant phenotypes.(a) Phenotypes of wt and gre1 plants screened on 15 or 30 μm glyphosate medium for 12 days under LI = 22 ± 2 μmol m⁻² sec⁻¹, 16 h light regime. Close-up pictures show shoot and root phenotypes of wt and gre1 plants. White scale bars represent 5 mm, and empty arrowheads point to the primary leaves.(b–d) Fresh weight, relative chlorophyll content and shikimate level of wt and gre1 plants in glyphosate dose–response studies. Fresh weight data were taken at day 12 and chlorophyll content and shikimate analyses were done at day 9. Each data point represents mean ± standard errors (SEs), with 15 plants analysed for fresh weight, and five plants for chlorophyll and shikimate content analyses. Double and single asterisks indicate P-values < 0.05 and < 0.1 respectively.

Figure 2

The glyphosate resistant phenotypes of gre1 are conferred by the knock-out mutation of PHYB gene. (a) TAIL-PCR revealed a T-DNA insertion (triangle) at the first exon of PHYB gene (At2g18790) in the gre1 plant. Arrow indicates the left border of T-DNA at the 1.45 kb downstream of the start codon.(b) RT-PCR analysis of PHYB expression in total RNA extracted from wt and gre1 plants. Expression of the housekeeping gene, UBIQUITIN10 (UBQ10) was used to normalize band intensities.(c) Comparison of glyphosate hypersensitive and resistance phenotypes of wt and gre1 plants with an allelic phyB mutant line (phyB-9), PHYB over-expressed lines in wt (pbc1) and in gre1 (gpbc1), a phyA deficient line (phyA-201), and plastid heme-oxygenase loss-of-function mutant (hy1-100), in 30 μm glyphosate treatments under a 16 h, 22 ± 2 μmol m⁻² sec⁻¹ light regime. Close-up pictures depicting shoot growth are placed below each corresponding line. Pictures were taken at day 12 of the treatment. Scale bars = 10 mm.

Figure 3

Low R:FR light causes glyphosate hypersensitivity. (a) Four-day-old wt and gre1 plants were tested under high (3.8 μmol m⁻² sec⁻¹) and low (0.09 μmol m⁻² sec⁻¹) R:FR light enriched with white light (total LI = 22 ± 2 μmol m⁻² sec⁻¹) in a 16 h light regime with (20 μm) or without (control) glyphosate treatments for 9 days. White scale bars are 10 mm.(b) Shikimate assay was done on glyphosate treated plants treated under the same conditions described above. Data represent the mean of five plants with ± standard error (SE). Asterisk indicates P-values < 0.05.(c) Shikimate pathway regulatory gene expression was performed by qPCR on 12-day-old, agar plate grown plants treated under white (W), high or low R:FR light conditions, or dark (D) condition for 2 h after the 8 h dark period. Relative transcript abundance was calculated using W light treated wt mRNA as the calibrator and normalized with respect to βACTIN7 gene transcript level. Error bars represent ± SE of the mean of three samples. Ratio of relative transcript numbers in high R:FR/W of both genotype are written next to the corresponding gene. Asterisks indicate a significant increase.

Figure 4

The shikimate pathway is affected by the circadian clock and phyB signaling.Twelve-day-old agar plate grown plants under LI = 22 ± 2 μmol m⁻² sec⁻¹ were used in these experiments. Values are means ± standard error (SE) of three replicates and times start from 0 h as day light begins. Shaded areas indicate the night cycle. Data are expressed as log2 and asterisks indicate significant differences.(a) Transcript accumulation of shikimate pathway components under regular day-night regime. Ratio of relative transcript numbers at 1 h/8 h and 16 h/8 h of both genotypes are written next to the corresponding gene. (b) Transcript abundance of circadian clock and phyB downstream signaling elements. Ratios of transcript levels of gre1/wt are written above the bars of the corresponding genes. (c) Transcript level of shikimate pathway and circadian clock components under continuous light condition with the ratios of 30 ZT/24 ZT plotted next to the corresponding genes.

Figure 5

Glyphosate effects are dependent on the time of day at which spraying occurs. (a) Comparison of plant survival rates after 200 μm glyphosate (0.2535 mg m⁻²) application at 5:00, 9:00, 13:00, 17:00 and 21:00 h on 20-day-old greenhouse grown plants and the control was a spray without glyphosate. Pictures were taken 12 days after the treatment.(b) Shoot length of glyphosate treated plants. Measurement was done at day 12 after the application (n = 10).

Figure 6

A model proposing controls of the shikimate pathway.PhyB exerts control over the pathway by changing its active and non-active, Pfr and Pr, conformations according to the light quality (R:FR). The R:FR increases gradually from the onset of light till midday and decreases towards evening. Expression of DAHPS1 and SK1 is repressed by the circadian component CCA1 during the day, while phyB (Pfr) promotes the accumulation of DHQS and EPSPS1 in response to high R:FR through the interaction with PIF4 and PIF5. During the evening, EC elements have a role in stabilizing PIF4 and PIF5. Arrows and blunt end lines indicate stimulatory and inhibitory effects respectively, and dashed arrows indicate transcriptional activation. The proposed feed-back regulation of the circadian clock and EC-dependent regulation are shown as embossed arrows.