Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel

Abstract

Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1.

Keywords: Bisulfite sequencing; DNA methylation; branched broomrape (Phelipanche ramosa); dormancy release; epigenetic regulation; parasitic plant; seed germination; strigolactone..

Fig. 1.

The conditioning period is not controlled by ABA. (A) Germination rate of P. ramosa seeds conditioned in IM supplemented or not with 250 µM ABA. After different lengths of conditioning, seeds were stimulated by 1nM GR24 and germination rates were determined after 3 days. Means are values ± SD (n = 6). (B) RT-PCR analysis of the PrCYP707A1 expression in seeds conditioned for different lengths of conditioning in IM supplemented or not with 1mM ABA. After conditioning, seeds were stimulated by 1nM GR24 and expression levels were determined after 6h. Means are values ± SD (n = 3). Means with the same letter are not significantly different from each other (Tukey test, P < 0.05).

Fig. 2.

A global DNA demethylation occurs during the conditioning period. (A) Germination rate of P. ramosa seeds conditioned in IM supplemented or not with 250 µM 5-azacytidine or 250 µM hydroxyurea. After different lengths of conditioning, seeds were stimulated by 1nM GR24 and germination rates were determined after 3 days. Means are values ± SD (n = 6). (B) Percentage of 5-mC in seeds conditioned for different lengths of conditioning in IM supplemented (black bars) or not with 1mM 5-azacytidine (white bars) or 1mM hydroxyurea (grey bars). Means are values ± SD (n = 3). Means with the same letter are not significantly different from each other (Tukey test, P < 0.05).

Fig. 3.

Global DNA methylation controls PrCYP707A1 expression. RT-PCR analysis of the PrCYP707A1 expression in seeds conditioned 2 (D2), 3 (D3), or 7 (D7) days in IM supplemented or not with 1mM 5-azacytidine. After conditioning, seeds were stimulated by 1nM GR24 (+GR24) or not (−GR24) and expression levels were determined after 6h. Means are values ± SD (n = 3). Means with the same letter are not significantly different from each other (Tukey test, P < 0.05).

Fig. 4.

The hypermethylating compound hydroxyurea applied during the conditioning period inhibits GR24-dependent up-regulation of PrCYP707A1 and subsequent germination. (A) Germination rates of P. ramosa seeds conditioned in IM supplemented or not with 250 µM hydroxyurea. After 7 days of conditioning, seeds were stimulated by 1nM GR24 (+GR24) or not (−GR24) and germination rates were determined after 3 days. Means are values ± SD (n = 6). (B) RT-PCR analysis of the expression of PrCYP707A1 in seeds conditioned 7 days in IM supplemented or not with 1mM hydroxyurea. After conditioning, seeds were stimulated (+GR24) or not (−GR24) by 1nM GR24 treatment and expression levels were determined after 6h. Means are values ± SD (n = 3). Means with the same letter are not significantly different from each other (Tukey test, P < 0.05). “Hydroxyurea + GR24” corresponds to a treatment with both compounds after conditioning.

Fig. 5.

DNA demethylation occurs in the promoter of PrCYP707A1. (A) Localization of CpG islands and primer sets (black arrow) used in MeDIP-PCR on the PrCYP707A1 promoter. Values correspond to island and primer positions in relation to the +1 transcription start site. (B) Methylation status of CpG islands on the PrCYP707A1 promoter assayed by MeDIP-PCR. Expression values after 0 or 7 days of conditioning were normalized with non-immunoprecipitated DNA. Values are percentages of immunoprecipitated DNA compared to initial DNA quantity used in the immunoprecipitation assay (input DNA %). Values are means ± SD (n = 3). An asterisk indicates a significant methylation difference between 0 and 7 days of conditioning (Tukey test, P < 0.05). (C) Nucleotide sequence of CpG island 2. Underlined nucleotides correspond to the −1851/−1753 region exhibiting a DNA demethylation as shown by MeDIP-PCR. Nucleotides in bold correspond to the −1839/−1762 sequence mostly demethylated during conditioning as demonstrated by bisulfite sequencing.

Fig. 6.

DNA demethylation occurs in a 78-bp region of the PrCYP707A1 promoter. (A) Cytosine methylation variations of the CpG island 2 in the PrCYP707A1 promoter during seed conditioning assayed by bisulfite sequencing. DNA from D0 and D7 seeds were treated with bisulfite and the CpG island 2 region was amplified. PCR products were cloned and a total of 20 clones per condition were sequenced. Negative numbers in the x-axis indicate cytosine position from the transcription start site. For a given cytosine, a vertical bar corresponds to the cytosine methylation variation, expressed as a percentage, calculated according to the following formula: (number of clones showing a methylated cytosine at D7 minus the number of clones showing a methylated cytosine at D0) divided by 20. Thus, a positive value indicates a cytosine methylation process, a negative value a cytosine demethylation process, and a null value no variation in the methylation status during the conditioning period. (B) Detailed methylation profiles for CpG island 2 at 0 (D0) and 7 days (D7) of conditioning. Potentially methylated sites CG, CHG, and asymmetric CHH (H = A, T, or C) are shown by diamonds, squares, and triangles, respectively. According to their methylation percentages, cytosine sites are considered as hypermethylated (% of methylated cytosine > 50%, filled symbols), or hypomethylated (% of methylated cytosine < 50%, open symbols) (Trap-Gentil et al., 2011). (C) CG methylation status of CpG island 2 at 0 (D0), 3 (D3), 4 (D4), and 7 days (D7) of conditioning assayed by MS-PCR and three primer sets (black arrow). The McrBC enzyme cuts in hypermethylated regions.