P1 Epigenetic Regulation in Leaves of High Altitude Maize Landraces: Effect of UV-B Radiation

Abstract

P1 is a R2R3-MYB transcription factor that regulates the accumulation of a specific group of flavonoids in maize floral tissues, such as flavones and phlobaphenes. P1 is also highly expressed in leaves of maize landraces adapted to high altitudes and higher levels of UV-B radiation. In this work, we analyzed the epigenetic regulation of the P1 gene by UV-B in leaves of different maize landraces. Our results demonstrate that DNA methylation in the P1 proximal promoter, intron1 and intron2 is decreased by UV-B in all lines analyzed; however, the basal DNA methylation levels are lower in the landraces than in B73, a low altitude inbred line. DNA demethylation by UV-B is accompanied by a decrease in H3 methylation at Lys 9 and 27, and by an increase in H3 acetylation. smRNAs complementary to specific regions of the proximal promoter and of intron 2 3' end are also decreased by UV-B; interestingly, P1 smRNA levels are lower in the landraces than in B73 both under control conditions and after UV-B exposure, suggesting that smRNAs regulate P1 expression by UV-B in maize leaves. Finally, we investigated if different P1 targets in flower tissues are also regulated by this transcription factor in response to UV-B. Some targets analyzed show an induction in maize landraces in response to UV-B, with higher basal expression levels in the landraces than in B73; however, not all the transcripts analyzed were found to be regulated by UV-B in leaves.

Keywords: P1 transcription factor; UV-B; epigenetic regulation; flavonoids; maize.

Figure 1

DNA methylation changes in three regions of the P1 gene in the B73 inbred line and Arrocillo (ARR) and Mishca (MIS) landraces after 8 h of UV-B exposure. Methylation percentage measured by qPCR of digested DNA from the P1 proximal promoter, introns 1 and 2 under control conditions and after UV-B exposure with HpaII/MspI restriction enzymes (A). Ratio of DNA methylation in UV-B exposed plants vs. DNA methylation in control plants (B). Primers were designed to amplify across the restriction sites (Table 1); therefore, amplification is expected if DNA is methylated and not digested. Three biological replicates were performed for each sample, and three qPCR experiments were done with each sample. Error bars are standard errors. Statistical significance was analyzed using ANOVA, Tukey test with P < 0.05; differences from the control are marked with different letters.

Figure 2

Methylation state of H3 K9 and K27 associated with the P1 proximal promoter, introns 1 and 2 of B73, Arrocillo (ARR), and Mishca (MIS). ChIP assays were done using antibodies specific for H3K9me2, H3K27me3, or total H3 in nuclei from leaves after an 8-h-UV-B treatment (UV-B) or kept under control conditions in the absence of UV-B (control). The immunoprecipitates were analyzed for the presence of the P1 proximal promoter (A), intron 1 (B), and intron2 (C) sequences by qPCR. Enriched fractions from UV-B treated vs. control plants were compared. ChIP data were normalized to input DNA before immunoprecipitation. The signal detected in samples incubated in the absence of any antibody as a control was less than 5% of the signal when antibodies were used. Error bars are standard errors. Statistical significance was analyzed using ANOVA, Tukey test with P < 0.05; differences from the control are marked with different letters. Three biological replicates of chromatin immunoprecipitation (ChIP) were performed from each genotype/treatment sample type, and three qPCR experiments were done with each sample.

Figure 3

Methylation state ratio of H3 K9 and K27 associated with the P1 proximal promoter, introns 1 and 2 of B73, Arrocillo (ARR) and Mishca (MIS) enriched fractions from UV-B treated vs. control conditions plants. P1 proximal promoter (A), intron 1 (B), and intron 2 (C) sequences were analyzed. ChIP data were normalized to input DNA before immunoprecipitation. Absolute values correspond to data shown in Figure 2. The signal detected in samples incubated in the absence of any antibody as a control was less than 5% of the signal when antibodies were used. Error bars are standard errors. Statistical significance was analyzed using ANOVA, Tukey test with P < 0.05; differences from the control are marked with different letters. Three biological replicates of chromatin immunoprecipitation (ChIP) were performed from each genotype/treatment sample type, and three qPCR experiments were done with each sample.

Figure 4

Acetylation state of H3 associated with P1 regions of B73, Arrocillo (ARR) and Mishca (MIS). ChIP assays were done using antibodies specific for N-terminal acetylated H3, in nuclei from leaves after an 8-h-UVB treatment (UV-B) or kept under control conditions in the absence of UV-B (control). The immunoprecipitates were analyzed for the presence of P1 sequences of the proximal promoter, introns 1 and 2. Enriched fractions from UV-B treated vs. control plants were compared. ChIP data were normalized to input DNA before immunoprecipitation. The signal detected in samples incubated in the absence of any antibody as a control was less than 5% of the signal when antibodies were used. Error bars are standard errors. Statistical significance was analyzed using ANOVA, Tukey test with P < 0.05; differences from the control are marked with different letters. Three biological replicates of chromatin immunoprecipitation (ChIP) were performed from each genotype/treatment sample type, and three qPCR experiments were done with each sample.

Figure 5

Expression of smRNAs complementary to the P1 proximal promoter and the 3′ end of intron 2 in leaves of B73, Arrocillo (ARR) and Mishca (MIS). Plants were irradiated with UV-B for 8 h or kept under control conditions in the absence of UV-B. (A) Northern blot analysis developed using ³²P-labeled smRNA probes complementary to the P1 proximal promoter (probes 4 and 5) and the 3′ end of intron 2 (probe 6), described in Table 3, or alternatively with an specific U6 probe. U6 mRNA was used as a control of equal loading of RNA in each lane. Each blot is representative of three individual experiments. (B) Densitometry analysis of replicated experiments normalized to U6. Different letters indicate significant differences between samples from control and UV-B irradiated plants (p < 0.05).

Figure 6

Expression analysis of P1 target genes under control conditions and after UV-B exposure in leaves of B73, Arrocillo (ARR) and Mishca (MIS). Transcript levels relative to the reference thr-like gene that is not regulated by UV-B are shown. Statistical significance was analyzed using ANOVA, for each sample analyzed, different letters indicate significant differences with P < 0.05.