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. 2024 Jul 18;22(11):3037–3050. doi: 10.1111/pbi.14426

Figure 2.

Figure 2

A 328‐bp insertion (InDel‐41) in TIP4;3 is significantly associated with maize chilling tolerance. (a) Relative expression levels of TIP2;1, TIP3;2 and TIP4;3 after cold treatment at 4 °C for 0, 6, 12 and 24 h. Samples from seedlings maintained at permissive conditions (25 °C) were collected as a 0‐h time point. (b) The gene expression of TIP4;3, TIP3;2 and TIP2;1 in hyper‐sensitive and hyper‐tolerant inbred lines at permissive condition (25 °C) or after cold treatment (4 °C) for 12 h. Different letters represent significant differences (P < 0.05, one‐way ANOVA). “n” represents the number of inbred lines. (c) Local Manhattan plot showing the association analysis of genetic variation at the TIP4;3 locus with chilling tolerance in maize and the pattern of pairwise linkage disequilibrium of DNA polymorphisms. A diagram of the TIP4;3 locus is shown. The white, light blue and red rectangles represent 1.3‐kb promoter, UTR and coding region, respectively. The most significant Indel (Indel‐41) in the promoter is connected to its location in the locus diagram by a dotted line. The transverse dotted line represents the threshold line. (d) Area of leaf injury (%) of inbred lines harbouring the Hap1 or Hap2 haplotypes of TIP4;3. (e) Relative TIP4;3 transcript levels in inbred lines harbouring the Hap1 or Hap2 haplotypes grown at 25 °C or exposed to 4 °C for 12 h. The 21 Hap1 and 16 Hap2 lines were randomly selected for gene expression analysis. (f) Dual luciferase (LUC) reporter assays, using firefly LUC controlled by the TIP4;3 B73, TIP4;3 CIMBL52 or TIP4;3 CIMBL52Δ promoter in maize protoplasts under 4 °C for 3 h. (g) Chromatin immunoprecipitation followed by quantitative PCR (ChIP‐qPCR) showing the chromatin state at the TIP4;3 4019 and TIP4;3 384‐2 promoters following cold stress treatment. Anti‐H3K27me3 (left) and anti‐H3K9me2 (right) antibodies were used for immunoprecipitation. Five regions (P1–P5) of the TIP4;3 promoter were tested; the maize Ubiquitin (Ubi) was used as a negative control.