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. 2024 Aug 7;14(1):18278.
doi: 10.1038/s41598-024-69373-9.

The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare)

Ewa Sybilska  1 , Anna Collin  1 , Bahareh Sadat Haddadi  2 , Luis A J Mur  2 , Manfred Beckmann  2 , Wenbin Guo  3 , Craig G Simpson  4 , Agata Daszkowska-Golec  5
Affiliations

The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare)

Ewa Sybilska et al. Sci Rep. .

Abstract

To decipher the molecular bases governing seed germination, this study presents the pivotal role of the cap-binding complex (CBC), comprising CBP20 and CBP80, in modulating the inhibitory effects of abscisic acid (ABA) in barley. Using both single and double barley mutants in genes encoding the CBC, we revealed that the double mutant hvcbp20.ab/hvcbp80.b displays ABA insensitivity, in stark contrast to the hypersensitivity observed in single mutants during germination. Our comprehensive transcriptome and metabolome analysis not only identified significant alterations in gene expression and splicing patterns but also underscored the regulatory nexus among CBC, ABA, and brassinosteroid (BR) signaling pathways.

Keywords: ABA; Alternative splicing; Barley; Cap-binding complex; Embryo; Germination; Transcriptome.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Germination of hvcbp20.ab, hvcbp80.b, hvcbp20.ab/hvcbp80.b and the WT in the presence of 75 µM ABA. (a) Seed germination percentage at 1 DAI. (b) Seed germination phenotypes at 1 DAI. Bar = 1 cm (c) Embryo germination phenotypes at 1 DAI. Bar = 3 mm. (d) Seed germination percentage at 7 DAI. (e) Seed germination phenotypes at 7 DAI in control conditions. (f) Seed germination phenotypes at 7 DAI in ABA presence. Bar = 1 cm DAI (day after imbibition). Statistical analyses were performed using one-way ANOVA (P ≤ 0.05) with post-hoc Tukey HSD (Honestly Significant Difference). Statistically significant differences (P ≤ 0.05) are marked by different lower-case letters.
Figure 2
Figure 2
Global analysis of differentially expressed genes (DEG) of hvcbp20.ab, hvcbp80.b, hvcbp20.ab/hvcbp80.b and WT after 75 µM ABA treatment compared to control conditions. (a) Co-expressed clusters of DEGs in response to ABA. (b) Heatmap of DEGs and enriched GO biological processes in the C8 cluster. (c) Heatmap of DEGs and enriched GO biological processes in the C11 cluster. (d) Venn diagram of DEGs displaying up- and downregulated exclusively expressed genes in each contrast group. (e) Overrepresented GO biological processes among upregulated and downregulated exclusive DEGs in hvcbp20.ab/hvcbp80.b.
Figure 3
Figure 3
Global analysis of genes with differential alternative splicing (DAS) genes and differentially expressed transcripts (DET) in hvcbp20.ab, hvcbp80.b, hvcbp20.ab/hvcbp80.b and WT after 75 µM ABA treatment compared to control conditions. (a) Venn diagram of DAS genes in each contrast group. (b) Venn diagrams displaying the amount of exclusive DAS genes with DTU transcripts. (c) Venn diagram of DETs displaying up- and downregulated exclusively expressed transcripts in each genotype. (d) Overrepresented GO biological processes among upregulated and downregulated exclusive DETs in hvcbp20.ab/hvcbp80.b. (e) The number of genes encoding transcripts of splicing factors (SF) with the corresponding number of transcripts of SFs among exclusive DET in each genotype.
Figure 4
Figure 4
Network of protein–protein in silico physical interaction with CBP20 (red) and CBP80 (red) among differentially expressed genes (DEG) and genes with differential alternative splicing (DAS) in hvcbp20.ab, hvcbp80.b, hvcbp20.ab/hvcbp80.b and WT after 75 µM ABA treatment compared to control conditions.
Figure 5
Figure 5
Metabolome profiling analysis of germinating embryos of hvcbp20.ab, hvcbp80.b, hvcbp20.ab/hvcbp80.b and WT after 75 µM ABA treatment. (a) Principal component analysis (PCA) score plot of the metabolic profiles. (b) Endogenous 6-alpha-hydroxy-6-deoxycastasterone (6-OH-6-deoxoCS) content.
Figure 6
Figure 6
Comparisons of BarkeRTD, Isoseq RTD, RNAseq RTD and BaRTv2.18 on (a) splice junctions and (b) intron combinations of multi-exon transcripts. Splice junctions or Intron combinations shared by multiple transcripts were only counted once.
Figure 7
Figure 7
Hypothetical model explaining the seed germination phenotype of the hvcbp20.ab/hvcbp80.b in the presence of ABA linked with BR signaling. In the presence of ABA in the hvcbp20.ab/hvcbp80.b double mutant, the unknown transcription factor from the CPP-family is exclusively upregulated. This transcription factor potentially binds SAPK10 and inhibits its expression. It leads to reduced phosphorylation of the TGA6 transcription factor by SAPK10 and reduced expression of the REM4.1 gene encoding a negative regulator of BR signaling. Additionally, in hvcbp20.ab/hvcbp80.b, in response to ABA, the BKI1 gene encoding the negative regulator of the BR signaling pathway is exclusively downregulated. All this together causes the seed germination process in the hvcbp20.ab/hvcbp80.b double mutant to be inhibited in the presence of ABA. The dashed lines represent possible interactions. The solid lines represent known interactions. The green color indicates exclusively differentially upregulated genes in hvcbp20.ab/hvcbp80.b in response to ABA. The red indicates exclusively differentially downregulated genes in hvcbp20.ab/hvcbp80.b in response to ABA. P- phosphorylation. Illustration created with BioRender.
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References

    1. Bradford, K. J. & Nonogaki, H. Seed Development, Dormancy and Germination (Blackwell Publishing Ltd, 2007). 10.1002/9780470988848.
    1. Boesewinkel, F. D. & Bouman, F. The seed: structure. In Embryology of Angiosperms (ed. Johri, B. M.) 567–610 (Springer Berlin Heidelberg, 1984). 10.1007/978-3-642-69302-1_12.
    1. Potokina, E., Sreenivasulu, N., Altschmied, L., Michalek, W. & Graner, A. Differential gene expression during seed germination in barley (Hordeumvulgare L.). Funct. Integr. Genom.2, 28–39 (2002). - PubMed
    1. Sreenivasulu, N. et al. Barley grain maturation and germination: Metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools. Plant Physiol.146, 1738–1758 (2008). - PMC - PubMed
    1. Soós, V. et al. Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1. BMC Plant Biol.10, 236 (2010). - PMC - PubMed

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