Abstract
Plants can regenerate new individuals under appropriate culture conditions. Although the molecular basis of shoot regeneration has steadily been unraveled, the role of age-dependent DNA methylation status in the regulation of explant regeneration remains practically unknown. Here, we established an effective auxin/cytokinin-induced shoot regeneration system for the resurrection plant Boea hygrometrica via direct organogenesis and observed that regeneration was postponed with increasing age of donor plants. Global transcriptome analysis revealed significant upregulation of genes required for hormone signaling and phenylpropanoid biosynthesis and downregulation of photosynthetic genes during regeneration. Transcriptional changes in the positive/negative regulators and cell wall-related proteins involved in plant regeneration, such as ELONGATED HYPOCOTYL5 (HY5), LATERAL ORGAN BOUNDARIES DOMAIN, SHOOT-MERISTEMLESS, and WUSCHEL, were associated with the regeneration process. Comparison of DNA methylation profiling between leaves from young seedlings (YL) and mature plants (ML) revealed increased asymmetrical methylation in ML, which was predominantly distributed in promoter regions of genes, such as HY5 and a member of ABA-responsive element (ABRE) binding protein/ABRE binding factor, as well as genes encoding glycine-rich cell wall structural protein, CENTRORADIALIS-like protein, and beta-glucosidase 40-like essential for shoot meristem and cell wall architecture. Their opposite transcription response in ML explants during regeneration compared with those from YL demonstrated the putative involvement of DNA methylation in regeneration. Moreover, a significant lower expression of DNA glycosylase-lyase required for DNA demethylation in ML was coincident with its postponed regeneration compared with those in YL. Taken together, our results suggest a role of promoter demethylation in B. hygrometrica regeneration.
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Abbreviations
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- 6-BA:
-
6-benzylaminopurine
- ABA:
-
abscisic acid
- BSP:
-
bisulfite sequencing PCR
- DEG:
-
differentially expressed gene
- DMG:
-
differentially methylated gene
- DMR:
-
differentially methylated region
- GO:
-
Gene Ontology
- KEGG:
-
Kyoto Encyclopedia of Genes and Genomes
- mC:
-
methylcytosine
- ML:
-
leaves from mature plants
- qPCR:
-
quantitative real-time PCR
- RNA-Seq:
-
high-throughput mRNA sequencing
- SIM:
-
shoot-inducing medium
- WGBS:
-
whole-genome bisulfite sequencing
- YL:
-
leaves from young seedlings
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This work was supported by the National Natural Science Foundation of China (grant numbers 31770293 and 31470361).
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XD conceived the experiments. EZ, JQ, and YL prepared the plant materials. RS analyzed the data and wrote the manuscript. XD and CL revised the manuscript. All authors have read and approved the manuscript.
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Fig. S1
Effects of different compositions and concentrations of plant hormones on shoot regeneration. (PNG 2602 kb)
Fig. S2
Young (a) and mature (b) donor plants used for comparison of regeneration capacity. (PNG 2438 kb)
Fig. S3
KEGG pathway enrichment analysis among up- and downregulated DEGs in YL explants during regeneration. (PNG 461 kb)
Fig. S4
Expression profile of transcription factors in YL explants during regeneration. (PNG 609 kb)
Fig. S5
Expression profile of genes involved in hormone signaling in YL explants during regeneration. (PNG 3389 kb)
Fig. S6
WGBS depth and saturation of YL (a) and ML (b). (PNG 382 kb)
Fig. S7
Distribution of the DNA methylation level in YL (a) and ML (b). (PNG 348 kb)
Fig. S8
Average mC levels of gene loci in YL (a) and ML (b) and a comparison between samples (c). (PNG 733 kb)
Fig. S9
Correlation analysis of DEGs in YL explants during regeneration and methylation differences in the corresponding YL and ML regions. (PNG 1254 kb)
Fig. S10
BSP validation of two representative DMRs between YL and ML. (PNG 279 kb)
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Sun, RZ., Zuo, EH., Qi, JF. et al. A role of age-dependent DNA methylation reprogramming in regulating the regeneration capacity of Boea hygrometrica leaves. Funct Integr Genomics 20, 133–149 (2020). https://doi.org/10.1007/s10142-019-00701-3
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DOI: https://doi.org/10.1007/s10142-019-00701-3