Characterization of expansin genes and their transcriptional regulation by histone modifications in strawberry.

MAIN CONCLUSION: The possible candidate expansin genes, which may be important for strawberry fruit softening, have been identified in the diploid woodland strawberry Fragaria vesca and the octoploid cultivated strawberry Fragaria × ananassa and their transcriptional regulation by histone modifications has been studied. Softening process greatly affects fruit texture and shelf life. Expansins (EXPs) are a group of structural proteins participating in cell wall loosening, which break the hydrogen bonding between cellulose microfibrils and hemicelluloses. However, our knowledge on how EXP genes are regulated in fruit ripening, especially in non-climacteric fleshy fruits, is limited. Here, we have identified the EXP genes in both the octoploid cultivated strawberry (Fragaria × ananassa) and one of its diploid progenitor species, woodland strawberry (Fragaria vesca). We found that EXP proteins in F. × ananassa were structurally more divergent than the ones in F. vesca. Transcriptome data suggested that FaEXP88, FaEXP114, FveEXP11 and FveEXP33 were the four candidate EXP genes more likely involved in fruit softening, whose transcript levels dramatically increased when firmness decreased during fruit maturation. Phylogenetic analyses showed that those candidate genes were closely clustered, indicating the presence of homoeolog expression dominance in the EXP gene family in strawberry. Moreover, we have performed chromatin immunoprecipitation (ChIP) experiments to investigate the distribution of histone modifications along the promoters and genic regions of the EXP genes in F. vesca. ChIP data revealed that the transcript levels of EXP genes were highly correlated with the enrichment of H3K9/K14 acetylation and H3K27 tri-methylation. Collectively, this study identifies the key EXP genes involved in strawberry fruit softening and reveals a regulatory role of histone modifications in their transcriptional regulation, which would facilitate functional studies of the EXP genes in the ripening of non-climacteric fruits.

A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits.

BACKGROUND: Covalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation. RESULTS: We describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated for the enriched regions. Compared to X-ChIP (cross-linked chromatin followed by immunoprecipitation), our optimized N-ChIP procedure has a higher signal-to-noise ratio and a lower background for both the active and the silent histone modifications. Furthermore, high-throughput sequencing following N-ChIP demonstrates that nearly 90% of the enriched H3K9/K14ac peaks are overlapped between biological replicates, indicating its remarkable consistency and reproducibility. CONCLUSIONS: An N-ChIP method suitable for the fleshy fruit tissues of woodland strawberry Fragaria vesca is described in this study. The efficiency and reproducibility of our optimized N-ChIP protocol are validated by both qRT-PCR and high-throughput sequencing. We conclude that N-ChIP is a more suitable method for strawberry fruit tissues relative to X-ChIP, which could be combined with high-throughput sequencing to investigate the impact of histone modifications in strawberry and potentially in other fruits with high content of polysaccharides.