Feature, expression, and subcellular localization analyses ofMdHSFA9b and MdHSFA1d
Phylogenetic tree analysis showed that Apple and Arabidopsis HSFs were divided into four groups(Ⅰ,Ⅱ,Ⅲ,Ⅳ), with MdHSFA1a/b/d/e in groupⅡ, and MdHSFA9a/b in groupⅠ(Figure 8a). We also examined the expression patterns ofMdHSFs in different tissues of several apple varieties (Figure 8b; Table S2). And the expression levels of MdHSFA1a/b/d/e
and MdHSFA9a/b showed significantly higher in buds.
The subcellular localizations of MdHSFA9b and MdHSFA1dwere studied by independently introducing 35S::MdHSFA9b -EGFP and 35S::MdHSFA1d -EGFP, respectively, into tobacco leaves (Figure 8c). Tobacco leaves transformed with the empty vector 35S::EGFP served as controls. In the tobacco leaves expressing 35S::MdHSFA9b -EGFP and 35S::MdHSFA1d -EGFP, the GFP signals were observed in both the nucleus and cytoplasm, while the GFP signal was detected throughout the control tobacco leaf cells, indicating that MdHSFA9b andMdHSFA1d localized to both the nucleus and cytoplasm. The co-localization of MdNup62 with both MdHSFA9b andMdHSFA1d further verified their interactions.
A tissue-specific expression analysis revealed that MdHSFA1d was expressed highest in flower buds and stems. The highest expression level of MdHSFA9b was in stems, but the expression levels in the other tissues were also high. Subsequently, the expression levels of MdHSFA9b and MdHSFA1d remained high during the flower bud developmental stages, while the highest was at 70 days after flowering (Figure 8d). These results indicated that MdHSFA9b and MdHSFA1d maintained high expression levels during flower bud induction, suggesting that they may be involved in the bud differentiation of apple.