3.6. Tissue-specific response to AZ activation through cell wall modification and hormone response signals
Next, we investigated target genes related to cell wall modification, which are substantially associated with AZ formation (Figure 5c). We selected one each of laccase and expansin families as the qRT-PCR target which were also present in the list of DEG analysis in 2018 (Supporting Information Table S1). Laccase 7 (LAC7 ) is involved in the lignin polymerization and discerns the non-separating cells from separating cells when plants undergo cell wall processing accompanied by the lignin brace during the abscission (Lee et al., 2018). The branch tissue showed the biggest gap in the change in expression from 6 to 18 hr after the initial cold shock. Expansins (EXPs ) are involved in cell wall loosening by disrupting the bond between celluloses and glycans (Ouyang et al., 2013). Here, in our study, both branch and pedicel maintained significantly up-regulated MdEXPA10 expression compared to that in control. JOINTLESS is responsible for AZ formation (Nakano et al., 2012). Within 6 hr, the expression of MdJb , a homolog of JOINTLESS, was significantly down-regulated in cold-stressed branch and pedicel, but it did not show a significant change at 18 hr.
Figure 6 illustrates the spatial relative expression of genes involved in hormone signal transduction including ethylene, auxin, and gibberellin. The expression of ACC synthase (MdACS1 ), a gene regulating ethylene biosynthesis, was significantly up-regulated in the cold-stressed subunits after 18 hr (Figure 6a). Additionally, both the branch and pedicel tissues revealed a significant difference in the expression of the ethylene receptor (MdETR2 ) at 18 hr. The expression of ethylene responsive factor 1 gene (MdERF1 ) was significantly up-regulated in fruit and branch tissues at 6 hr.
Auxin-related genes were down-regulated as a late response (Figure 6b). The expression of auxin efflux carrier MdPIN1 was significantly down-regulated in fruit and branch after 18 hr. Auxin/indole-3-acetic acid (Aux/IAA) family has been shown to repress the transcription of auxin-responsive factors, and also participate in abiotic stress tolerance (Shani et al., 2017). We selected MdIAA9 andMdIAA20 as target genes because MdIAA9 is thought to be involved in drought tolerance and MdIAA20 is related to chilling tolerance in apple (Huang et al., 2019). Interestingly, the cold-stressed fruit and pedicel tissues were found to maintain a significantly down-regulated MdIAA9 expression and MdIAA20was significantly down-regulated in branch and pedicel after 18 hr.
We compared the expression of GA 20-oxidase (MdGA20ox ) which regulates gibberellin (GA) biosynthesis (Figure 6c). The MdGA20oxexpression was found to be down-regulated in the cold-stressed fruit after 6 and 18 hr. RGLs which encode DELLA protein family are thought to be associated with cold stress response in apple (Wisniewski et al., 2015), and the expression of MdRGL2 was up-regulated in cold-stress exposed branch and pedicel at 6 hr, however, the pedicel rather maintained the significant up-regulation at 18 hr.