3.5. Tissue-specific response to cold activates ABA signal transduction to potentially induce early fruit abscission
To understand the molecular mechanism underlying early cold response and abscission signals, we conducted qRT-PCR with excised tree subunits and compared tissue-specific responses. Within 6 hr of incubation at 25 °C followed by initial cold, the expression of ABA biosynthesis geneMdNCED1 was up-regulated in pedicel tissues (Figure 4). However, the expression decreased dramatically within the next 12 hr, whereas there was a delayed up-regulation of MdNCED1 in neighbouring fruit and branch. The significant up-regulation of MdCYP707A , the ABA 8’ hydroxylase which is a key regulator of ABA metabolism (Saito et al., 2004), was observed after 18 hr in cold-stressed pedicel.
Next, we investigated the spatial expression pattern of the ABA receptor genes of the PYL family (MdPYL3 , MdPYL8 ). After 18 hr of incubation at 25 °C, the expression of MdPYL8 , a mediator of the drought stress response (Lee et al., 2015), increased significantly in cold-stressed pedicel but neither in fruit or branch. In contrast,MdPYL3 , which is involved in cold and drought stress response (Lenka et al., 2018), was significantly up-regulated in both fruit and branch tissues. In the cold-stressed pedicel, the down-regulation of ABA repressor MdPP2C was sustained at both 6 and 18 hr of incubation, whereas the branch tissue did not show any significant difference. The ABA Insensitive 5 gene (MdABI5 ) plays a key role in regulating the core ABA metabolism signalling (Yan et al., 2012; reviewed by Skubacz et al., 2016). MdABI5 was significantly up-regulated in fruit within 6 hr. On the other hand, its expression was significantly down-regulated in the pedicel after 18 hr. Interestingly, the expression of MdWRKY40 , the repressor of ABA downstream response, was up-regulated in all tissue types during the early response (6 hr), but it was all significantly down-regulated within the next 12 hr.
The CBF family is a key regulator of cold stress response which can also be activated by ABA (Knight et al., 2004). In Figure 5a, MdCBF2gene expression, which is known to play a key role and highly responsive to cold in CBF regulon (Novillo et al., 2007; Tacken et al., 2010), increased significantly after 18 hr in fruit and branch, while, there was no significant change in the pedicel. As a consequence of cold response, not only is the expression of COR genes activated by CBF signals, but the dehydration response is induced as well (reviewed by Chinnusamy et al., 2007). We selected CBF downstream targets including one of COR genes, Cold shock protein 120 (CS120 )- like and a couple of drought-responsive genes which differed in expression among tissue types between cold shock treatment and control (Figure 5b). Within 6 hr after the cold shock, the expression of MdCS120-like was up-regulated in all tissue types but more extensively in the branch and pedicel. The expression of response to dehydration 22 (MdRD22 ) gene, which is responsive to both ABA and drought stress (Matus et al., 2014), was significantly up-regulated in fruit and the pedicel within 6 hr, as well as in the branch, 18 hr later. In contrast, the MdWRKY57 gene was up-regulated after 18 hr in the pedicle and rather suppressed in both fruit and branch at the same time point.