4.3. Responses in cell wall modification contribute to AZ formation with the cytoplasmic stream of vesicles
From 2019 ex-vivo experiment, we observed that both branch and pedicel showed the up-regulation of the cell wall loosening (MdEXPA10 ) and the lignin polymerization (MdLAC7 ) signals for the substantial AZ formation after 18 hr of initial cold shock (Figure 5c). This is possible because the abscission signal is affected by the cold response. A similar tendency was observed in the RNAseq and metabolomics data from the previous year. Based on LCMS result, metabolites related to lignin biosynthesis (quinic acid and ferulic acid) accumulated more in fruit/pedicel mixed tissues (Figure 2). Transcriptomics data revealed an up-regulated SHR homologMDP04G1046000 , which is related to the lignin signalling pathway for abscission induction (Supporting Information Table S1). SHRmediates the formation of Casparian strips in endodermis (Lee et al., 2018). When the neighbouring cells undergo organ separation during abscission, the lignin brace is constructed in the secession cells. Therefore, such SHR gene, which mediates the formation of lignin structure, would be positively regulated in fruit pedicel as a consequence of abscission induction following cold stress.
Another member participating in the AZ development is pectin, one of the main cell wall components. A number of pectinesterases were differentially expressed, as shown in Supporting Information Table S1. Pectin is accumulated during AZ formation, and changes in its methylesterification occur before cell separation (Roongsattham et al., 2016). Furthermore, AZ cells show the association of cytoplasmic vesicles during cell separation process (reviewed by Roberts et al., 2002). We observed the accumulation of vesicles in the cytoplasm of AZ cortical cells as a result of cold shock in this study (Figure 3b). Several of them appeared to be bound to the cell wall, and the cytoplasmic streaming of vesicles seemed strongly associated with the modification of cell wall by carrying molecules to be used in cell wall metabolism or by removing these from the cell wall.