Feature, expression, and subcellular localization analyses ofMdNup62
We initially performed a simple bioinformatics analysis ofMdNup62 . A phylogenetic tree of Nup62 from six Rosaceae plants (Rosa chinensis , Pyrus communis ,Prunus persica , M. domestica , Rubus occidentalis , and Fragaria vesca ) was constructed using MEGA-X. MdNup62was most closely related to the Nup62 of pear (Figure 2a). The aligned protein sequences revealed a conserved Nsp1_C domain (Figure 2b). The subcellular localization of MdNup62 was determined by introducing 35S::MdNup62 -GFP into tobacco leaves (Figure 2c). Tobacco leaves transformed with the empty vector 35S::GFP were used as controls. In the tobacco leaves expressing 35S::MdNup62 -GFP, the GFP signal was observed only in the nuclear pore, while the GFP signal was detected throughout the control tobacco leaf cells, indicating thatMdNup62 localized to the nuclear pore.
The transcript levels of MdNup62 in different tissues were determined using qRT-PCR (Figure 2d). The highest expression level was in flower buds. An MdNup62 expression analysis during the flower bud developmental stages revealed that the expression level was stable at 30 to 60 days after flowering and reached its highest level at 70 days after flowering (Figure 2e). Thus, MdNup62 maintained a high expression level during flower bud induction, indicating that it may be related to bud differentiation in apple.
We exposed apple tissue-cultured seedlings to a heat treatment. The reactive oxygen species (ROS) accumulation in leaves increased from 0 to 6 h under heat-treatment conditions (Figure 2f). Moreover, the expression level of MdNup62 was determined at different times during the high-temperature treatment (Figure 2g). MdNup62 was significantly induced by high temperature, and its expression level was highest at 1 h after exposure to the high temperature. Thus,MdNup62 may be involved in the heat-resistance pathway of apple.