Characterization of apple NADPH oxidase genes and their expression associated with oxidative stress in shoot culture in vitro

Genes encoding Rboh (respiratory burst oxidase homolog) have been described in a variety of plant species. Transcriptional regulation of rboh genes has been shown during plant response to stress or phytohormone treatment. In vitro conditions often induce plant stress that leads to slow plant growth, early senescence, or even recalcitrance to in vitro growth. Production of reactive oxygen species (ROS) is an important manifestation of plant environmental stress. Expression of rboh orthologs in plant tissues grown under in vitro conditions and their role in response to abiotic stress is not fully understood. Therefore the aim of this study was to identify rboh homologues in apple ( Mdrboh ) and to characterize their expression during the senescence of apple shoot culture in vitro. Similarity searches using Arabidopsis Rboh (AtRboh) protein sequences revealed nine homologous rboh genes in the apple genome. Phylogenetic analysis using conservative N-terminal half region sequences of the apple, Arabidopsis , pear, peach and wild strawberry Rboh proteins revealed four groups of related sequences that were linked to AtRboh D, E, F and H. A homologue with unique sequence was annotated as Rboh K in plants of the Rosaceae family. Expression of MdrbohD1 – 3 and F was detected in apple leaves, in vitro cultures of shoots and cell suspension, and MdrbohE2 and H1 – 2 varied among the tissues. The MdrbohD1 – 2 and F genes were differentially expressed during transfer, growth and senescence stages of apple in vitro shoot culture having varying levels of oxidative stress damage, suggesting transcriptional regulation of the Mdrboh genes in the apple shoot culture, and a potential for distinctive functions of the three Rboh D orthologs.