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Self-incompatibility (SI) is an important mechanism in higher plants that promotes outcrossing and prevents self-fertilization. ‘Banpeiyu’ ( Citrus maxima ) and ‘Hyuganatsu’ ( Citrus tamurana ), two of the  Citrus  cultivars distributed in Kyusyu, Japan, show gametophytic SI. In this study, we used the  Citrus  mature pollen culture system and stylar crude protein extracts to simulate compatible (C) and SI responses in ‘Banpeiyu’ pollen tubes. We analyzed the protein changes in pollen tubes with the C- and SI-like treatments by nano-liquid chromatography–mass spectrometry (nano-LC-MS); 14 and 27 proteins were identified in C- and SI-like treatments, respectively. We picked up some candidate genes that were particularly prevalent in SI-like treatment and analyzed their expression level changes during C- and SI-like treatments in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes. The expression levels of  copper/zinc superoxide dismutase  ( Cu/Zn SOD ),  manganese SOD  ( Mn SOD ),  catalase  ( CAT ), and  cysteine protease  ( CYP ) increased after SI-like treatment. We used a fluorescent probe to visualize reactive oxygen species (ROS) level changes in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes after C- and SI-like treatments and found that 2-hour SI-like treatment induced ROS levels to increase in the pollen tubes of both cultivars. These results suggest that an ROS increase could be one of the key phenomena in the SI response of  Citrus  and that gene expression changes were responses to ROS generation.

In Vitro Self-incompatible-like Response Applied for Protein Identification and Gene Expression Analysis in Citrus Cultivars, Banpeiyu and Hyuganatsu