Comprehensive Analysis of Expressed Proteins in the Different Stages of the Style Development of Self-incompatible 'Hyuganatsu' (Citrus tamurana hort. ex Tanaka)

In Citrus, self-incompatibility (SI) regulation is gametophytic, and this phenomenon is an economically critical problem for some Citrus cultivars without high parthenocarpic ability. Few molecular biological studies of SI in Citrus have been performed, and the molecular mechanism of SI has not been clarified. To investigate the effect of different stages of style development on pollen tube behavior, flower buds of self-incompatible ‘Hyuganatsu’ (Citrus tamurana hort. ex Tanaka) were histologically assayed. When the flower buds were observed 168 hours after pollination, pollen tubes in the self-pollinated flower buds 1 and 3 days before anthesis (DBA) were arrested in the upper part of the styles, while those in flower buds self-pollinated 5 DBA reached the base of the styles. These results revealed that SI in ‘Hyuganatsu’ has not yet occurred in the flower buds 5 DBA, but generated in the flower buds 3 DBA. To search SI-related pistil proteins in Citrus, we profiled a number of protein expressions in ‘Hyuganatsu’ styles of 1, 3, and 5 DBA by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. One-hundred thirty-eight protein spots were significantly different in abundance among the three stages, and 17 up-regulated and 26 down-regulated protein spots could be identified. Among the 17 up-regulated proteins, nine up-regulated proteins exhibited the expression pattern of 1 DBA ≥ 3 DBA > 5 DBA, a pattern which reflected the transmission from SC to SI, evaluated by pollen tube growth. BLASTP homology search against the peptide sequences of these proteins was carried out and predicted the proteins related to the maintenance of cell shape, the signaling of pollen tube growth, the flavonol biosynthesis, the responses to various stresses, photosynthesis, and the methionine metabolic process. Among the nine proteins, some may be the SI-related pistil proteins; however, it was not possible to determine the SI-related key protein of the style in Citrus.