Citrus is a genus with gametophytic self-incompatibility, which prevents fertilization by self-pollination. Polyamines (PAs), as a class of active small molecules, widely participate in various cellular activities. To investigate the effects of the changes in PA contents [putrescine, spermidine (SPD), and spermine] on pollen tube growth in self-incompatible (SI) plants, we cultured pollen of Citrus maxima ‘Banpeiyu’ and C. tamurana ‘Hyuganatsu’ in a Citrus mature pollen culture system and detected the PA content changes in pollen by high-performance liquid chromatography (HPLC) analysis during germination and after treatment with crude protein extracts of compatible or incompatible styles. We profiled the PA content changes during the germination course in pollen of ‘Banpeiyu’ and ‘Hyuganatsu’ by HPLC, and identified increases of free and perchloric-acid-insoluble SPD contents in pollen tubes in the incompatible treatment. Exogenous SPD treatment obviously increased the SPD contents in the pollen tube, inhibited pollen tube elongation, and resulted in some abnormal morphological alterations, such as tip inflation, tube content leakage and tip twist, which were observed in both ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes exposed to incompatible treatment. This finding implied that SPD might participate in the SI response in Citrus.
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.
Interspecific hybridization is useful in raspberry (Rubus idaeus L. ssp. idaeus) breeding to introgression of traits such as heat or cold tolerance, and excellent fruit qualities. Rubus L. wild species in Asia, including Rubus parvifolius L., have been attracting a great deal of attention as sources of new traits in breeding raspberry and blackberry (Rubus fruticosus Agg.). We previously developed and selected IPI-1 and IPI-3 first backcross (BC1) hybrids, [‘Indian Summer’ (R. idaeus ssp. idaeus) × R. parvifolius] × ‘Indian Summer’, as raspberry cultivars adapted to the warm climate in parts of Japan. In this study, we investigated the growth, morphological traits, and fruit qualities, such as sugar, organic acid, anthocyanins, and carotenoids, of each of these IPI lines over a 2-year period to discern their potential as commercial raspberry cultivars. IPI lines had the characteristic of primocane fruit with overflowing from side buds while the parent, IP-1 (‘Indian Summer’ × R. parvifolius), did not. IPI lines showed significantly lower values in anthocyanin content than red raspberry ‘Skeena’, while showing higher carotenoid contents. This study is the first research about fruit qualities such as anthocyanin and carotenoid content of BC1 hybrids using Japanese wild Rubus species.