Function diversification of CONSTANS-like genes in Pyrus and regulatory mechanisms in response to different light quality.

Pear (Pyrus L.) is a significant commercial fruit globally, with diverse species exhibiting variations in their flowering periods due to environmental factors. CONSTANS-like (COL) genes, known from previous studies in Arabidopsis, are key regulators of flowering time by sensing photoperiod. However, the evolutionary history and functions of COL genes in different pear species remain unclear. In this study, we identified a total of 79 COL genes in different pear species, including 12 COL genes in Pyrus bretschneideri 'DangshanSuli', 9 in Pyrus ussuriensis × hybrid 'Zhongai 1', 11 in Pyrus communis 'Bartlett', 13 in Pyrus betulifolia, 18 in Pyrus pyrifolia 'Cuiguan', 16 in Pyrus pyrifolia 'Nijisseiki'. Analysis of gene structure, phylogenetic tree, and multiple sequences provided valuable insights into the fundamental understanding of COL genes in pear. The impact of selection pressure on the PbrCOLs in Chinese white pear was assessed using Ka/Ks, revealing that the evolution rate of PbrCOLs was influenced by purification selection factors. The study also revealed different tissue-specific expression patterns of PbrCOLs under varying light quality. Real-time quantitative PCR revealed that under natural light conditions, the expression patterns of PbrCOL2, PbrCOL3, and PbrCOL4 are similar to previous studies on CONSTANS gene in Arabidopsis, with increased expression levels during the day and decreased levels at night. However, PbrCOL1, PbrCOL6, and PbrCOL9 exhibit different expression patterns, with decreased expression levels both during the day and at night. After red light treatment, high expression of PbrCOL3 and PbrCOL4 was observed at night, while the expression patterns of the other four genes did not show significant changes. Following blue light treatment, the expression peaks of PbrCOL1 and PbrCOL6 occurred during the night, showing opposite expression patterns compared to the study in Arabidopsis. The overexpression of PbrCOL3 significantly increase the chlorophyll content in pear seedlings, and its expression significantly affected the expression of other key flowering-related genes. Also, overexpression of PbrCOL3 resulted in a late-flowering phenotype in Arabidopsis. These findings indicate diverse responsive mechanisms and functions of PbrCOL genes on flowering time in pear. In conclusion, this study established a foundation for a deeper understanding of the specific roles of PbrCOLs in regulating the reproductive development of pear, particularly in the context of the photoperiodic flowering process.