关键词: 棉花种子, 种子衰老, 种子活力, ATP合成酶, mRNA完整性

Abstract:

【Objective】Seed aging is a complex biological process, previous studies have been used to elucidate the events. However, the mechanism of seed aging is still unclear. The naturally aged cotton seeds were used as experimental materials, and the physiological and biochemical changes as well as the changes in ATP synthase mRNA integrity that occurred in cotton seed during storage were investigated in order to provide a foundation for further illuminating the aging mechanism of cotton seeds.【Method】In this study, a collection of seeds (cultivar Xinluzao 74) that had been stored for 3 and 5 years served as the experimental materials, the newly harvested seeds were used as the control (CK). The germination percentage, water absorption and viability of cotton seeds were valued by germination test between paper, low constant temperature over method, and TTC staining method, respectively; The acid value and respiratory rate of cotton seeds were determined by the acid-base titration method, and the ATP synthase activity was detected with plant ATP synthase ELISA Kit. The mRNA integrity of ATP synthase subunit α, β, γ, ε, and δ in cotton embryo was analyzed by reverse transcription blocking-double primer amplification method.【Result】Our data suggest that seed vigor dramatically decreased over storage time. After 3 and 5 years of storage, the germination percentage of cotton seeds was significantly decreased from 98.7% to 84.0% and 58.0%, respectively (P<0.05). At the initial stage of seed imbibition (the first 4 h), the water absorption rate of seeds was significantly decreased by 11.0% and 26.9%, respectively. The results of TTC staining showed that only the radicle was slightly stained in seeds preserved 5 years but not the cotyledons and other organs stained; The acid value of seeds was significantly increased by 28.4% and 40.0%, respectively (P<0.05), this indicated that severe hydrolysis of lipid occurred in seeds. Seed respiration rate and ATP synthase activity showed an increasing trend during imbibition, but the increasement was significantly decreased (P<0.05); The respiration rate of seeds was reduced by 33.3% and 49.2% after 24 hours of imbibition, and the activity of ATP synthase was decreased by 17.9% and 73.4% after 12 hours of imbibition, respectively. The results of reverse transcription blocking-double primer amplification showed that the R value of ATP synthase subunits α, β, γ, and δ mRNAs stored in seeds were significantly decreased, but the subunit ε mRNA was significantly increased. These results indicated that the integrity of the ATP synthase subunits mRNA decreased to varying degrees during the natural storage process.【Conclusion】These results showed that a prolonged storage time could reduce seed vigor; The integrity loss of ATP synthase subunit mRNAs stored in seed embryos would cause ATP synthase subunit to be impaired and ATP synthase activity declined, thus lead to a decreased production of ATP and affect seed germination capacity. This might be one of the important reasons for cotton seed aging.

Key words: cotton seed, seed senescence, seed vigor, ATP synthase, mRNA integrity