Real-time quantitative PCR analysis of apple seeds

Apple seeds were subjected to accelerated aging. After 7, 14, and 21 days of aging, embryos were isolated. Part of the embryos were shortly fumigated with nitric oxide (NO). After 48 h of embryos culture (aged embryos or aged embryos treated with NO), embryonic axes were used to extract the total RNA. RT-qPCR were done to analyze the changes in the expression of genes related to seed aging. Short-term (3 h) treatment of embryos isolated from accelerated aged apple seeds (Malus domestica Borkh.) with NO partially reduced the effects of aging. The aim of the study was to investigate the impact of the short-term NO treatment of embryos isolated from apple seeds subjected to accelerated aging on the expression of genes potentially linked to the regulation of seed aging. Apple seeds were artificially aged for 7, 14, or 21 days. Then the embryos were isolated from the seeds, treated with NO, and cultured for 48 h. Progression of seeds aging was associated with the decreased transcript levels of most of the analyzed genes (Lea1, Lea2a, Lea4, Hsp70b, Hsp20a, Hsp20b, ClpB1, ClpB4, Cpn60a, Cpn60b, Raptor, and Saur). The role of NO in the mitigation of seed aging depended on the duration of the aging. After 7 and 14 days of seed aging, a decreased expression of genes potentially associated with the promotion of aging (Tor, Raptor, Saur) was noted. NO-dependent regulation of seed aging was associated with the stimulation of the expression of genes encoding chaperones and proteins involved in the repair of damaged proteins. After NO application, the greatest upregulation of ClpB, Pimt was noted in the embryos isolated from seeds subjected to 7-day long accelerated aging, Hsp70b, Hsp70c, Cpn in the embryos of seeds aged for 14 days, and Lea2a in the embryos of seeds after 21 days of aging. The expression of genes (Lea1, Lea2a, Lea2b, Lea4, Hsp90, Hsp70a, Hsp70b, Hsp70c, Hsp20a, Hsp20b, ClpB1, ClpB4, Cpn60a, Cpn60b, Tor, Raptor, Pimt, and Saur) were analyzed in the axes of apple embryos after 7, 14, and 21 days of aging and in the axes of apple embryos after 7, 14, and 21 days of aging, treated with NO. The expression levels were normalized using two reference genes and calculated using the method described by Vandesompele et al. (2002) and Hellemans et al. (2007). The experiments were done in three biological replicates, in three technical repetitions.