Accumulated Endogenous Abscisic Acid Contributes to the Cold Tolerance of Pre-planted Cultivated Tobacco

Pre-planted cultivation is widely used in tobacco seedlings in south China to avoid damage from the cold. However, the underlying mechanisms of pre-planted cultivation that can improve the cold tolerance of tobacco are still unknown. In the present study, hormone measurement was performed on seedlings with both pre-planted cultivation and floating cultivation. The results showed that pre-planted tobacco seedlings accumulated more ABA than floating ones at the same growth stage. Further exogenous application of ABA inhibitors on pre-planted seedlings decreased their freezing tolerance. However, exogenous application of ABA enhanced the cold tolerance of floating-cultivated seedlings. To explore the ABA-responsive AREB/ABF members, we conducted a genome-wide analysis of the AREB/ABF family in tobacco and identified 16 members. Duplicated NtAREB/ABF genes showed high structural conservation and contributed to gene family expansion. Amino acid sequence alignments showed high sequence similarity among the gene products, especially in the basic leucine zipper (bZIP) region, indicating that they could possess functional redundancy. Gene expression profiles revealed most of the NtAREB/ABF members showed significantly improved expression after ABA treatment by qPCR. Three NtAREB/ABFs (Ntab0169850, Ntab0398220, and Ntab0577270) were randomly selected for further analysis. Subcellular localization analysis and electrophoretic mobility shift assays (EMSAs) demonstrated that they are nuclear-localized proteins and have an affinity to ABRE in vitro. Finally, transactivation activity assays revealed that Ntab0169850 and Ntab0398220 possess transcriptional activity in yeast. Collectively, these data demonstrate why pre-planted tobacco seedlings showed enhanced cold tolerance and provide a framework for future functional analysis of the AREB/ABF subfamily in tobacco.