Submergence-inducible and circadian rhythmic basic helix–loop–helix protein gene in Nicotiana tabacum

Submergence stress leads to diverse changes in transcription and translation of genes involved in developmental and physiological metabolisms of plants. The basic helix–loop–helix (bHLH) protein family is one of the largest transcriptional factor families in plants, and has been shown to play pivotal roles in diverse biological responses. However, there has been no report on bHLH protein related to submergence stress response. In this study, a novel bHLH gene, NtbHLH, was isolated from tobacco (Nicotiana tabacum) by differential screening of a submergence-stress-induced cDNA library. NtbHLH cDNA is 1027 bp in length, with an open reading frame (ORF) of 702 nucleotides encoding 233 amino acid residues that contain the bHLH domain. RNA-blot analyses showed that transcription of NtbHLH was induced by submergence stress, while cold, heat shock, and drought decreased its expression. The gene expression was down-regulated by gibberellins, but ABA and ethylene seemed not to affect it. It was also apparent that NtbHLH expression follows circadian rhythmicity. The electrophoretic mobility shift and chemical cross-linking assays showed that NtbHLH specifically binds to G-box and forms homo-dimers.