Transcriptome-Wide Identification and Expression Analysis of bHLH Family Genes in Iris domestica under Drought and Cu Stress

Abstract

The role of bHLH transcription factors in plant response to abiotic stress and regulation of flavonoid metabolism is well documented. However, to date, the bHLH transcription factor family in Iris domestica remains unreported, impeding further research on flavonoid metabolism in this plant. To address this knowledge gap, we employed bioinformatics to identify 39 IdbHLH genes and characterised their phylogenetic relationships and gene expression patterns under both drought and copper stress conditions. Our evolutionary tree analysis classified the 39 IdbHLHs into 17 subfamilies. Expression pattern analysis revealed that different IdbHLH transcription factors had distinct expression trends in various organs, suggesting that they might be involved in diverse biological processes. We found that IdbHLH36 was highly expressed in all organs (Transcripts Per Million (TPM) > 10), while only 12 IdbHLH genes in the rhizome and four in the root were significantly upregulated under drought stress. Of these, four genes (IdbHLH05, -37, -38, -39) were co-upregulated in both the rhizome and root, indicating their potential role in drought resistance. With regards to copper stress, we found that only 12 genes were upregulated. Further co-expression analysis revealed that most bHLH genes were significantly correlated with key enzyme genes involved in isoflavone biosynthesis. Thereinto, IdbHLH06 showed a significant positive correlation with IdC4H1 and Id4CL1 (p < 0.05). Furthermore, a transient expression assay confirmed that the IdbHLH06 protein was localised in the nucleus. Our findings provide new insights into the molecular basis and regulatory mechanisms of bHLH transcription factors in isoflavone biosynthesis in I. domestica.