CRISPR/Cas9 edited StbHLH47 lines exhibit altered expression profiling of iron regulating genes and increased iron content in Solanum tuberosum

Abstract

Iron is an essential plant nutrient, and a continuous supply of it is required as it is a key factor in various metabolic processes, including photosynthesis, chlorophyll synthesis, and respiration. Various transcription factors are known to regulate iron homeostasis in plants, and the bHLH transcription factor family is one of them. The StbHLH47 is a homologue of the Arabidopsis POPEYE (PYE), which is known to repress iron homeostasis-related genes in Arabidopsis. Potato is the most consumed vegetable in the world and is low in iron content. We have generated CRISPR/Cas9-edited StbHLH47 lines and performed a detailed analysis of these lines. The analysis revealed that the roots of StbHLH47 edited lines have decreased ferric chelate reductase (FCR) activity compared to the roots of the wild-type (WT) plant. We also observed that CRISPR/Cas9 edited lines have fewer trichomes when compared to the WT plant. The expression of genes associated with iron homeostasis was also measured. Compared to the control, the expression of StbHLH47 was downregulated in the edited lines, while the expression of StNAS4, StOPT3, and StFRO3 was upregulated. This suggests the negative regulation of StbHLH47 in modulating iron. The iron content was also quantified using inductively coupled plasma mass spectrometry (ICP-MS) and found to be increased in the generated transgenic lines when compared to WT plants. Overall, this study reveals that StbHLH47 negatively regulates the expression of iron homeostasis-related genes. StbHLH47 edited lines exhibited decreased FCR activity, changes in phenotype, and increased iron content in the potato plants.