Responses of a Triple Mutant Defective in Three Iron Deficiency-Induced <i>BASIC HELIX-LOOP-HELIX</i> Genes of the Subgroup Ib(2) to Iron Deficiency and Salicylic Acid

<div><p>Plants are sessile organisms that adapt to external stress by inducing molecular and physiological responses that serve to better cope with the adverse growth condition. Upon low supply of the micronutrient iron, plants actively increase the acquisition of soil iron into the root and its mobilization from internal stores. The subgroup Ib(2) <i>BHLH</i> genes function as regulators in this response, however their concrete functions are not fully understood. Here, we analyzed a triple loss of function mutant of <i>BHLH39</i>, <i>BHLH100</i> and <i>BHLH101</i> (<i>3xbhlh</i> mutant). We found that this mutant did not have any iron uptake phenotype if iron was provided. However, under iron deficiency the mutant displayed a more severe leaf chlorosis than the wild type. Microarray-based transcriptome analysis revealed that this mutant phenotype resulted in the mis-regulation of 198 genes, out of which only 15% were associated with iron deficiency regulation itself. A detailed analysis revealed potential targets of the bHLH transcription factors as well as genes reflecting an exaggerated iron deficiency response phenotype. Since the <i>BHLH</i> genes of this subgroup have been brought into the context of the plant hormone salicylic acid, we investigated whether the <i>3xbhlh</i> mutant might have been affected by this plant signaling molecule. Although a very high number of genes responded to SA, also in a differential manner between mutant and wild type, we did not find any indication for an association of the <i>BHLH</i> gene functions in SA responses upon iron deficiency. In summary, our study indicates that the bHLH subgroup Ib(2) transcription factors do not only act in iron acquisition into roots but in other aspects of the adaptation to iron deficiency in roots and leaves.</p>