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

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) BHLH 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 BHLH39, BHLH100 and BHLH101 (3xbhlh 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 BHLH genes of this subgroup have been brought into the context of the plant hormone salicylic acid, we investigated whether the 3xbhlh 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 BHLH 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.植物是固着生长的生物，通过激活分子与生理响应以更好地应对不利生长环境。当微量营养元素铁供应不足时，植物会主动提升土壤中铁向根部的吸收效率，并调动体内储存的铁元素。碱性螺旋-环-螺旋（basic Helix-Loop-Helix, bHLH）基因Ib(2)亚家族在该响应中发挥调控作用，但其具体功能尚未完全阐明。本研究针对BHLH39、BHLH100与BHLH101的三功能缺失突变体（3xbhlh突变体）展开分析。研究发现，在铁供应充足的条件下，该突变体未表现出铁吸收相关的异常表型。但在缺铁环境下，该突变体的叶片失绿黄化程度较野生型更为严重。基于基因芯片的转录组分析显示，该突变体的表型导致198个基因的表达失调，其中仅15%的基因与缺铁调控直接相关。进一步的详细分析揭示了该bHLH转录因子的潜在靶基因，以及反映过度缺铁响应表型的相关基因。鉴于该亚家族的BHLH基因已被证实与植物激素水杨酸（salicylic acid, SA）相关，本研究进一步探究了3xbhlh突变体是否受该植物信号分子的影响。尽管有大量基因对水杨酸产生响应，且突变体与野生型间的响应模式存在差异，但本研究未发现任何证据表明该类BHLH基因在缺铁条件下参与水杨酸响应通路。综上，本研究表明，bHLH Ib(2)亚家族转录因子不仅参与根部的铁吸收过程，还在根与叶片的缺铁适应的其他方面发挥作用。