Iron (Fe) deficiency is a common problem in citrus production. As the second largest superfamily of transcription factors (TFs), the basic/helix-loop-helix (bHLH) proteins have been shown to participate in the regulation of Fe homeostasis and a series of other biological and developmental processes in plants. However, this family of members in citrus and their functions in citrus Fe deficiency are still largely unknown. In this study, we identified a total of 128 CgbHLHs from pummelo (Citrus grandis) genome that were classified into 18 subfamilies by phylogenetic comparison with Arabidopsis thaliana bHLH proteins. All of these CgbHLHs were randomly distributed on nine known (125 genes) and one unknown (3 genes) chromosomes, and 12 and 47 of them were identified to be tandem and segmental duplicated genes, respectively. Sequence analysis showed detailed characteristics of their intron-exon structures, bHLH domain and conserved motifs. Gene ontology (GO) analysis suggested that most of CgbHLHs were annotated to the nucleus, DNA-binding transcription factor activity, response to abiotic stimulus, reproduction, post-embryonic development, flower development and photosynthesis. In addition, 27 CgbHLH proteins were predicted to have direct or indirect protein-protein interactions. Based on GO annotation, RNA sequencing data in public database and qRT-PCR results, several of CgbHLHs were identified as the key candidates that respond to iron deficiency. In total, 128 CgbHLH proteins were identified from pummelo, and their detailed sequence and structure characteristics and putative functions were analyzed. This study provides comprehensive information for further functional elucidation of CgbHLH genes in citrus.

中文翻译：

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系统分析柚（Citrus grandis）的基本/螺旋-环-螺旋（bHLH）转录因子家族，并鉴定参与铁缺乏反应的关键成员

铁（Fe）缺乏症是柑橘生产中的常见问题。作为转录因子（TF）的第二大超家族，碱性/螺旋-环-螺旋（bHLH）蛋白已显示出参与植物体内铁稳态和其他一系列生物学和发育过程的调控。然而，这个柑橘族成员家族及其在柑橘铁缺乏症中的功能仍是未知之数。在这项研究中，我们从柚子（Citrus grandis）基因组中鉴定出总共128个CgbHLH，通过与拟南芥bHLH蛋白的系统发育比较将其分为18个亚家族。所有这些CgbHLHs随机分布在9个已知（125个基因）和1个未知（3个基因）染色体上，其中12个和47个分别被鉴定为串联和分段重复基因。序列分析显示了其内含子-外显子结构，bHLH结构域和保守基序的详细特征。基因本体论（GO）分析表明，大多数CgbHLHs均标注了细胞核，DNA结合转录因子活性，对非生物刺激的响应，繁殖，胚后发育，花发育和光合作用。此外，预计27种CgbHLH蛋白具有直接或间接的蛋白-蛋白相互作用。根据GO注释，公共数据库中的RNA测序数据和qRT-PCR结果，确定了几种CgbHLHs作为对铁缺乏症作出反应的关键候选药物。总共从柚子中鉴定出128种CgbHLH蛋白，并分析了它们的详细序列，结构特征和推定功能。