Table 3_ASR gene family: a case of tandem-drive evolution.xlsx

IntroductionABA, Stress, and Ripening (ASR) proteins are characterized by the presence of the ABA/WDS domain and are involved in plant development processes and tolerance to abiotic and biotic stresses. Despite their importance as transcription factors or molecular chaperones, a complete understanding of their biological roles is limited by a lack of information on their mechanisms of action, protein structure, and evolutionary relationships between family members. Our previous molecular dynamics simulation analysis of rice OsASR5 suggested that H91, R92, H93, and K94, are the main residues involved in the interaction with DNA, essential for the transcription factor activity of this protein. However, the presence and conservation of the DNA-binding domain among ASR family members remain unknown. Likewise, there is a lack of phylogenetic analyses evaluating the evolutionary history of ASR proteins across major taxonomic groups, outside just the Solanum species.
MethodsTo address these gaps, we conducted a phylogenetic study and protein sequence analyses to gain insights into the evolution of ASR genes in plants. We performed a genome-wide identification of ASR genes via HMMER, using the ABA/WDS domain, in 163 Archaeplastida genomes.
Results and discussionOur results reveal that the potential origin of the ASR gene occurred in the common ancestor of Streptophytes (Charophytes and Embryophytes). Moreover, our study identifies ASR genes in seedless plants. The evolutionary relationship between 465 ASR homologs, found in 76 species, was estimated through maximum likelihood analysis. The results reinforce the rapid and dynamic evolution of the ASR gene family, reflected by the low support in the deep nodes of the phylogeny and the great variation in the number of ASRs in the genomes evaluated, and in some cases their complete absence. As for diversification, tandem duplications seem to be the main mechanism involved. Regarding the conservation of residues in the domain, only two of the 78 are widely conserved, such as E79 and H93. By analyzing the three-dimensional model, we noticed the interaction between them and we hypothesize that they are essential for the stabilization of the domain during interaction with DNA.

引言
脱落酸、胁迫与成熟（ABA, Stress, and Ripening, ASR）蛋白以携带ABA/WDS结构域（ABA/WDS domain）为典型特征，参与植物发育进程以及对非生物与生物胁迫的耐受过程。尽管ASR蛋白作为转录因子或分子伴侣发挥着关键作用，但目前对其完整生物学功能的认知仍受限于作用机制、蛋白质结构以及家族成员间进化关系相关数据的缺失。我们此前针对水稻OsASR5开展的分子动力学模拟分析表明，H91、R92、H93与K94是参与DNA结合的核心残基，而该结合过程对该蛋白的转录因子活性至关重要。不过，ASR家族成员中DNA结合结构域（DNA-binding domain）的存在与保守性仍未明确。同样，目前尚无针对除茄属（Solanum）物种之外的主要分类群中ASR蛋白进化历史的系统发育分析研究。

方法
为填补上述研究空白，我们开展了系统发育研究与蛋白质序列分析，以深入解析植物中ASR基因的进化历程。我们借助隐马尔可夫模型搜索工具HMMER，以ABA/WDS结构域为靶标，对163个古质体类（Archaeplastida）基因组完成了ASR基因的全基因组鉴定。

结果与讨论
本研究结果显示，ASR基因可能起源于链型植物（Streptophytes，包含轮藻（Charophytes）与有胚植物（Embryophytes））的共同祖先。此外，我们在无籽植物中也鉴定到了ASR基因。通过最大似然分析（maximum likelihood analysis），我们对76个物种中的465个ASR同源基因的进化关系进行了推断。研究结果印证了ASR基因家族快速且动态的进化特征：系统发育树深层节点的支持度普遍较低，所评估基因组中ASR基因的数量差异显著，部分类群甚至完全缺失该基因家族。就基因多样化进程而言，串联重复（tandem duplications）似乎是主要的进化驱动机制。针对结构域内残基的保守性分析显示，78个残基中仅2个具有广泛保守性，即E79与H93。通过三维结构模型分析，我们观察到这两个残基间存在相互作用，并据此推测其对该结构域与DNA结合过程中的稳定性至关重要。