Table_1_Heme oxygenase-independent bilin biosynthesis revealed by a hmox1 suppressor screening in Chlamydomonas reinhardtii.XLSX

Bilins are open-chain tetrapyrroles synthesized in phototrophs by successive enzymic reactions catalyzed by heme oxygenases (HMOXs/HOs) and ferredoxin-dependent biliverdin reductases (FDBRs) that typically serve as chromophore cofactors for phytochromes and phycobiliproteins. Chlamydomonas reinhardtii lacks both phycobiliproteins and phytochromes. Nonetheless, the activity and stability of photosystem I (PSI) and the catalytic subunit of magnesium chelatase (MgCh) named CHLH1 are significantly reduced and phototropic growth is significantly attenuated in a hmox1 mutant that is deficient in bilin biosynthesis. Consistent with these findings, previous studies on hmox1 uncovered an essential role for bilins in chloroplast retrograde signaling, maintenance of a functional photosynthetic apparatus, and the direct regulation of chlorophyll biosynthesis. In this study, we generated and screened a collection of insertional mutants in a hmox1 genetic background for suppressor mutants with phototropic growth restored to rates observed in wild-type 4A+ C. reinhardtii cells. Here, we characterized a suppressor of hmox1 named ho1su1 with phototrophic growth rates and levels of CHLH1 and PSI proteins similar to 4A+. Tetrad analysis indicated that a plasmid insertion co-segregated with the suppressor phenotype of ho1su1. Results from TAIL-PCR and plasmid rescue experiments demonstrated that the plasmid insertion was located in exon 1 of the HMOX1 locus. Heterologous expression of the bilin-binding reporter Nostoc punctiforme NpF2164g5 in the chloroplast of ho1su1 indicated that bilin accumulated in the chloroplast of ho1su1 despite the absence of the HMOX1 protein. Collectively, our study reveals the presence of an alternative bilin biosynthetic pathway independent of HMOX1 in the chloroplasts of Chlamydomonas cells.

生物素是一种由光能生物通过连续的酶促反应合成的开环四吡咯，这些反应由血红素氧合酶（HMOXs/HOs）和铁氧还蛋白依赖性胆绿素还原酶（FDBRs）催化，这些酶通常作为植物色素和蓝藻蛋白的色素辅因子。莱因哈德氏衣藻（Chlamydomonas reinhardtii）缺乏植物色素和光敏色素。尽管如此，hmox1突变体中光合系统I（PSI）和镁螯合酶催化亚基CHLH1的活性和稳定性显著降低，光向性生长显著减弱，而hmox1突变体在生物素生物合成中存在缺陷。与这些发现一致，先前对hmox1的研究揭示了生物素在叶绿体逆向信号传导、维持功能性光合装置以及直接调控叶绿素生物合成中的关键作用。在本研究中，我们针对hmox1基因背景生成并筛选了一系列插入突变体，以寻找能够恢复野生型4A+ C. reinhardtii细胞光向性生长速率的抑制突变体。在此，我们表征了一种名为ho1su1的hmox1抑制突变体，其光向性生长速率以及CHLH1和PSI蛋白水平与4A+相似。四分体分析表明，质粒插入与ho1su1的抑制表型共分离。TAI-PCR和质粒拯救实验的结果表明，质粒插入位于HMOX1位点的第1外显子。在ho1su1的叶绿体中异源表达生物素结合报告基因Nostoc punctiforme NpF2164g5表明，尽管缺乏HMOX1蛋白，生物素仍然在ho1su1的叶绿体中积累。总体而言，我们的研究揭示了在莱因哈德氏衣藻细胞叶绿体中存在一种独立于HMOX1的替代生物素生物合成途径。