Data_Sheet_1_Heme oxygenase-independent bilin biosynthesis revealed by a hmox1 suppressor screening in Chlamydomonas reinhardtii.PDF

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.

生物素是一类由光合生物通过连续的酶促反应合成的开链四吡咯，这些反应由血红素氧合酶（HMOXs/HOs）和依赖于铁氧还蛋白的胆绿素还原酶（FDBRs）催化，这些酶通常作为植物色素和藻蓝蛋白的色素辅因子。莱茵衣藻（Chlamydomonas reinhardtii）缺乏藻蓝蛋白和植物色素。尽管如此，hmox1突变体中光系统I（PSI）和镁螯合酶催化亚基CHLH1的活性和稳定性显著降低，光向性生长也显著减弱。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、维持功能性光合作用装置以及直接调节叶绿素生物合成中的重要作用。在本研究中，我们生成并筛选了hmox1遗传背景下的插入突变体集合，以寻找能够将光向性生长恢复至野生型4A+ C. reinhardtii细胞水平的抑制子突变体。在此，我们鉴定了一种名为ho1su1的hmox1抑制子，其光能生长速率、CHLH1和PSI蛋白水平与4A+相似。四倍体分析表明，质粒插入与ho1su1的抑制子表型共分离。TAI-PCR和质粒拯救实验的结果表明，质粒插入位于HMOX1基因座的第一外显子。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，生物素仍积累在ho1su1的叶绿体中。综上所述，我们的研究揭示了莱茵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。