Orozco-Nunnelly Danielle A, Muhammad Durreshahwar, Mezzich Raquel, Lee Bao-Shiang, Jayathilaka Lasanthi, Kaufman Lon S, Warpeha Katherine M
Molecular, Cell and Developmental Group, Department of Biological Sciences, Department of Biological Sciences, University of Illinois at Chicago (UIC), Chicago, Illinois, United States of America.
Protein Research Laboratory, University of Illinois at Chicago (UIC), Chicago, Illinois, United States of America.
PLoS One. 2014 Apr 4;9(4):e93371. doi: 10.1371/journal.pone.0093371. eCollection 2014.
Pirins are cupin-fold proteins, implicated in apoptosis and cellular stress in eukaryotic organisms. Pirin1 (PRN1) plays a role in seed germination and transcription of a light- and ABA-regulated gene under specific conditions in the model plant system Arabidopsis thaliana. Herein, we describe that PRN1 possesses previously unreported functions that can profoundly affect early growth, development, and stress responses. In vitro-translated PRN1 possesses quercetinase activity. When PRN1 was incubated with G-protein-α subunit (GPA1) in the inactive conformation (GDP-bound), quercetinase activity was observed. Quercetinase activity was not observed when PRN1 was incubated with GPA1 in the active form (GTP-bound). Dark-grown prn1 mutant seedlings produced more quercetin after UV (317 nm) induction, compared to levels observed in wild type (WT) seedlings. prn1 mutant seedlings survived a dose of high-energy UV (254 nm) radiation that killed WT seedlings. prn1 mutant seedlings grown for 3 days in continuous white light display disoriented hypocotyl growth compared to WT, but hypocotyls of dark-grown prn1 seedlings appeared like WT. prn1 mutant seedlings transformed with GFP constructs containing the native PRN1 promoter and full ORF (PRN1::PRN1-GFP) were restored to WT responses, in that they did not survive UV (254 nm), and there was no significant hypocotyl disorientation in response to white light. prn1 mutants transformed with PRN1::PRN1-GFP were observed by confocal microscopy, where expression in the cotyledon epidermis was largely localized to the nucleus, adjacent to the nucleus, and diffuse and punctate expression occurred within some cells. WT seedlings transformed with the 35S::PRN1-GFP construct exhibited widespread expression in the epidermis of the cotyledon, also with localization in the nucleus. PRN1 may play a critical role in cellular quercetin levels and influence light- or hormonal-directed early development.
梨蛋白是具有杯状折叠结构的蛋白质，与真核生物中的细胞凋亡和细胞应激有关。在模式植物拟南芥中，梨蛋白1（PRN1）在特定条件下对种子萌发以及光和脱落酸调节基因的转录发挥作用。在此，我们描述了PRN1具有以前未报道的功能，这些功能可深刻影响早期生长、发育和应激反应。体外翻译的PRN1具有槲皮素酶活性。当PRN1与处于无活性构象（结合GDP）的G蛋白α亚基（GPA1）一起孵育时，可观察到槲皮素酶活性。当PRN1与处于活性形式（结合GTP）的GPA1一起孵育时，未观察到槲皮素酶活性。与野生型（WT）幼苗相比，黑暗中生长的prn1突变体幼苗在紫外线（317 nm）诱导后产生了更多的槲皮素。prn1突变体幼苗在能杀死WT幼苗的高能量紫外线（254 nm）辐射剂量下存活了下来。与WT相比，在连续白光下生长3天的prn1突变体幼苗显示下胚轴生长方向紊乱，但黑暗中生长的prn1幼苗的下胚轴看起来与WT相似。用含有天然PRN1启动子和完整开放阅读框（PRN1::PRN1-GFP）的GFP构建体转化的prn1突变体幼苗恢复到WT反应，即它们不能在紫外线（254 nm）下存活，并且在白光照射下没有明显的下胚轴方向紊乱。通过共聚焦显微镜观察用PRN1::PRN1-GFP转化的prn1突变体，子叶表皮中的表达主要定位于细胞核、细胞核附近，并且在一些细胞内出现弥散和点状表达。用35S::PRN1-GFP构建体转化的WT幼苗在子叶表皮中表现出广泛表达，也定位于细胞核。PRN1可能在细胞槲皮素水平中起关键作用，并影响光或激素导向的早期发育。
