Abstract
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.
Highlights
Flavonoids are a class of phenylpropanoids, that are induced in germinating seedlings by UV and blue-light (B) [1], and are reported to play many diverse, but not fully understood, roles in plant physiology [2,3]
PRN1 Possesses Quercetinase Activity Based on reports that in vitro-translated human and bacterial
Pirin could act as quercetinases [12], in vitro-translated PRN1 was analyzed for the ability to cleave quercetin (Figure 1A)
Summary
Flavonoids are a class of phenylpropanoids, that are induced in germinating seedlings by UV and blue-light (B) [1], and are reported to play many diverse, but not fully understood, roles in plant physiology [2,3]. Quercetin (a flavonol, a sub-class of the flavonoids) accumulates in young Arabidopsis seedlings of 4–7 days (d) old, in the cotyledonary node, hypocotyl/root transition zone and root tip, with glycosylated flavonoids in the cotyledon [4]. In vivo evidence indicates that flavonols regulate auxin accumulation in the transparent testa 4 (tt; makes no flavonols, including kaempferol and quercetin) mutants [5,6]. It has been reported that auxin accumulates in rol (repressor of lrx1) mutant seedlings due to flavonol-induced changes to auxin transport [7]. Saslowsky et al demonstrated that flavonoids and specific biosynthetic enzymes of flavonoids were present both in the nucleus and cytosol [10], and Peer et al, found quercetin in the nuclear region, endomembrane system and plasma membrane [4]
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