Structural and Functional Characteristics of Two Molecular Variants of the Nitrogen Sensor PII in Maritime Pine

Abstract

High levels of nitrogen are stored as arginine during the last stages of seed formation in maritime pine (Pinus pinaster Aiton). The protein sensor PII regulates the feedback inhibition of arginine biosynthesis through interaction with the key enzyme N-acetylglutamate kinase (NAGK). In this study, the structural and functional characteristics of PII have been investigated in maritime pine to get insights into the regulation of arginine metabolism. Two different forms of PII have been identified, PpPIIa and PpPIIb, which differ in their amino acid sequence and most likely correspond to splicing variants of a single gene in the pine genome. Two PII variants are also present in other pine species but not in other conifers such as spruces. PpPIIa and PpPIIb are trimeric proteins for which structural modeling predicts similar tridimensional protein core structures. Both are located in the chloroplast, where the PII-target enzyme PpNAGK is also found. PpPIIa, PpPIIb, and PpNAGK have been recombinantly produced to investigate the formation of NAGK-PII complexes. The interaction of PpPIIa/PpPIIb and PpNAGK may be enhanced by glutamine and contribute to relieve the feedback inhibition of PpNAGK by arginine. Expression analysis of PpPII genes revealed that PpIIa transcripts were predominant during embryogenesis and germination. The potential roles of PpPIIa and PpPIIb in the regulation of arginine metabolism of maritime pine are discussed.

Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation

“…In Arabidopsis cells, PII was detected for the first time in chloroplasts by immunolocalization in a dotted pattern [ 35 ]. GFP fusions of two PII isoforms from maritime pine ( Pinus pinaster ) expressed in tobacco cells also revealed a similar localization pattern [ 36 ]. Focal aggregation of PII has even been observed in evolutionary distant organisms such as the cyanobacterium Synechococcus elongatus [ 37 ].…”

Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation

“…Revisiting former localization studies of plant PII proteins revealed that the aggregation of this protein in plastids had been indeed observed before, but did not attract further interest: In Arabidopsis cells PII was detected for the first time in chloroplasts by immunolocalization in a dotted pattern [35]. GFP fusions of two PII isoforms from maritime pine (Pinus pinaster) expressed in tobacco cells also revealed a similar localization pattern [36]. Focal aggregation of PII has even been observed in distant organisms like the cyanobacterium Synechococcus elongatus [37].…”

Identification of Metabolic Pathways Differentially Regulated in Somatic and Zygotic Embryos of Maritime Pine

“…However, when high levels of nitrogen (N) are available, the protein sensor PII interacts with NAGK to relieve this feedback control, increasing the metabolic flux through the pathway for N storage ( Llaìcer et al, 2008 ). Two isoproteins of PII, PIIa, and PIIb, have been described in maritime pine, with PIIa being the predominant form in developing embryos ( Llebrés et al, 2020 ). Compared to zygotic embryos, the enhanced expression of ASS and ARG genes in somatic embryos ( Figure 7 ) is consistent with the simultaneous synthesis and degradation of arginine, as previously suggested ( Pérez-Rodríguez et al, 2006 ; Llebrés et al, 2018b ).…”