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

María Teresa Llebrés,Francisco M Cánovas,Fernando N De La Torre,María Belén Pascual,Carolina Valle,Concepción Avila,Luis Gómez,Luis Gómez,José Miguel Valderrama-Martin

doi:10.3389/fpls.2020.00823

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.

Highlights

Plants, like other organisms, have developed mechanisms to detect and respond to changes in carbon and nitrogen levels
GFP–PpPIIa constructs containing the chloroplastic transit peptide (cTP) were appropriately targeted to the chloroplasts (Figure 2B2), as it was observed for control constructs with cTP but lacking the PpPIIa coding sequence (Figure 2B4)
Upon removal of the putative cTP, mature PpIIa and PpIIb proteins would have equal number of amino acids their sequences differ in 15 amino acid residues (∼90% identity), with all the amino acid changes spread over the first half of the amino acid sequence of the mature putative proteins (Figure 1)

Summary

Introduction

Like other organisms, have developed mechanisms to detect and respond to changes in carbon and nitrogen levels These mechanisms regulate the activity of proteins involved in the transport and metabolism of nitrogen and carbon compounds, allowing plants to optimize their energy sources. These processes are important in trees due to their long life cycles (Cánovas et al, 2018). They decrease the rate of nitrogen assimilation when the levels of photosynthetic products are low or the internal levels of nitrogen are relatively high These mechanisms allow plants to adjust their energy and metabolite allocation to a shifting carbon and nitrogen availability (Coruzzi and Zhou, 2001; Gent and Forde, 2017)

Methods

Results

Discussion

Conclusion