From cyanobacteria to plants: conservation of PII functions during plastid evolution
This article reviews the current state-of-the-art concerning the functions of the signal processing protein PII in cyanobacteria and plants, with a special focus on evolutionary aspects. We start out with a general introduction to PII proteins, their distribution, and their evolution. We also discuss PII-like proteins and domains, in particular, the similarity between ATP-phosphoribosyltransferase (ATP-PRT) and its PII-like domain and the complex between N-acetyl-l-glutamate kinase (NAGK) and its PII activator protein from oxygenic phototrophs. The structural basis of the function of PII as an ATP/ADP/2-oxoglutarate signal processor is described for Synechococcus elongatus PII. In both cyanobacteria and plants, a major target of PII regulation is NAGK, which catalyzes the committed step of arginine biosynthesis. The common principles of NAGK regulation by PII are outlined. Based on the observation that PII proteins from cyanobacteria and plants can functionally replace each other, the hypothesis that PII-dependent NAGK control was under selective pressure during the evolution of plastids of Chloroplastida and Rhodophyta is tested by bioinformatics approaches. It is noteworthy that two lineages of heterokont algae, diatoms and brown algae, also possess NAGK, albeit lacking PII; their NAGK however appears to have descended from an alphaproteobacterium and not from a cyanobacterium as in plants. We end this article by coming to the conclusion that during the evolution of plastids, PII lost its function in coordinating gene expression through the PipX-NtcA network but preserved its role in nitrogen (arginine) storage metabolism, and subsequently took over the fine-tuned regulation of carbon (fatty acid) storage metabolism, which is important in certain developmental stages of plants.
KeywordsChloroplast CLANS NAGK Oxygenic phototroph PII signaling Synechococcus elongatus
Introduction to PII signal processors: general properties and evolution of canonical PII proteins
PII proteins constitute a superfamily of the most widely distributed signaling proteins in nature, represented in all domains of life (Sant’Anna et al. 2009; Huergo et al. 2012). Members of this superfamily are present in almost all taxonomic groups of bacteria and are ubiquitous in nitrogen-fixing methanogens of the archaeal kingdom; however, in eukaryotes they are only found in oxygenic phototrophs (Arcondéguy et al. 2001; Forchhammer 2008). In all cases studied so far, PII proteins are involved in the control of anabolic nitrogen metabolism. They detect the metabolite state of the cell by interdependent binding of ATP and 2-oxoglutarate (2-OG) or ADP in a highly conserved manner, and thereby regulate the activity of transcription factors or key metabolic enzymes (Fokina et al. 2010a, b; Truan et al. 2010; Litz et al. 2011; Radchenko and Merrick 2011; Zeth et al. 2012). Interestingly, these PII-regulated target proteins are distinct in different phylogenetic groups of organisms.
Based on the widespread occurrence of the PII superfamily member GlnK in diverse prokaryotes and its conserved genetic coupling with the ammonium transport protein AmtB, it has been hypothesized that modern trimeric PII proteins may have arisen from an ancient trimeric PII protein that originated early in the evolution of prokaryotes in conjunction with the trimeric ammonium transporters to control ammonium uptake in response to the metabolite state of the cells (Thomas et al. 2000; Sant’Anna et al. 2009). Other PII paralogues, GlnB and NifI, may have evolved subsequently from this primordial GlnK protein by gene duplication and functional diversification. These paralogues are implicated in the regulation of nitrogen-dependent gene expression, in the activity regulation of glutamine synthetase, and in the control of nitrogen fixation through a stupendous variety of mechanisms (Huergo et al. 2012; Leigh and Dodsworth 2007; Luque and Forchhammer 2007; Masepohl and Forchhammer 2007).
In cyanobacteria, PII proteins are present in all known species. While most cyanobacteria harbor one PII protein, some strains encode a second or even a third paralogue (Laichoubi et al. 2011). In contrast to many bacteria, where PII proteins (mainly of the GlnB subfamily) are involved in regulation of glutamine synthetase at various levels (Ninfa and Atkinson 2000; Leigh and Dodsworth 2007), cyanobacterial PII proteins have evolved to regulate the ornithine pathway, which leads to arginine and polyamine synthesis, and to the modulation of nitrogen-dependent transcription. In eukaryotes, PII homologues have only been identified in Chloroplastida (green algae and land plants), where they are nuclear-encoded, and in Rhodophyta, where they are coded by the plastid genome (Uhrig et al. 2009). In both these groups, PII is localized in the chloroplast (Hsieh et al. 1998; Ermilova et al. 2012) and appears to control the key step in arginine synthesis, as in cyanobacteria.
PII-like proteins: witnesses of a widely distributed signal processing mode
The PII architecture: from structure to function
Modification of PII proteins
The E. coli GlnB protein was the first PII protein to be carefully studied. As in many other proteobacteria, E. coli PII proteins (GlnB and GlnK) are subject to covalent UMP-modification (uridylylation) at Tyr51, located at the apex of the T-loop (Adler et al. 1975; Atkinson et al. 1994). In fact, uridylylation was long regarded as the hallmark of PII signaling (Arcondeguy et al. 2000; Ninfa and Atkinson 2000). Uridylylation is brought about by the bifunctional enzyme GlnD (Uridylyltransferase/uridylyl-removase), whose activity is regulated by glutamine, with low glutamine levels favoring PII uridylylation and high glutamine levels favoring PII deuridylylation. By these means, the PII protein becomes a highly sensitive transmitter of the cellular glutamine status (Jiang and Ninfa 2011). However, uridylylation is not a general trait of PII signaling. The PII protein in the cyanobacterium S. elongatus was shown to be phosphorylated at Ser49, a position adjacent to the uridylylation site of E. coli PII (Forchhammer and Tandeau de Marsac 1994). In vivo, the phosphorylation status of PII depends on the carbon/nitrogen supply of the cells: nitrogen-limiting conditions favor PII phosphorylation, excess nitrogen, preferably in the form of ammonia, causes PII dephosphorylation. With S. elongatus cell-free extracts, phosphorylation could be achieved in the presence of millimolar concentrations of 2-OG and ATP. The PII kinase is still unknown; however, the phosphatase of PII-P, PphA, could be identified in Synechocystis PCC 6803 (Irmler and Forchhammer 2001) and has since then been thoroughly studied (Ruppert et al. 2002; Kloft and Forchhammer 2005; Schlicker et al. 2008; Su et al. 2011; Su and Forchhammer 2011, 2012). PphA is a Ser/Thr phosphatase of the PP2C family and the crystal structure of the PphA homologue from the thermophilic cyanobacterium Thermosynechococcus has been solved (Schlicker et al. 2008). In addition to the binuclear metal center, a third metal, which is occasionally observed in bacterial PP2C homologues, was shown to be an essential part of the catalytic center (Su et al. 2011). Recognition of the phosphorylated PII protein involves a flap subdomain, which shields the catalytic center of PphA (Su and Forchhammer 2011). The formation of a precisely fitted substrate-enzyme complex is a prerequisite for dephosphorylation. The conformation of the T-loop plays a critical role in this process: only when PII is non-ligated with 2-OG, PphA is able to dephosphorylate PII. As long as PII-P resides in the Mg-ATP/2-OG ligated state, it is protected from dephosphorylation (Ruppert et al. 2002; Su and Forchhammer 2011), implying that the 2-OG induced conformation of the T-loop does not fit into the catalytic crevice of PphA. Ser49 phosphorylation seems not to be generally conserved in cyanobacteria. In Prochlorococcus marinus, an abundant marine prochlorophyte, the evidences indicate absence of PII phosphorylation (Palinska et al. 2002), in agreement with the lack of a PphA homologue gene (Cyanobase: http://genome.kazusa.or.jp/cyanobase). In filamentous cyanobacteria, the situation is less clear. No PII phosphorylation could be detected in Nostoc punctiforme extracts but the N. punctiforme PII protein could be phosphorylated in vitro by S. elongatus cell extracts (Hanson et al. 1998). Mass spectroscopic analysis of the PII protein from Anabaena extracts revealed Tyr51 to be subjected to nitration under diazotrophic conditions while no phosphorylation at Ser49 was detected (Zhang et al. 2007). Absence of PII phosphorylation in the Nostocales is, however, in contrast to the presence of a PphA homologue. Mutation of the PphA homologue gene resulted in altered PII functions in Anabaena (Laurent et al. 2004). In plants, potential PII phosphorylation was investigated in A. thaliana. Its PII protein has conserved the seryl-phosphorylation site, but no phosphorylation could be identified. Recently, the PII protein from Chlamydomonas reinhardtii was characterized. It has a potentially phosphorylatable threonyl residue at the corresponding position, but like in Arabidopsis, protein phosphorylation analysis revealed only non-phosphorylated PII protein (Ermilova et al. 2012). At a first glance, it seems odd that in spite of conservation of this site, phosphorylation of PII seems not to be conserved. However, conservation of this site could be due to its pivotal role in PII-NAGK interaction.
PII-mediated regulation of the arginine pathway in cyanobacteria and plants
Yeast-two hybrid screening for PII-interaction partners in cyanobacteria and plants using genomic DNA from S. elongatus and A. thaliana identified the enzyme N-acetyl-l-glutamate kinase (NAGK) as a novel PII receptor (Burillo et al. 2004; Heinrich et al. 2004; Sugiyama et al. 2004). In plants and cyanobacteria, NAGK catalyzes the committed step of arginine biosynthesis and in agreement, the enzyme is feedback-inhibited by arginine. In S. elongatus and A. thaliana, PII modulates the catalytic properties of NAGK. Initial experiments (Heinrich et al. 2004; Chen et al. 2006) yielded some inconsistent results due to the use of a non-optimized assay buffer, which impaired 2-OG effects and which lacked a reducing agent, necessary for high NAGK activities (Beez et al. 2009). When tested under optimized conditions, the following common properties between the proteins from S. elongatus and A. thaliana became evident (Beez et al. 2009): (1) PII activates the overall catalytic efficiency (k cat/K m) of NAGK, for S. elongatus 8-fold and for A. thaliana 1.5-fold. In the latter case, activation is mainly an effect on k cat. (2) PII relieves NAGK from arginine feed-back inhibition. It also increases the half maximal inhibitory concentration of arginine (IC50) from 20 to 200 μM for S. elongatus, and from 1 to 6 mM for A. thaliana NAGK. Arginine inhibits NAGK by increasing the K m for the substrate NAG, an effect, which is counteracted by PII. (3) 2-OG antagonizes the protection of NAGK by PII from arginine inhibition.
Conservation of PII-NAGK interaction during plastid evolution
The high degree of conservation in sequence, structure, and function between S. elongatus and A. thaliana PII-NAGK complexes implies a strong selective pressure for maintaining PII-regulated arginine biosynthesis in the evolution of plastids from an ancestral cyanobacterium. If this is indeed the case, the phylogeny of PII and NAGK sequences should be similar to each other, and should also reflect the evolution of plants. Hitherto studies have only focused on the phylogenetic analysis of the PII superfamily and they have not been conclusive on whether plant PII proteins are of cyanobacterial origin or not (Osanai and Tanaka 2007; Uhrig et al. 2009; Sant’Anna et al. 2009). Since PII proteins are short and highly similar in sequence, reliable inference of their phylogeny is difficult. Also, to our knowledge, a comprehensive phylogenetic analysis of NAGK proteins has not been performed yet. Spurred by this, we decided to revisit the analysis of PII and NAGK proteins using cluster analysis and maximum likelihood-based phylogenetic reconstruction. In cluster analysis, sequences are treated as point masses in a virtual multi-dimensional space which attract or repel each other depending on the statistical significance of their pairwise sequence similarities. Sequences find their equilibrium position in the map not only by attraction to similar sequences but also by repulsion of different ones. Unlike phylogenetic methods, which have exponential computational complexity and only allow calculation of trees with a few thousand sequences at most, the computational complexity of cluster analyses only increases approximately quadratically with the number of sequences, making calculation of maps with several thousand sequences within a reasonable time possible. In fact, cluster maps become more accurate with an increasing number of sequences as the larger number of pairwise relationships average out the random error arising from simpler pairwise similarity-based comparisons.
Further functions of PII signaling in oxygenic phototrophs
The above-mentioned PII receptors are absent in plants; however, a new PII receptor, the chloroplast acetyl-CoA carboxylase (ACCase), a key enzyme in fatty acid synthesis in plastids, was identified by pull-down experiments with A. thaliana extracts (Feria Bourrellier et al. 2010). ACCase activity was repressed by PII, but this repression was antagonized by 2-OG and oxaloacetate and to a lesser extent by pyruvate. So far this interaction has not been characterized in great detail and also it needs to be confirmed if this interaction can be generalized for all plants. Nevertheless, the regulation of ACCase by PII represents an intriguing link to carbon storage metabolism. Physiological analyses in A. thaliana support the function of PII in storage metabolism. The PII gene is upregulated in early seed maturation by the transcription factor WRINKLED1 (Baud et al. 2010), and in seeds of PII deficient mutants, a transient increase of fatty acid production and an alteration in fatty acid composition were observed. From these results, a regulatory role of PII in the fine-tuning of fatty acid biosynthesis and partitioning in seeds had been inferred (Baud et al. 2010). Furthermore, in A. thaliana PII mutants, nitrite uptake in chloroplast is enhanced (Ferrario-Mery et al. 2008), resembling the regulatory defect of nitrite/nitrate uptake in cyanobacterial PII mutants (Kloft and Forchhammer 2005). Altogether, the data indicate that during the evolution of plastids, PII lost its primary function of coordinating gene expression through interactions with PipX, but preserved its role in nitrogen (arginine) storage metabolism, and eventually took over fine-tuned regulation of carbon (fatty acid) storage metabolism. Currently, PII is known to play a role in early seed maturation, but it is unclear if it also has roles in other developmental stages of plants. Studies of phylogenetically more ancient plants and of unicellular green algae will be necessary to unravel the roles of PII in the metabolic pathways of Chloroplastida.
We thank Andrei Lupas for helpful discussions. This study was supported by DFG grant Fo195/9 and by institutional funds from the Max Planck Society.
Conflict of interest
The authors declare that they have no conflict of interest.