Nitrogen is one of the most important nutrients for plant growth and development. Plants can utilize different forms of nitrogen including nitrate, ammonium, and amino acids. Most plants use inorganic nitrogen nitrate as the primary nitrogen source. Nitrate taken up from the soil will be reduced to ammonium by nitrate reductase and nitrite reductase. Ammonium derived from nitrate or remobilized from the other nitrogen-containing compounds can be assimilated into glutamine and glutamate via the glutamine synthetase (GS)/glutamine-oxoglutarate aminotransferase (GOGAT) cycle. Glutamine and glutamate are the major amino donors for the synthesis of the other amino acids and nitrogen-containing compounds in plants . In addition to their roles in protein synthesis and metabolism, glutamine and glutamate may also serve as signaling molecules in plants [2–6].
The synthesis of glutamine and glutamate also depends on the availability of α-ketoglutarate. In bacteria, the carbon skeleton of ammonia assimilation, α-ketoglutarate, signals nitrogen deficiency, whereas glutamine, the fully aminated product, often signals nitrogen sufficiency . In E. coli, the expression of glutamine synthetase gene and its enzyme activity are regulated by the availability of glutamine and α-ketoglutarate [7–10]. In response to low glutamine/α-ketoglutarate, the E. coli PII protein (encoded by glnB) is uridylylated by GlnD, an uridylyltransferase/uridylyl-removing enzyme [11, 12]. The uridylylated PII interacts with an adenylyltransferase to deadenylylate and activate the GS enzyme (encoded by glnA) [11, 13]. In addition, the NtrB/NtrC two-component system will activate the expression of glnA under nitrogen-limiting conditions [9, 14–19]. By contrast, in response to high glutamine/α-ketoglutarate, the uridylylated PII is deuridylylated by GlnD. The unmodified PII protein interacts with adenylyltransferase thereby causing the adenylylation and inactivation of the GS enzyme [11, 12]. The unmodified PII protein also interacts with the NtrB/NtrC two-component system to inactivate the expression of glnA [9, 14–19]. Thus bacterial PII proteins are sensors of α-ketoglutarate and adenylate energy charge, whereas GlnD is the sensor of glutamine [20, 21].
Little is known about amino acid sensing and signaling in plants. PII-like proteins have been identified in Arabidopsis and rice [22, 23]. However, bacterial GlnD homologs have yet to be identified in plants. The E. coli sensor protein GlnD is composed of a nucleotide transferase domain, a nucleotide hydrolase domain, and two C-terminal ACT domains. It has been shown that the C-terminal ACT domains of GlnD may regulate its activity through the binding of glutamine .
The ACT domain, named after bacterial aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase), is a regulatory domain that serves as an amino acid-binding site in feedback-regulated amino acid metabolic enzymes [24–28]. For instance, the E. coli 3-phosphoglycerate dehydrogenase (PGDH), a key enzyme in serine biosynthesis, is feedback regulated by serine. The C-terminal ACT domain of E. coli PGDH is the binding site for its allosteric effector serine [24, 29, 30]. The other amino acid metabolic enzymes such as acetohydroxyacid synthase , threonine deaminase [32, 33], and phenylalanine hydroxylase  also contain the regulatory ACT domain. In addition, the ACT domain is also found in several transcription factors [35–39].
We previously identified a novel type of ACT domain-containing protein family in Arabidopsis, whose members contain four ACT domain repeats (the "ACR" protein family) . Other than the ACT domain, the amino acid sequences of the ACR proteins do not have homology to any known enzymes or motifs in the database (http://www.ebi.ac.uk/Tools/InterProScan/). Although proteins homologous to the ACR family have been identified in rice [41–43], the functions of these ACR proteins are largely unknown.
In this report, we have identified four additional ACT domain-containing proteins in Arabidopsis. These proteins are composed of three or two copies of the ACT domain. The amino acid sequences of these proteins do not have any recognizable motifs except the ACT domain. These novel ACT domain-containing proteins are classified as new members of the ACR family. We showed that the newly identified ACR11 and ACR12 proteins are localized to the chloroplast. Interestingly, the expression of ACR11 is co-regulated with GLN2 that encodes a chloroplastic glutamine synthetase (GS). The possible functions of Arabidopsis ACR11 are discussed herein.