The ACR11 encodes a novel type of chloroplastic ACT domain repeat protein that is coordinately expressed with GLN2 in Arabidopsis

Abstract

Background: 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. We have previously identified a novel type of ACT domain-containing protein family, the ACT domain repeat (ACR) protein family, in Arabidopsis. Members of the ACR family, ACR1 to ACR8, contain four copies of the ACT domain that extend throughout the entire polypeptide. Here, we describe the identification of four novel ACT domain-containing proteins, namely ACR9 to ACR12, in Arabidopsis. The ACR9 and ACR10 proteins contain three copies of the ACT domain, whereas the ACR11 and ACR12 proteins have a putative transit peptide followed by two copies of the ACT domain. The functions of these plant ACR proteins are largely unknown.

Results: The ACR11 and ACR12 proteins are predicted to target to chloroplasts. We used protoplast transient expression assay to demonstrate that the Arabidopsis ACR11- and ACR12-green fluorescent fusion proteins are localized to the chloroplast. Analysis of an ACR11 promoter-β-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity was mainly detected in mature leaves and sepals. Interestingly, coexpression analysis revealed that the GLN2, which encodes a chloroplastic glutamine synthetase, has the highest mutual rank in the coexpressed gene network connected to ACR11. We used RNA gel blot analysis to confirm that the expression pattern of ACR11 is similar to that of GLN2 in various organs from 6-week-old Arabidopsis. Moreover, the expression of ACR11 and GLN2 is highly co-regulated by sucrose and light/dark treatments in 2-week-old Arabidopsis seedlings.

Conclusions: This study reports the identification of four novel ACT domain repeat proteins, ACR9 to ACR12, in Arabidopsis. The ACR11 and ACR12 proteins are localized to the chloroplast, and the expression of ACR11 and GLN2 is highly coordinated. These results suggest that the ACR11 and GLN2 genes may belong to the same functional module. The Arabidopsis ACR11 protein may function as a regulatory protein that is related to glutamine metabolism or signaling in the chloroplast.

Figure 1

Sequence analysis of the Arabidopsis ACR family. (A) Phylogenetic relationships of Arabidopsis ACR proteins and the C-terminal ACT domains of E. coli GlnD. Full-length amino acid sequences of Arabidopsis ACR1 to ACR12 and amino acid residues 708-890 of E. coli GlnD were aligned by ClustalW2 and the neighbor-joining algorithm was used to obtain the phylogenetic tree. (B) Schematic gene structures of Arabidopsis ACR9 to ACR12. Exons are shown as black boxes and introns are indicated as solid lines. (C) Schematic diagram of Arabidopsis ACR9 to ACR12 proteins. The black boxes indicate the ACT domains.

Figure 2

Amino acid sequence alignments of ACR proteins and ACT domains. (A) Sequence alignment of Arabidopsis ACR11 and ACR12 proteins. ACT domains are indicated with solid lines above the sequences. Arrowheads indicate the predicted cleavage sites of chloroplast transit peptides. Asterisks shown below the sequences denote the putative ligand-binding sites. (B) Sequence alignment of ACT consensus sequence (ACTc) from Pfam01842, and ACT domains from ACR11 (ACR11.1 and ACR11.2), ACR12 (ACR12.1 and ACR12.2) and GlnD (GlnD1 and GlnD2). The predicted secondary structure of the ACTc is shown above the sequences. Arrow indicates the conserved glycine residue in the β1-α1 loop region. Identical and similar amino acid residues are shaded in black and gray, respectively.

Figure 3

The Arabidopsis ACR11- and ACR12-GFP fusion proteins are localized to the chloroplast. Arabidopsis mesophyll protoplasts were transformed with ACR11- and ACR12-GFP constructs, which encode the full-length ACR11 protein, and the first 94 amino acids of ACR12 fused to GFP, respectively. Chloroplasts were visualized by red chlorophyll autofluorescence. The green fluorescent signals of ACR11- and ACR12-GFP colocalized with the red fluorescent signals of chlorophyll (merge). Arabidopsis protoplasts transformed with the empty vector are shown as controls for the subcellular localization of GFP. Scale bars are 10 μm.

Figure 4

Coexpressed gene networks around Arabidopsis ACR11 and ACR12. (A) The three genes having the highest coexpression mutual rank (MR) with ACR11 are At5g35630 (encoding glutamine synthetase 2; MR = 1.0), At4g15545 (encoding an unknown protein; MR = 8.5) and At5g64460 (encoding an unknown protein; MR = 9.2). The ACR11 (At1g16880) is annotated as an uridylyltransferase-related protein in the database. (B) The three genes having the highest coexpression mutual rank (MR) with ACR12 are At3g29350 (encodes AHP2, histidine-containing phosphotransmitter2; MR = 2.2), At1g10200 (encodes WLIM1, a member of the Arabidopsis LIM proteins; MR = 6.2), and At1g49820 (encodes MTK1, 5-methylthioribose kinase1; MR = 7.5). The coexpression gene networks of ACR11 and ACR12 can be obtained at the ATTED-II website (http://atted.jp/data/locus/At1g16880.shtml and http://atted.jp/data/locus/At5g04740.shtml).

Figure 5

Expression patterns of ACR11 and GLN2 in Arabidopsis. Total RNA (10 μg) from roots (R), leaves (L), stems (St), flowers (F), and siliques (Si) of 6-week-old Arabidopsis grown in soils was used for RNA gel blot analysis. The ethidium bromide-stained agarose gel of the same samples is shown at the bottom.

Figure 6

The expression of Arabidopsis ACR11 and GLN2 is co-regulated by sucrose and light/dark treatments. Total RNA (10 μg) from 14-day-old Arabidopsis plants treated with complete darkness or continuous light for 48 h was used for RNA gel blot analysis. During the dark or light treatment, plants were grown on MS media containing 0% sucrose, 3% sucrose, or 3% mannitol. The expression of ACR11 and GLN2 is up-regulated by sucrose and light.

Figure 7

GUS activity in transgenic Arabidopsis containing ACR11 promoter-GUS fusion. (A) 3-day-old, (B) 5-day-old, (C) 7-day-old, (D) 10-day-old, (E) 14-day-old seedlings. (F) Flower buds and mature flowers. (G) Close-up of a mature flower. (H) A mature silique.