Molecular characterization of a novel gene family encoding ACT domain repeat proteins in Arabidopsis

Abstract

In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the "ACR" protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-beta-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.

Figure 1

Sequence alignment of Arabidopsis ACR proteins. Identical and similar amino acid residues are shaded in black and gray, respectively. The locations of the ACT domains are indicated with solid lines above the sequences. Asterisks shown above the sequences denote the putative nuclear localization sequence of ACR1. The amino acid sequences of ACR1 and ACR3 to ACR8 are deduced from the cDNAs reported here. The amino acid sequence of ACR2 is from accession number NM_122441.

Figure 2

A, Schematic diagram of Arabidopsis ACR and E. coli GlnD proteins. The gray boxes indicate the ACT domains. Arabidopsis ACR3 is shown as a representative of the ACR protein family. It contains four ACT domains and two conserved regions (ovals). E. coli GlnD has two ACT domains that show high similarity to those in the Arabidopsis ACR proteins. NT, Nucleotide transferase domain; HD, nucleotide hydrolase domain. B, Sequence alignment of ACT consensus sequence (ACTc) from Pfam01842, four ACT domains (ACT1–ACT4) from Arabidopsis ACR3, and two ACT domains (GlnD1 and GlnD2) from E. coli GlnD. Identical and similar amino acid residues are shaded in black and gray, respectively. The secondary structure of the ACTc predicted by a computer program (http://www.aber.ac.uk/∼phiwww/prof/) is shown above the sequences. The most conserved portion of the ACT domain is the region at the interface between the first strand and the first helix, which corresponds to the ligand-binding site. C, Amino acid sequence similarities among the ACT domains aligned in B.

Figure 3

A, Chromosomal locations of the Arabidopsis ACR genes. The relative sizes of five Arabidopsis chromosomes are derived from the National Center for Biotechnology Information database. B, Schematic gene structures of the ACR gene family. Exons are shown as gray boxes and solid lines indicate introns.

Figure 4

ACR gene family expression in various organs of 6-week-old Arabidopsis. Total RNA (10 μg) from roots (R), leaves (L), stems (St), flowers (F), and siliques (Si) was used for northern-blot analysis and hybridized with gene-specific probes of ACR1 to ACR8. The ACR2 transcript was not detectable (not shown). The ethidium bromide-stained agarose gel of the same samples is shown at the bottom.

Figure 5

GUS activity in transgenic Arabidopsis containing the ACR3p-GUS fusion. A, Two-week-old seedlings; B, close-up of the emerging leaves and shoot apex in A; C, roots from 2-week-old seedlings; D, young flower buds and a developing cauline leaf; E, flower buds and a mature flower; and F, a developing silique.

Figure 6

Plant hormones differentially regulate steady-state levels of ACR mRNA. Total RNA (10 μg) from 2-week-old Arabidopsis seedlings treated with plant hormones for 1 h (A) or 4 h (B) was used for northern analysis and hybridized with ACR1 to ACR8 gene-specific probes. Levels of ACR8 mRNA were dramatically increased by ABA (compared with C, control). B, Levels of ACR3 and ACR4 mRNA were increased by treatment with BA for 4 h. The transcripts of ACR2 and ACR6 were not detectable. The ethidium bromide-stained gel is shown at the bottom.

Figure 7

The levels of ACR mRNA are differentially regulated by salt, cold, and light/dark treatment. Total RNA (10 μg) from 2-week-old Arabidopsis seedlings treated with 250 mm NaCl for 0, 1, or 4 h (A), 4°C for 2 h (B), or light/dark for 48 h (C) was used for northern analysis to detect levels of ACR1 to ACR8 mRNA. C, Control; CS, cold stress; L, light; D, dark. The transcripts of ACR2 and ACR6 were not detectable. The ethidium bromide-stained gel is shown at the bottom.

Figure 8

A, Schematic diagram of ACT domains in Arabidopsis amino acid metabolic enzymes. The gray boxes indicate the conserved regulatory ACT domains. The white boxes represent the catalytic domains. S, substrate-binding domain; N, nucleotide-binding domain; PDH, prephenate dehydrogenase. B, Schematic diagram of novel ACT domain-containing proteins in Arabidopsis. The gray boxes indicate the ACT domains. AY070086, AY045670, and AY072401 are representatives of the ACT domain-containing protein kinase family and two unknown protein families, respectively. PK, Protein kinase domain (Pfam00069).