Data mining the Arabidopsis genome reveals fifteen 14-3-3 genes. Expression is demonstrated for two out of five novel genes

Abstract

In plants, 14-3-3 proteins are key regulators of primary metabolism and membrane transport. Although the current dogma states that 14-3-3 isoforms are not very specific with regard to target proteins, recent data suggest that the specificity may be high. Therefore, identification and characterization of all 14-3-3 (GF14) isoforms in the model plant Arabidopsis are important. Using the information now available from The Arabidopsis Information Resource, we found three new GF14 genes. The potential expression of these three genes, and of two additional novel GF14 genes (Rosenquist et al., 2000), in leaves, roots, and flowers was examined using reverse transcriptase-polymerase chain reaction and cDNA library polymerase chain reaction screening. Under normal growth conditions, two of these genes were found to be transcribed. These genes were named grf11and grf12, and the corresponding new 14-3-3 isoforms were named GF14omicron and GF14iota, respectively. The gene coding for GF14omicron was expressed in leaves, roots, and flowers, whereas the gene coding for GF14iota was only expressed in flowers. Gene structures and relationships between all members of the GF14 gene family were deduced from data available through The Arabidopsis Information Resource. The data clearly support the theory that two 14-3-3 genes were present when eudicotyledons diverged from monocotyledons. In total, there are 15 14-3-3 genes (grfs 1-15) in Arabidopsis, of which 12 (grfs 1-12) now have been shown to be expressed.

Figure 1

Gene maps of all Arabidopsis 14-3-3 (GF14) genes. Exons are indicated as black boxes and introns as white boxes. Exon and intron sizes are indicated with the number of bases within in each box. The names of the resulting proteins are within parentheses. The genes can be divided into two groups based on exon patterns: grfs 1 through 8, and grfs 9 through 14, excluding the severely truncated grf15 that does not readily fit into either group. The five novel genes discussed in this paper are grf11, grf12, grf13, grf14, and grf15, of which grf11 and grf15 were already reported by Rosenquist et al. (2000). The abbreviation grf stands for G box regulating factor.

Figure 2

Detection of novel 14-3-3 gene transcripts in flower (A, B, and F), leaf (C and E), and root (D) tissues by RT-PCR and cDNA library PCR. Primary RT-PCR reactions were performed with primers corresponding to exon 2 (see Fig. 1) of grf11 (omicron; A and C), grf12 (iota; A and C), grf13 (A, C, and D), and grf14 (B, C, and D). Subsequent RT-PCR reactions were performed with full-length primers for grf11 on leaf (E) and root (D) cDNA, and for grf12 on flower (F) and root (D) cDNA. Products were run on 1.5% (w/v) agarose gel (inverted image) and bands of interest are indicated with black arrowheads. The identities of these bands were confirmed by sequencing. Sizes of standard (std) markers are indicated to the left. Note that the band grf12 (iota) exon2 (A) only corresponds to 290 bp of the exon due to optimization of primer positions, and therefore is 17 bp shorter than grf11 (omicron) exon2.

Figure 3

DNA sequences of the two expressed novel Arabidopsis 14-3-3 (GF14) genes, grf11 and grf12, and corresponding amino acid sequences for the proteins, GF14omicron and GF14iota, respectively. Conserved identity between all expressed GF14s is indicated with gray boxes, and conserved homology is indicated with white boxes.

Figure 4

A phylogenetic tree with topology representative for the Arabidopsis 14-3-3 (GF14) protein family. The putative gene products of grf13 and grf14 are designated Grf13 and Grf14, respectively. A heuristic search using the maximum parsimony method was done on the alignment of GF14 protein sequences using the PAUP 4.0b4 software (Sinauer Associates, Inc. Publishers, Sunderland, MA), with gaps treated as missing data. Bootstrap values for 12,000 replicates are indicated on each branch. The hypothetical 14-3-3-like protein coded by grf15 (Table I; Fig. 1) was excluded from the analysis due to strong deviance in both sequence and length. The 82 amino acids encoded by grf15 show highest homology (44% identity) to GF14psi and upsilon, and correspond to the C terminus of a functional 14-3-3 (data not shown).

Figure 5

Arabidopsis chromosome map. BAC clones harboring 14-3-3 (GF14) genes are indicated with black lines. Gene names, with corresponding gene products written in parentheses, are indicated by each BAC. Centromeres are indicated with white circles. The gray dashed arrow indicates a major duplication event that took place 170 million years ago, and the two gray arrows indicate another major duplication event that took place 50 million years ago (Vision et al., 2000). Thin dashed and dotted lines connect relatively closely related genes and hence suggest other duplication events. The gene grf14 (T16E15.9) is located 1,329 bases from grf10 (T16E15.8) within the same BAC clone. Given the low homology of the severely truncated 14-3-3 gene grf15 in F12P23, no connection to other genes is indicated.