Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase

Abstract

The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short integuments due to early cessation of cell division in these structures. SIN2 was isolated and encodes a putative GTPase sharing features found in the novel DAR GTPase family. DAR proteins share a signature DAR motif and a unique arrangement of the four conserved GTPase G motifs. We found that DAR GTPases are present in all examined prokaryotes and eukaryotes and that they have diversified into four paralogous lineages in higher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases have been found to localize to mitochondria and are related to eubacterial proteins that facilitate essential steps in biogenesis of the large ribosomal subunit. We propose a similar role for SIN2 in mitochondria. A sin2 insertional allele has ovule effects similar to sin2-1, but more pronounced pleiotropic effects on vegetative and floral development. The diverse developmental effects of the mitochondrial SIN2 GTPase support a mitochondrial role in the regulation of multiple developmental pathways.

Figure 1.—

Scanning electron micrographs of stage 3-VI ovules (anthesis; stages from Schneitz et al. 1995). (A) Wild-type Ler. (B) sin2-1. (C and D) sin2-2 plants. In D, the gradation in severity of effects of sin2-2 from the base to the apex of the carpel is apparent. f, funiculus; ii, inner integument; n, nucellus; oi, outer integument. Bar, 50 μm (A–C) and 100 μm (D).

Figure 2.—

Identification and expression of the SIN2 gene. (A) The chromosomal region surrounding the SIN2 locus with the molecular markers used to map SIN2 shown above, and the number of recombination events between markers found in 918 F₂ plants indicated below the top horizontal line. The BAC T1K18 spans part of the region between the closest flanking markers. Cosmid subclones .105, .136, and .107, derived from T1K18 and used in the complementation test, are illustrated. (B) The 4.0-kb EcoRI fragment spanning a single gene, At2G41670, derived from cJBT1K18.136, which was able to complement the sin2-1 mutant phenotype. The exons in this region and the transcriptional start site as determined by RT–PCR are shown as boxes and a bent arrow, respectively. Arrows indicate the positions of primers used to screen T-DNA lines for additional sin2 alleles. The positions of the missense (sin2-1) and insertional (sin2-2) mutations are indicated. Numbering is relative to the translational start site. (C) RT–PCR with total RNA from several plant parts used as template shows that SIN2 RNA was present in all structures assayed.

Figure 3.—

DAR protein alignment. An alignment of the seven Arabidopsis DAR proteins, SIN2 and DGP2–7, and the human SIN2 ortholog, human DGP1, highlights the six conserved motifs of this family. Residues found in all of these proteins are shown in white on a black background. These include amino acids within the GTPase motifs G1–G4 and the DAR motif, labeled and underscored below each sequence. There is also a region C-terminal to the recognizable GTPase motifs, which appears loosely conserved and includes a L(X)₅G motif. Amino acids conserved between SIN2 and the closely related proteins DGP2 (38% identity, 57% similarity) and human DGP1 (31% identity, 47% similarity) are shaded. Residues conserved between SIN2 and DGP2 or HsDGP1 are shown in black or white letters, respectively. The positions corresponding to the sin2-1 S150F transition and the sin2-2 insertion are indicated above the sequence.

Figure 4.—

Phylogenetic analysis of DAR GTPases. (A) The single tree generated by parsimony analysis of a culled alignment was modified to collapse branches that were not supported in both culling and elision analyses by bootstrap values >50%. Four main clades are resolved, designated clades 1–4. Eukaryotes are represented in each clade. Prokaryotic sequences are found in clade 1 and two orphan branches (including Methanococcus and Pyrobaculus sequences). Arabidopsis proteins are shaded and SIN2 is outlined in black. Numbers on the branches indicate bootstrap values based on 500 bootstrap replicates. Branch lengths are relative to distance as calculated using the transformed BLOSUM45 matrix. Clade designations are shown above the respective branches. Proteins without previous names were given “DGP” designations. (B–D) DAR proteins share conserved sequences and secondary structure with Era family members. (B) An alignment of the G1 and G2 motifs of SIN2, E. coli Era, ThdF, EngA1, and EngA2 demonstrates that the SIN2 G1 motif is similar to that of Era family members (Era, ThdF, and EngA). (C) A region of EngA similar to a DAR motif was found between EngA1 G3 and EngA1 G4. (D) A comparison of the clade 1 consensus secondary structure predicted by the Jpred server (Cuff et al. 1998) with that derived from the E. coli EngA and E. coli Era crystal structures (Chen et al. 1999; Robinson et al. 2002). EngA structure (arrows and cylinders indicate β-sheet and α-helix regions, respectively) appears to derive from a direct repeat of two Era-like units. The DAR GTPases, represented by SIN2, resemble the central (G4–G3) region of the EngA proteins that include DAR motifs.

Figure 5.—

Localization of SIN2:GFP in Arabidopsis. Arabidopsis hypocotyl cells transformed with 35S:SIN2:GFP. Fluorescence appears white. (A) Green fluorescent protein is localized to small particles within the cells that correspond to mitochondria stained with MitoFluor 589 dye, shown in B.