SGR2, a phospholipase-like protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravitropism of Arabidopsis

Abstract

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid-preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism.

Figure 1.

Shoot Gravitropism of sgr2 and zig/sgr4. (A) to (C) Gravitropic response of inflorescence stems of 5-week-old plants after 90 min of horizontal gravistimulation. (A) Wild type (Col). (B) sgr2-1. (C) zig-1. (D) to (F) Gravitropism of 3-day-old etiolated seedlings. (D) Wild type (Col). (E) sgr2-1. (F) zig-1. The arrow indicates the direction of gravity (g).

Figure 2.

Morphology of Primary and Lateral Shoots. Aerial parts of wild type (Col) (A), sgr2-1 (B), and zig-1 (C) plants.

Figure 3.

Time Course of the Gravitropic Response of Inflorescence Stems. (A) The gravitropic response of sgr2 alleles: wild-type (Col; open circles), sgr2-1 (closed circles), sgr2-3 (open squares), sgr2-5 (closed triangles), sgr2-6 (open triangles), and sgr2-10 (closed squares) stem segments. (B) The gravitropic response of zig/sgr4 alleles: wild-type (Col; open circles), zig-1 (open triangles), zig-2 (open squares), wild-type (Wassilewskija; closed circles), and zig-3 (closed triangles) stem segments. Stem segments were gravistimulated by being placed horizontally at 23°C in the dark. The curvature was measured at the times indicated.

Figure 4.

sgr2 Seed and Seedlings. (A) and (C) The mature seed in a >1.2-cm-long sheath. Typically shaped seed are shown magnified in the insets. (A)Wild-type seed are uniformly oval. (C) A few sgr2 seed are abnormally shaped or shrunken (arrowhead). (B) and (D) Seven- to 10-day-old seedlings. (B) Wild-type seedling. (D) A seedling with three cotyledons (sgr2-1). Bars in (A) and (C) = 300 μm; bars in (B) and (D) = 1 mm.

Figure 5.

Molecular Cloning of the SGR2 Gene (A) SGR2 was mapped to lie between the m254 and 7G6 markers on chromosome 1. The numbers of recombinants are indicated under these markers (recombinant chromosomes/analyzed chromosomes). Probe X (XbaI–XbaI fragment derived from F11B17) was used as a probe in DNA gel blot analysis. (B) DNA gel blot analysis of three sgr2 alleles that were generated by radiation mutagenization. These genomic DNAs, digested with XbaI, were loaded and blotted. A polymorphism was detected between sgr2-10 and Col. Probe X DNA fragment was used as probe. (C) The structure of the SGR2 gene and the mutation sites of the sgr2 alleles examined. Boxes represent exons: closed boxes, translated regions; open boxes, untranslated regions. SGR2 is encoded by an ∼5.8-kb genome fragment that contains 22 exons and 21 introns. Each mutation of 10 sgr2 alleles is mapped in the genome region. (D) Complementation of sgr2 gravitropism by the wild-type SGR2 genomic region. (E) RNA gel blot analysis of SGR2 mRNA levels in each wild-type organ. Each lane was loaded with 5 μg of total RNA. R, roots of a 7-day-old seedling; H, hypocotyl of an etiolated seedling grown in the dark for 3 days; L, mature rosette leaves; S, inflorescence stems; B, floral buds, including inflorescence meristem; Si, siliques.

Figure 6.

Structure of the SGR2 Protein and Its Homologs. (A) Scheme of SGR2 (Arabidopsis), PA-PLA1 (B. taurus), MO3A1.6 (C. elegans), and p125 (H. sapiens). Boxes represent conserved sequences. aa, amino acids. (B) Multiple sequence alignment of two regions that are conserved between SGR2 and its homologs. The horizontal line shows the lipase consensus sequence (GXSXG). Two boldface letters indicate the residues that are changed in sgr2-3 and sgr2-6.

Figure 7.

Histological Analysis of the zig Mutant. (A) and (C) Epidermal cell layers of wild-type (A) and zig-1 (C) inflorescence stems. (B) and (D) Longitudinal sections of wild-type (B) and zig-1 (D) inflorescence stems. Bars = 50 μm.

Figure 8.

Molecular Cloning of the ZIG Gene. (A) Initial mapping placed the zig mutation between the nga76 and DFR markers. Using newly generated PCR markers, the position of the ZIG locus was narrowed to an ∼380-kb region. The numbers of recombinants among 2422 chromosomes tested are indicated (recombinant chromosomes/analyzed chromosomes). DNA gel blot analysis using the K16M23 TAC clone as a probe showed that deletions had occurred in both zig-1 and zig-2 in the region that contains MUL8.15. (B) The structure of the ZIG gene and the mutation sites of the zig alleles examined. Boxes represent exons: closed boxes, translated regions; open boxes, untranslated regions. zig-2 had lost the ZIG gene completely as well as some neighboring genes (data not shown). (C) Molecular lesion in each allele shown on the protein. The X shows the chromosomal discontinuity in zig-1. Black and gray boxes indicate a predicted transmembrane domain and a coiled-coil domain, respectively, of ZIG/AtVTI11. (D) and (E) Complementation of zig by the wild-type ZIG genomic region. The 5-kb genomic DNA fragment that includes MUL8.15 (A) was transformed into zig-1 plants. (D) The resulting 4-week-old transgenic plants. (E) Plants placed horizontally for 2 hr at 23°C in the dark. The arrow indicates the direction of gravity (g).