myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

Abstract

myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all null mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution.

FIGURE 1.

Expression pattern of MIPS1. A, MIPS1 transcript levels in different plant parts detected by RNA blot analysis. Ten μg of total RNA from roots, stems, leaves, flowers, and siliques were loaded per lane. B–G, MIPS1::GUS expression patterns in whole seedling (B), cotyledon (C), root (D), mature leaf (E), floral organ (F), and silique (G). GUS staining was performed overnight.

FIGURE 2.

Seedling phenotypes of the mips1 mutants. A, structure of the MIPS1 gene. Boxes represent exons. The sites of T-DNA insertions are indicated. B, MIPS1 transcript levels in wild type and mips1 mutants detected with the RNA blotting analysis. mips1-2 (SALK_023626) and mips1-5 (CS851587) are in the Col-0 background, and mips1-4 is in the C24 background. C–G, Col-0 seedling (C) and representative mips1-2 seedlings defective in cotyledon development (D–G). H and I, root length of 10-day-old wild type and mips1 seedlings on an MS plate without or with 100, 500, or 1000 μm inositol. Data are means ± S.E. (n = 20). J, levels of phosphatidylinositol species in 10-day-old Col-0 and mips1-2 seedlings. Data are means ± S.E. (n = 5 biological replicates). Bars, 1 mm (C) and 2 mm (D–G). **, p < 0.05; *, p < 0.01.

FIGURE 3.

Embryo development and the DR5 reporter expression pattern in mips1 mutants. A–H, embryos at the early globular stage of WT Col-0 (A) and mips1-2 (B), heart stage of WT (C) and mips1 (D), torpedo stage of WT (E) and mips1 (F), and mature embryo of WT (G) and mips1 (H). I–L, DR5-GFP in embryos at the later globular stage of WT (I) and mips1 (J), torpedo stage of WT (K) and mips1 (L). M–P, multiple restricted DR5-GUS maxima in cotyledons of 2-day-old mips1-2 grown in the dark (M) or in the light (O) compared with one diffusive DR5-GUS maximum in cotyledons of 2-day-old Col-0 grown in the dark (N) or in the light (P).

FIGURE 4.

Altered cotyledon vein patterning in mips1. A–F, differential interference contrast (DIC) images of cleared 7-day-old seedling cotyledons of Col-0 (A) and mips1-2 (B–F).

FIGURE 5.

Root gravitropism and columella cell patterning in mips1. A, root curling assay of the wild type and mips1 seedlings. The pictures were taken 2 days after the plate was placed horizontally. B, root curvature of 5-day-old wild type and mips1 roots. Shown are means ± S.E. (n = 33). C and D, Lugol's solution stained Col-0 (C) and mips1-2 (D) root tips. E and F, high magnification of Col-0 (E) and mips1-2 (F) root cap stained with Lugol's solution. G and H, propidium iodide-stained Col-0 (G) and mips1-2 (H) root tips. QC, quiescent center; ci, columella initials; t1, t2, t3, the first three columella cell layers of the root cap. I, quantitative analyses of root re-orientation of mips1, eir1, aux1 single and double mutants. Root angles were determined as the deviation from 0° representing complete re-orientation to the vertical and grouped in 12 sectors of 30°. Bars represent relative numbers of roots as percentage of the total (n). J, inflorescence of 6-week-old Col-0, aux1–7, mips1-2, and mips1 aux1 double mutant.

FIGURE 6.

Impaired polar auxin transport and altered auxin response in mips1. A and B, relative rate of basipetal (A) and acropetal (B) auxin transport in mips1 roots. Data are means ± S.E. (n = 3). C and D, DR5-GUS activity (A.U., arbitrary units) in Col-0 and mips1-2 seedlings treated with different concentrations of 2,4-D (C) or NAA (D). Five-day-old seedlings were treated with the indicated concentrations of auxins for 24 h before extracting the protein for GUS assay. Data are means ± S.E. (n = 3).

FIGURE 7.

Impaired vesicle trafficking in mips1 mutants. Shown are fluorescence images of FM4-64 uptake in the root tips of Col-0 (A–D), mips1-2 (E–H), C24 (I–L), and mips1-4 (M–P). Roots of 5-day-old seedlings were incubated in a half-strength MS medium containing 5 μm FM4-64 for 5 min on ice. Pictures were taken immediately or after 30 or 60 min of incubation in a half-strength MS solution at 22 °C. For BFA-induced compartments, 5-day-old seedlings were incubated in 50 μm BFA for 2 h before incubation in 50 μm BFA plus 5 μm FM4-64 for 30 min. Pictures were taken immediately after the FM4-64 treatment. More than 30 seedlings were examined for each treatment. Bars, 25 μm.

FIGURE 8.

Altered sorting of membrane proteins in mips1 mutants. A–J, PIN2 localization in root tips of Col-0 (A–E) and mips1-2 (F–J). PIN2-GFP in root epidermal and cortical cells of Col-0 (A) and mips1 (F) without BFA treatment. PIN2 accumulation after 30, 60, and 90 min of BFA treatment in Col-0 (B–D) and mips1 (G–I). Note that as compared with one or two large PIN2 compartments in the Col-0 after 90 min of BFA treatment, multiple smaller PIN2 compartments are evident in mips1. Two hours after BFA washout, PIN2 polar localization is reestablished in Col-0 (E), although considerable amount of PIN2 is still in the cytosol in mips1-2 (J). K–N, PIP2 localization in root tips of Col-0 (K and L) and mips1-2 (M and N). PIP2-GFP in epidermal cells of Col-0 (K) and mips1-2 (M) without BFA treatment. PIP2 accumulation after 90 min of BFA treatment in Col-0 (L) and mips1-2 (N). More than 30 seedlings were examined for each treatment. Bars, 20 μm.