Fig. 1: A type II MCA depletion model affects seed physiology and vacuolar morphology.
a Schematic diagram of MCAs (top left) and phylogeny of type I and II MCAs (top right) in Arabidopsis and the nomenclature used. Bottom cartoon, chromosomal locations MCAs in the 5 chromosomes. Pro, prodomain; p20 and p10, large and small subunits; H, C, catalytic histidine-cysteine dyad. b Representative immunoblot probed with α-MCA-II-a from seedlings of the mca-II-a mutant and the mca-II-KOc (lines #93 and #30) at 7 days post germination (DPG). α-ACTIN was used as a loading control; the black arrowhead indicates MCA-II-a band (biological replicates N = 3, n = 1 technical replicate with 20 seedlings per lane). c Immunoblot probed with α-TSN from WT, mca-II-df double mutant (background used to generate the CRISPR mutants), and mca-II-KOc mutant seedlings. α-TUBULIN was used as a loading control; the black arrowhead indicates TSN band (N = 3, n = 1 with 20 seedlings 7 DPG per lane). Right: relative quantification of TSN band (black arrowhead, full length). P-values were calculated by one-way ANOVA (N = 3, n = 1 wells). d Representative images showing the growth of seedlings germinated from freshly collected WT and mca-II-KOc seeds at 3 and 7 DPG (N = 3, n = 1 with ≥ 40 seedlings in total). e Growth of seedlings germinated from WT and mca-II-KOc (3, 6, and 9 month-old) seeds at 3 and 7 DPG (N = 3, n = 1 with ≥ 40 seedlings in total). f Germination rate (%) from freshly collected or 9 month-old seeds of WT and mca-II-KOc. P-values were calculated by one-way ANOVA (N = 3, n = 2 replicates with a total of ≥ 278 seedlings). The first P-value corresponds to the 1 to 3rd point interval, while the second P-value corresponds to the 4th point; the magenta and light-green areas around the individual points (means) represent ± s.d. g Visualization of vacuoles in embryonic roots from WT and mca-II-KOc plants after a 2 day stratification (hydration) counter-stained with the pH-sensitive lumen dye BCECF. The styryl dye FM4-64 was used to visualize the plasma membrane (cell contours, magenta). Yellow arrowheads point to vacuole-free regions in the cytoplasm that compress the vacuolar membrane in the mca-II-KOc (corresponding to lipid droplets; Fig. 5b). Scale bars, 10 μm. Source data are provided as a Source Data file.
