Suspensor length determines developmental progression of the embryo in Arabidopsis

Abstract

The first structure that differentiates during plant embryogenesis is the extra-embryonic suspensor that positions the embryo in the lumen of the seed. A central role in nutrient transport has been ascribed to the suspensor in species with prominent suspensor structures. Little is known, however, about what impact the size of the rather simple Arabidopsis (Arabidopsis thaliana) suspensor has on embryogenesis. Here, we describe mutations in the predicted exo-polygalacturonase gene NIMNA (NMA) that lead to cell elongation defects in the early embryo and markedly reduced suspensor length. Mutant nma embryos develop slower than wild-type embryos, and we could observe a similar developmental delay in another mutant with shorter suspensors. Interestingly, for both genes, the paternal allele has a stronger influence on the embryonic phenotype. We conclude that the length of the suspensor is crucial for fast developmental progression of the embryo in Arabidopsis.

Figure 1.

Embryonic phenotype of nma mutants compared with the wild type. A and B, Embryonic phenotype at one-cell stage of nma (A) and the wild type (B). Apical cells are false colored in yellow; basal cells are false colored in green. Measurements are given as average with sd in micrometers. C and D, Embryonic phenotype at 16-cell stage of nma (C) and the wild type (D). Suspensor cells are highlighted by accompanying asterisks. E and F, Late globular stage embryos of nma (E) and the wild type (F). G and H, Heart stage embryos of nma (G) and the wild type (H). I and J, Developing seed of nma (I) and the wild type (J) 7 dap. Bars = 20 µm. K, Measurements of embryo proper and suspensor length at different developmental stages. Number of analyzed embryos at one-cell stage: nma, n = 26 and the wild type, n = 26. Number at eight-cell stage: nma, n = 101 and the wild type, n = 98. Number at globular stage: nma, n = 36 and the wild type, n = 29. Number at triangular stage: nma, n = 54 and the wild type, n = 33. Mean values with sd are shown. Significant differences were determined in pairwise comparison by Mann-Whitney U test (* = P < 0.05, ** = P < 0.01, *** = P < 0.001, and / = P > 0.05). WT, Wild type.

Figure 2.

Developmental stages of embryos. A, Wild-type and nma embryos 4 dap were classified into six developmental stages: midglobular (mg), late globular (lg), triangular (tr), early heart (eh), late heart (lh), and early torpedo (et). Schematic depictions of the developmental stage are given below the graphs. Mean values of three independent biological replicates with sd are shown. B, Average developmental stage of embryos 4 dap in nma-1, Ler, ssp-2, and Col-0. Embryos were classified in six developmental stages as in Figure 2A. Numerical values were assigned to each stage. Averages and sds of three biological replicates are shown as bar graphs. Asterisks indicate sds in pairwise comparison (Mann-Whitney U test; P < 0.001). C, Average developmental stage of embryos 30 h after pollination in nma-1, Ler, ssp-2, and Col-0. Embryos were classified in three developmental stages: zygote (Z), one-cell (oc), and two- and four-cell (tc). Numerical values were assigned to each stage. Averages and sds of three biological replicates are shown as bar graphs.

Figure 3.

Expression analysis of NMA promoter::reporter gene constructs. A to H, Fluorescence micrographs of transgenic plants carrying a transcriptional fusion of a NMA promoter fragment to nuclear-localized triple Venus-YFP. A, Embryo after the first zygotic division. B, Globular-stage embryo, inset shows 4′,6-diamidino-2-phenylindole (DAPI) staining. C, Heart-stage embryo. D, Developing seed 2 dap. E, Pollen, arrows mark sperm cell nuclei and arrowhead indicates vegetative cell nucleus; inset shows DAPI staining. F, Female gametophyte after meiosis. G, Female gametophyte at eight-nuclei stage. H, Mature female gametophyte. Bars = 10 μm. I and J, GUS activity staining in transgenic plants carrying a transcriptional pNMA::GUS fusion construct. I, Inflorescence. J, Seedling. Insets in I and J show transgenic control plants without GUS gene. Bars = 2 mm.

Figure 4.

Transient expression of YFP-NMA fusion proteins in protoplasts. A to C, 24 h after transformation. D to F, 72 h after transformation. A and D, p35S::YFP-NMA fusion construct with intact SP sequence. Arrows in A point to dotted signals on protoplast surface. B and E, p35S::YFP-NMA dSP fusion construct with disrupted SP sequence. C and F, Untransformed control. Bars = 10 μm.