Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination

Abstract

The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.

Figure 1.

PME activities during germination of Arabidopsis wild-type seeds in the absence (control [CON]; green bars) and presence of 1 μm ABA (ABA; brown bars). Data represent averages ± se of four biological replicates of about 100 seeds each. Seeds were weighed before imbibition, and the activity shown is relative to the starting seed weight. The percentages over the bars list the proportions of seeds that had achieved testa rupture (TR) and endosperm rupture (ER). Note that both control seeds and ABA-treated seeds have reached 100% testa rupture at 32 h. Control seeds proceed to endosperm rupture, while ABA-treated seeds remain at the testa rupture stage for several days.

Figure 2.

Transcript abundance of PMEI5 in different organs and at different life cycle stages of Arabidopsis (ecotype Columbia wild type) as determined by qRT-PCR. Transcript levels were normalized to the mean of two reference genes, EF1a and ACT7. Averages of four biological replicates ± se are shown.

Figure 3.

Effect of PMEI5 OE on PMEI5 transcript levels and PME activity. A, Transcript abundance of PMEI5 in the leaves of wild-type plants and in the leaves of five independent PMEI OE lines (1–5) as determined by qRT-PCR. Transcript levels were normalized to the mean of two reference genes, EF1a and ACT7. B, PME activities in different tissues of adult Arabidopsis plants and in seeds and seedlings. The leaves and stems were of plants harvested 24 d after sowing. The seeds were sampled after 16 h of imbibition (i.e. during phase II of germination), and seedlings were 5 d post germination. WT, Wild-type or nontransgenic controls. The data in B are based on average PME activities determined from five wild-type lines and five PMEI5 OE lines that represented independent insertions ± se.

Figure 4.

Cell wall sugars and methylester content. A, Sugar composition of cell walls of wild-type (WT) and PMEI OE seeds (2 h after imbibition). The data are based on averages of five replicates ± se. B, Estimated methylester content as based on methanol associated with saponified cell wall materials. The data are based on averages of three replicates ± se.

Figure 5.

Effects of PMEI5 OE on cell size and the degree of pectin methylesterification of the embryo, endosperm, and testa. A to F, Immunolabeling of representative sections derived from PMEI OE and wild-type (WT) seeds with the JIM7 antibody (A and B), JIM5 antibody (C and D), and 2F4 antibody (E and F). G and H, Staining of sections. All images were taken at the same magnification for the WT and PMEI OE. Note that the cells of PMEI OE seeds are larger. cot, Cotyledons; es, endosperm; mu, mucilage; rad, radical; te, testa. Bars = 65 μm.

Figure 6.

Effects of PMEI5 OE on seed germination characteristics. A, Testa rupture (TR) and endosperm rupture (ER) in seeds of the PMEI OE lines as compared with the wild type (WT). Data represent averages ± se of three replicates of 50 seeds each. B, Effects of different concentrations of ABA on the germination (endosperm rupture) of PMEI OE lines versus the WT. Data represent averages ± se of three replicates of 50 seeds each. C, PME activities of protein extracts derived from PMEI OE lines during germination in the absence (control [CON]; blue bars) and presence of 1 μm ABA (ABA; gray bars). Data are based on averages ± se of four biological replicates of about 100 seeds each. Seeds were weighed before imbibition, and the activity shown is relative to the starting seed weight. The percentages over the bars list the proportion of seeds that had achieved testa rupture and endosperm rupture.

Figure 7.

Testa rupture (TR) and endosperm rupture (ER) of wild-type (WT) and PMEI OE seeds. A, Light microscopy images of WT and PMEI OE seeds. B, eSEM images. Arrows show how the endosperm of PMEI OE seeds is stretched more extensively and retained as a collar-like structure of cells that clings to the radicle after endosperm rupture. Note the clear rupture lines of the testa shown for WT seeds in B.

Figure 8.

Morphological phenotypes of PMEI OE seeds and siliques. A, Siliques of PMEI OE plants are wrinkled and shorter than those of wild-type plants. Also note the twisted growth of the stem. B, eSEM image of dry seeds of the wild type (WT; left) and PMEI OE (right). Note the larger size of PMEI OE testa cells. C, Quantitative differences between the characteristics of seeds, siliques, and plant growth of wild-type and PMEI OE lines.