ERECTA receptor-kinases play a key role in the appropriate timing of seed germination under changing salinity

Abstract

Appropriate timing of seed germination is crucial for the survival and propagation of plants, and for crop yield, especially in environments prone to salinity or drought. However, the exact mechanisms by which seeds perceive changes in soil conditions and integrate them to trigger germination remain elusive, especially once the seeds are non-dormant. In this study, we determined that the Arabidopsis ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2) leucine-rich-repeat receptor-like kinases regulate seed germination and its sensitivity to changes in salt and osmotic stress levels. Loss of ER alone, or in combination with ERL1 and/or ERL2, slows down the initiation of germination and its progression to completion, or arrests it altogether under saline conditions, until better conditions return. This function is maternally controlled via the tissues surrounding the embryo, with a primary role being played by the properties of the seed coat and its mucilage. These relate to both seed-coat expansion and subsequent differentiation and to salinity-dependent interactions between the mucilage, subtending seed coat layers and seed interior in the germinating seed. Salt-hypersensitive er105, er105 erl1.2, er105 erl2.1 and triple-mutant seeds also exhibit increased sensitivity to exogenous ABA during germination, and under salinity show an enhanced up-regulation of the germination repressors and inducers of dormancy ABA-insensitive-3, ABA-insensitive-5, DELLA-encoding RGL2, and Delay-Of-Germination-1. These findings reveal a novel role of the ERECTA receptor-kinases in the sensing of conditions at the seed surface and the integration of developmental, dormancy and stress signalling pathways in seeds. They also open novel avenues for the genetic improvement of plant adaptation to changing drought and salinity patterns.

Keywords: ERECTA genes; Abiotic stress signalling; cell wall; environmental sensing; mucilage; osmotic stress; receptor-kinases; salinity; seed dormancy; seed germination; seed size.

Fig. 1.

The three Arabidopsis ERECTA family members synergistically control the timing and speed of seed germination under salinity. (A, B) T₅₀ values (h post-stratification to rupture in 50% of seeds) for testa rupture (A) and endosperm rupture (B). (C) Time interval between testa rupture and endosperm rupture. The experiment was repeated five times with different seed batches and similar results were obtained. WT, wild-type (Col-0). As the triple-mutant is sterile, the segregating progeny of er105 erl1.2+/– erl2.1 plants were used to investigate germination, and is referred to as er erl1.2/seg erl2.1 (note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure). Data are means (±SE) from n=4 plates, with 30 seeds per genotype per plate. Different letters indicate significant differences as determined by two-way ANOVA and Tukey’s HSD pair-wise tests (P<0.001).

Fig. 2.

Germination-specific functions of the ERECTA genes in the control of germination sensitivity to salinity. (A–D) Time-course of endosperm rupture for the wild-type (WT), er105, er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 seeds over a 10-d incubation period on agar media containing either 0 mM NaCl (A, C) or 150 mM NaCl (B, D) following imbibition and stratification either directly on the media (A, B) or in water prior to plating (C, D). (E) Seedling relative expansion rates on 0 mM or 150 mM NaCl media. Seeds were first germinated on NaCl-free media and then transferred to fresh 0 mM or 150 mM NaCl plates and seedling expansion was then measured over the next 72 h. Measurements of the whole-seedling projected area were made on captured images using the ImageJ software. Note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure. All data are means (±SE), n=7. Different letters indicate significant differences as determined by two-way ANOVA and Tukey’s HSD pair-wise tests (P<0.001).

Fig. 3.

Transcripts of Arabidopsis ER, ERL1 and ERL2 genes are present in mature dry seeds and de novo transcription is activated early during germination. Gene expression was measured in dry seeds and in germinating seeds at the end of the stratification period (stage I), then 20 h later (stage II, testa rupture), and then after an additional 52 h (stage III-G, when endosperm rupture had completed on control media). Seeds on 150 mM NaCl that had not germinated by stage III were sampled and analysed separately, and are labelled as III-NG. Data are means (±SE) from n=4 pooled samples of 300 seeds per genotype and treatment. The experiment was repeated three times with similar results. Different letters indicate significant differences as determined using two-way ANOVA and Tukey’s HSD pair-wise tests (P<0.001).

Fig. 4.

The Arabidopsis ERECTA family regulates seed germination sensitivity to salinity mostly via interactions with ionic effects, but is also involved in the control of germination under osmotic stress. (A) Percentage of seeds with endosperm rupture for the wild-type (WT) and the salt-hypersensitive mutants er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 at different times post-stratification as a function of the osmotic potential of the media (π _e), which was varied using PEG8000 at concentrations ranging from 0–171 g l⁻¹. Note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure. Data are means, n=3 plates. (B) Germination response over an extended range of PEG concentrations, in an independent experiment with a different seed batch. Data are means of n=3 plates, and show the percentage of seeds exhibiting endosperm rupture at 6 d post-stratification. (C) Kinetics of seed germination under 0.99 MPa π _e induced by NaCl, using the same seed batch as in (B). The arrow indicates germination scores on day 6 when at least 90% of seeds had germinated under the same osmotic conditions induced by PEG, as shown in (B). Data are means of n=3 plates, with 30 seeds per plate and per genotype. The experiments were repeated three times with similar results.

Fig. 5.

Seed germination in the salt-hypersensitive Arabidopsis mutants er105, er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 is more sensitive to external NaCl than KCl under iso-osmotic conditions. Time-courses of germination under two different osmotic potentials (π _e) are shown, induced by supplementation of the media with either NaCl or KCl. Data are means (±SE) of n=3 plates, with 30 seeds per plate and per genotype. The experiments were carried out twice with similar results. Note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure.

Fig. 6.

Seed germination in the salt-hypersensitive Arabidopsis mutants er105, er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 readily resumes upon removal of the stress. Time-course of seed germination on media containing 150 mM NaCl (0–490 h) followed by transfer to NaCl-free media (arrow). The experiment was repeated three times with similar results. Data are means (±SE) of n=4 plates, with 30 seeds per genotype per plate. Different letters indicate significant differences as determined by one-way ANOVA and Tukey’s HSD pair-wise tests (P<0.05; NS, not significant). Note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure.

Fig. 7.

The Arabidopsis ERECTA genes interact with the sensitivity of seed germination to exogenous ABA and with the expression of major ABA and GA signalling genes. Wild-type (WT) and the salt-hypersensitive Arabidopsis mutants er105, er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 were examined (note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure). (A) Germination response to exogenous ABA application as indicated by T₅₀ values (h post-stratification to rupture in 50% of seeds) for the testa (A) and the endosperm (B). Data are means (±SE) of n=3 plates, with 30 seeds of each genotype per plate. The experiment was repeated three times with similar results. (C) Expression of ABI3, ABI5, RGL2, and DOG1 genes in dry seeds and seeds sampled at the end of stratification (stage I), then 20 h later (stage II, testa rupture), and then after an additional 52 h (stage III-G, when endosperm rupture had completed on control media). Seeds on 150 mM NaCl that had not germinated by stage III were sampled and analysed separately, and are labelled as III-NG. Different letters indicate significant differences as determined using two-way ANOVA and Tukey’s HSD pair-wise tests (P<0.05).

Fig. 8.

The function of Arabidopsis ERECTA genes in seed germination sensitivity to salinity is maternally controlled and shows partial overlap with a function in the determination of seed size. The wild-type (WT) and the mutants er105, erl1.2, erl2.1, er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 were examined (note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure). (A) Seed projected area. Data are means (±SE) of n≥400 seeds per genotype from 11 siliques. Different letters indicate significant differences as determined using one-way ANOVA and Tukey’s HSD pair-wise tests (P<0.001. (B) Relative expansion rate (mm² mm⁻² d⁻¹) of excised mature embryos over a 72-h incubation period on either 0 mM or 150 mM NaCl media (n=7). Different letters indicate significant differences as determined using two-way ANOVA and Tukey’s HSD pair-wise tests (P<0.001. (C) Time-course of germination for WT and er erl1.2 seeds, and F1 seeds generated from their reciprocal crosses. Similar results were obtained from crosses between WT and er erl2.1 flowers (data not shown). (D) Size of F1 seeds from reciprocal crosses between WT and er erl1.2+/– erl2.1 flowers (n=86–143 seeds per cross). Different letters indicate significant differences as determined using one-way ANOVA and Tukey’s HSD pair-wise tests (P<0.001). Crosses in (C, D) were made between flowers at similar positions on the main inflorescence and seeds were harvested at the same time, 3 weeks after crossing.

Fig. 9.

The Arabidopsis ERECTA genes are involved in the control of seed-coat permeability and mucilage composition, and play a salinity-dependent role in the regulation of germination speed. The wild-type (WT) and the mutants er105, erl1.2, erl2.1, er105 erl1.2, er105 erl2.1, and er105 erl1.2/seg erl2.1 were examined (note that for simplicity, er105, erl1-2, and erl2-1 are abbreviated to er, erl1, and erl2, respectively, in the figure). (A) Seed-coat permeability to Tetrazolium Red. Data are means (±SE) of n=4 replicates of 100 seeds each. Significant differences were determined using two-way ANOVA and Scheffe’s post hoc test (*P<0.05; **P<0.01). (B) Seed sodium content of seeds at 24 h post-stratification on 0 mM or 150 mM NaCl media. Data are means (±SE) of n=3 pools of 10 mg mature seeds each. Different letters indicate significant differences as determined using two-way ANOVA and Tukey’s HSD pair-wise tests; P=0.42 for genotype effect under control conditions and P=0.39 under salt treatment. (C) Correlations between mass of water-soluble mucilage per seed and seed size. Data for mucilage are means of n=4 seed samples of 40 mg per genotype and data for weight are means of n=5 pools of a known number of seeds (20–40) corresponding to seeds from five siliques of same age. Regression lines: 0 mM NaCl, y=36.6x–0.20, r² =0.84; 150 mM NaCl, y=36.3x+0.002, r² =0.81. The experiment was repeated three times with similar results. Similar results were also obtained with size expressed as area (data not shown). (D) Relationship between ratios of galacturonic acid/galactose (GalUA/Gal) and rhamnose/xylose (Rhm/Xyl). Different letters next to data points indicate significant differences in GalUA/Gal as determined using one-way ANOVA and Tukey’s post-hoc tests, compared to all unlabelled data points (P<0.05). The difference in Rhm/Xyl between er erl1.2/seg erl2.1 and the WT is significant at P=0.08. (E) T₅₀ values (h post-stratification to rupture in 50% of seeds) for testa rupture (TeR) and endosperm rupture (EnR) for intact seeds and seeds with the outer water-soluble mucilage removed (‘demucilaged’). Data are means of n=3 plates, with 30 seeds per genotype per plate. Labelled points highlight genotypes where removal of the mucilage significantly advanced germination on 150 mM NaCl media. The 1:1 line represents no effect of mucilage removal. (F) Expression of TCH3 in the WT and er erl1.2/seg erl2.1 in dry and imbibed seeds during the three germination phases Data are means (±SE) of n=4 samples of 300 seeds each per genotype and treatment.