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. 2009 Dec;21(12):3803-22.
doi: 10.1105/tpc.109.070201. Epub 2009 Dec 18.

Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana

Ada Linkies  1 , Kerstin Müller, Karl Morris, Veronika Turecková, Meike Wenk, Cassandra S C Cadman, Françoise Corbineau, Miroslav Strnad, James R Lynn, William E Finch-Savage, Gerhard Leubner-Metzger
Affiliations

Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana

Ada Linkies et al. Plant Cell. 2009 Dec.

Abstract

The micropylar endosperm cap covering the radicle in the mature seeds of most angiosperms acts as a constraint that regulates seed germination. Here, we report on a comparative seed biology study with the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana showing that ethylene biosynthesis and signaling regulate seed germination by a mechanism that requires the coordinated action of the radicle and the endosperm cap. The larger seed size of Lepidium allows direct tissue-specific biomechanical, biochemical, and transcriptome analyses. We show that ethylene promotes endosperm cap weakening of Lepidium and endosperm rupture of both species and that it counteracts the inhibitory action of abscisic acid (ABA) on these two processes. Cross-species microarrays of the Lepidium micropylar endosperm cap and the radicle show that the ethylene-ABA antagonism involves both tissues and has the micropylar endosperm cap as a major target. Ethylene counteracts the ABA-induced inhibition without affecting seed ABA levels. The Arabidopsis loss-of-function mutants ACC oxidase2 (aco2; ethylene biosynthesis) and constitutive triple response1 (ethylene signaling) are impaired in the 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination. Ethylene production by the ACC oxidase orthologs Lepidium ACO2 and Arabidopsis ACO2 appears to be a key regulatory step. Endosperm cap weakening and rupture are promoted by ethylene and inhibited by ABA to regulate germination in a process conserved across the Brassicaceae.

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Figures

Figure 1.
Figure 1.
Time Course of Endosperm Cap Weakening and Endosperm Rupture of L. sativum FR1 and the Effect of ABA. (A) Endosperm cap weakening and rupture of seeds incubated without (CON) or with 10 μM ABA added to the medium in continuous light at 18°C. Endosperm cap weakening was determined by measuring tissue resistance by the puncture force method at the times indicated. The time points (after the start of imbibition) at which tissues were dissected for the microarrays are indicated in gray. All seeds selected for the puncture force measurements and for the microarrays had completed testa rupture but intact endosperm caps. ABA did not affect testa rupture. (B) The CON and ABA seed populations showed a very similar relationship between decreasing endosperm cap puncture force and increasing percentage of seeds showing endosperm rupture. Mean values ± se of 3 × 50 seeds for the endosperm rupture [%] and at least 50 endosperm caps for the puncture force (mN) measurements are presented.
Figure 2.
Figure 2.
Endosperm Cap Hole Formation of L. sativum Is a Developmentally and Hormonally Regulated Process. (A) to (D) Endosperm cap hole formation was investigated by dissecting micropylar endosperm caps ([A]; isolated caps) from L. sativum FR1 seeds imbibed for 18 h in CON medium. (B) and (C) Subsequent incubation of isolated caps (radicle removed) for several days on CON medium (B) or on medium with 1 mM ACC resulted in endosperm cap hole formation (arrow), which was inhibited when the isolated caps were incubated in the presence of the ethylene action inhibitor NBD ([C]; 100 μL/L applied via the gas phase). (D) Prolonged incubation of caps with holes on CON medium leads in many cases to abscission of the cap tip (arrow). (E) and (F) Percentage of isolated caps that formed holes by 3 and 12 d, respectively. ACC promoted cap hole formation and completely reverted the inhibiting effects of NBD and 3 μM ABA when added in combination to the incubation medium of the isolated caps. Mean values ± se from two independent experiments with five and 12 isolated caps, respectively, are presented. All incubation conditions were continuous light at 18°C. Similar results were obtained for L. sativum FR14.
Figure 3.
Figure 3.
The Results of PCA Applied to CON and ABA Microarray Data of L. sativum FR1. (A) PCA was applied to the expression of all informative genes on the CON microarrays (22,025 genes; see Supplemental Data Set 1 online) in various tissues and at various times after the start of imbibition, which is before and near the end of endosperm weakening, respectively (see Figure 1A), to look for global patterns of similarities and differences between the samples. PC1 and 2 accounted for 46 and 26% of the variance in gene expression, respectively. (B) The results of PCA for the ABA microarrays (19,794 genes; see Supplemental Data Set 2 online) in various tissues and at various times, as indicated. Samples at 8, 18, and 30 h were taken before the start of endosperm weakening, and the sample at 96 h was taken near the end of endosperm weakening and before radicle emergence (see Figure 1A). PC1 and 2 accounted for 42 and 20% of the variance in gene expression, respectively.
Figure 4.
Figure 4.
Ethylene-Related Regulated Transcripts in L. sativum FR1 Seed Tissues. The regulated transcript data sets of the Lepidium seed arrays (see Supplemental Data Sets 5 [CON, 1350 transcripts] and 6 [ABA, 3530 transcripts] online) were analyzed in two ways. First, TAGGIT analysis (see Supplemental Figure 1 online) was performed, and transcripts of the functional category “ethylene” were indentified in this analysis. Second, comparison to Arabidopsis transcriptome data sets known to be regulated in seedlings by ethylene and/or ABA (Nemhauser et al., 2006) provided transcript subsets that are regulated in seeds as well as by ethylene and/or ABA in seedlings (see Supplemental Data Sets 7 [CON array] and 8 [ABA array] online; SDS7 and SDS8 in Figure 4). These ethylene-related transcripts were considered further in (A) to (C). Examples of these ethylene-related transcripts shown in (A) and (B) are from the CON array and ABA array, as indicated. Those that are not marked are regulated in seedlings by ethylene and/or ABA and therefore appear on the subset lists SDS7 and SDS8, whereas those that are marked with an asterisk are not and therefore do not appear in subset lists (see Venn diagrams in SDS7 and SDS8). (A) Key steps in ethylene biosynthesis include the oxygen-requiring conversion of ACC to ethylene by ACO. (B) Key steps in ethylene signaling include ethylene binding to the receptors, which can be blocked by the ethylene action inhibitor NBD. In the absence of ethylene or with NBD bound to the ethylene receptors activating CTR1, a negative regulator of the downstream signaling pathway and the ethylene responses are blocked. Upon ethylene binding, the receptors and consequently CTR1 are inactive and the downstream signaling pathway factors (EIN2, EIN3, and ERFs) become active and mediate the expression of genes that facilitate the ethylene responses. (C) Normalized expression values for transcripts of selected ethylene responsive genes that are regulated in seeds (CON and ABA arrays; see Supplemental Data Sets 3 and 4 online, respectively). ACS, ACC synthase; ERE, ethylene-responsive element.
Figure 5.
Figure 5.
The Effect of Ethylene, ACC, and ABA on the Time Course of Endosperm Rupture and on the Germination Rates GRX% of L. sativum FR14 Seeds. (A) and (B) The times to reach 15 or 50% endosperm rupture (t15% or t50%) of the seed population were determined from the time courses of endosperm rupture in medium without (CON) and with additions (NBD, ABA, ACC, C2H4, or combinations). Germination rates GR15% or GR50% were then calculated (GRX% = 1/tX%) and used in subsequent analyses (gray arrows). The effect of ACC on time course (A) and GR15% (B) of seeds treated without (CON) or with NBD or ABA added. (C) The effect of C2H4 on GR15% and GR50% of seeds imbibed without (CON) or with NBD or ABA. Numbers above the columns are fold increases in GRX% from the addition of ACC or C2H4 (GRX% ratios ±ACC or ±C2H4). Medium additions, as indicated: 5 μM ABA, 1 mM ACC, 100 μL/L NBD, and 70 μL/L ethylene. Mean values ± se of 3 × 50 seeds are presented.
Figure 6.
Figure 6.
The Effect of Ethylene and ABA on the Germination of Arabidopsis: Wild type (Col) and Ethylene-Related Mutants (aco2 and ctr1). (A) The effect of the ethylene precursor ACC on testa and endosperm rupture of wild-type seeds incubated on medium without (CON) or with NBD or ABA for 38 h. (B) The effect of ACC on the germination rates GR50% (= 1/t50%) of wild-type and ethylene-related mutants incubated on medium with ABA. Numbers above the columns are GR50% ratios (ABA±ACC). Inset: GRX% ratios at other percentages of endosperm rupture. Incubation conditions: continuous light, 24°C, no cold stratification. Medium additions, as indicated: 1 μM ABA, 1 mM ACC, and 100 μL/L NBD. Mean values ± se of 3 × 50 seeds are presented. For detailed time courses, see Supplemental Figure 2 online.
Figure 7.
Figure 7.
Analysis of ACO Enzyme Activity and Transcript Expression in Specific Seed Tissues of L. sativum during Germination. The framed box shows symbols used in (A) and (E) to (G). Mean values ± se ([A] to [C]) or +se (qRT-PCR; [E] to [G]) are presented for three ([A] to [C]) or four ([E] to [G]) biologically independent samples each with 50 seeds (C), 100 to 200 seed parts ([A] and [B]) or 1000 endosperm caps or 100 radicles ([E] to [G]) from seeds with ruptured testa but intact endosperm ([A], [B], and [E] to [G]). L. sativum FR14 ([A] to [D]) or FR1 ([E] to [G]) seeds were incubated at 24°C in continuous light. Note that the qRT-PCR and the microarrays were performed as independent experiments. (A) Time course of in vivo ACO enzyme activities in endosperm caps and radicles of seeds incubated without (CON) and with 10 μM ABA; for comparison, the kinetics of endosperm rupture are presented. In vivo ACO enzyme activities of endosperm caps and radicles dissected from seeds at different time points were measured by subsequent organ incubation in medium with ACC (plus ABA for the ABA series). (B) The effect of ambient and reduced oxygen atmospheres on the in vivo ACO enzyme activities of isolated seed parts compared with intact seeds. Note that only seeds in the testa rupture (TR) state were selected for the measurements (i.e., seeds with intact endosperm). No wound-induced ethylene production by the radicle/embryo was evident. (C) The effect of oxygen on the promotion of endosperm rupture by ACC and the reversion of the ABA inhibition of endosperm rupture by ACC. (D) Molecular phylogenetic analysis of Lepidium ACO and Arabidopsis ACO cDNA sequences. The bar (0.07) defines the number of substitutions per 100 amino acids. Lepidium ACO cDNA sequences and comparisons to the Arabidopsis orthologs are presented in Supplemental Figure 3 online. (E) to (G) LesaACO transcript expression pattern determined by qRT-PCR in endosperm cap and radicle during incubation on medium without (CON) or with 10 μM ABA added. Relative ΔΔCt expression values based on the comparison with validated constitutive transcripts are presented.
Figure 8.
Figure 8.
The Effect of ACC, NBD, or NBD+ACC on Endogenous ABA Contents and ACO2 Transcript Expression during Germination of L. sativum FR14. (A) Endogenous ABA concentrations of whole seeds incubated without (CON) or with 1 mM ACC or 100 μL/L NBD or ACC+NBD added to the medium in continuous light at 18°C. FW, fresh weight. (B) Lepidium ACO2 transcript expression pattern determined by qRT-PCR in whole seeds. Relative ΔΔCt expression values based on the comparison with validated constitutive transcripts are presented. Mean values ± se (A) or +se (B) are presented for three biologically independent samples each with 50 seeds.
Figure 9.
Figure 9.
The Effect of ACC, NBD, ABA, or Their Combinations on Endosperm Cap Weakening and Endosperm Rupture of L. sativum FR14. Endosperm cap weakening (columns) and rupture (percentages above columns) of seeds incubated without (CON) or with 1 mM ACC or 10 μM ABA added to the medium in continuous light at 24°C ([A] and [C]) or 14°C (B). Endosperm cap weakening was determined by measuring tissue resistance by the puncture force method at the times indicated. All the seeds selected for the puncture force measurements had completed testa rupture but still had intact endosperm caps; ACC, NBD, and ABA did not appreciably affect testa rupture. Mean values ± se of 3 × 50 seeds for the endosperm rupture and at least 50 endosperm caps for the puncture force measurements are presented. Note that the puncture force values measured for the endosperm cap weakening of L. sativum FR14 were approximately twofold compared with L. sativum FR1 (Figure 1). These higher values are accession-specific absolute differences, but the relative decreases of the two accessions are similar.
Figure 10.
Figure 10.
Proposed Model for the Hormonal Regulation of Endosperm Cap Weakening and Rupture. According to our working model, endosperm cap weakening is required during the transition from testa rupture (TR) to endosperm rupture (ER), and endosperm cap weakening in turn requires ethylene biosynthesis and signaling. Endosperm cap weakening is a developmental process that is regulated by interactions between the cap and the radicle. GA, as an embryo signal, releases coat dormancy (if present) and induces the cap weakening process. Thereafter, weakening is a cap-autonomous process, and the rate of this process is regulated by the GA-ABA and ethylene-ABA antagonisms. The involvement of GA as antagonist of ABA in seed germination is well established in different Brassicaceae (e.g., Yamaguchi et al., 2001; Ogawa et al., 2003; Chiwocha et al., 2005; Müller et al., 2006; Iglesias-Fernandez and Matilla, 2009). GA biosynthesis may be more important in the early phase of germination, and ethylene biosynthesis may be more important for the late phase of the process. Ethylene biosynthesis in germinating seeds is regulated differently in the radicle and the endosperm cap, and this effect is mediated by ACO2 gene expression in the following way: although both ACO2 and ACO1 are active in the radicle and endosperm cap, the total activity is greater in the former. The radicle produces ethylene in excess, and this is targeted at the endosperm cap. ABA delays the ACO activity in the radicle and inhibits ACO1 transcript accumulation, but ABA does not inhibit ACO2 transcript accumulation. The later increase in ACO activity in the radicle of ABA-treated seeds is therefore due to ACO2, and the ethylene produced promotes endosperm cap weakening by antagonizing the ABA inhibition. In the endosperm cap, ABA inhibits ACO2 and ACO1 transcript accumulation. A basal level of ethylene signaling is required to maintain ACO2 transcript levels in seeds. Ethylene does not affect the seed ABA levels and therefore must counteract the ABA-induced inhibition of endosperm rupture by interfering with ABA signaling. High degrees of GA and ethylene sensitivity of the endosperm cap are associated with the after-ripened (post coat dormancy) seed state and are prerequisites for ethylene-enhanced expression of ABA-inhibitable downstream weakening genes in the cap. The products of the weakening genes cause endosperm cap weakening by cell wall loosening and cell separation that finally leads to endosperm rupture and radicle emergence.
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