Screening for candidate ABA transporters in seeds

To select candidate ABA transporters that regulate seed germination, we first performed an in silico analysis (Arabidopsis eFP browser; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) of all 43 members of the ABCG subfamily of ABC transporters, and identified 10 genes that are highly expressed in mature seeds. To determine whether any of these candidates may function as ABA transporters within seeds, we screened the mutants for altered patterns of seed germination (Fig. 1). Six of these ten lines did not have an altered germination phenotype. In contrast, the mutant lines of two candidate genes, AtABCG30 and AtABCG31, exhibited clear differences in germination when compared with wild-type seeds. Seeds from freshly collected atabcg31 and atabcg30 mutants germinated earlier than the corresponding wild-type seeds on imbibition without stratification (Fig. 1a,c,e), whereas seed stratification abolished the difference between the mutants and wild type (Fig. 1f). To further investigate whether atabcg31 and atabcg30 mutant seeds exhibit deficiencies in ABA transport, we compared their responses with exogenously added ABA and found that germination of stratified atabcg31 and atabcg30 seeds was less inhibited by 0.1–1.0 μM ABA than was that of the corresponding wild-type seeds (Supplementary Fig. 1a–e). Similarly, we examined whether AtABCG25 and AtABCG40 function as ABA transporters in seeds. Indeed, atabcg40 seeds exhibited a higher germination rate than the wild type on medium containing 0.1 μM ABA (Supplementary Fig. 1b), suggesting that ABA uptake was reduced in these seeds, consistent with a previous report40. Furthermore, seeds from freshly collected, non-stratified atabcg40 mutants also germinated earlier than those from the corresponding wild type (Fig. 1d,e). In contrast, the germination of non-stratified, freshly collected atabcg25 seeds was similar to that of wild-type seeds (Fig. 1b,e), even when grown on medium containing exogenous ABA (Supplementary Fig. 1f). This finding contradicts a recent report that showed that the germination of atabcg25 seeds is delayed on medium containing ABA and that AtABCG25 acts as an ABA exporter39. This discrepancy may be explained by the ecotype difference and mild nature of the mutant’s phenotype, which may depend on plant growth conditions. Given the convincing data presented previously39, we continued to include atabcg25 in our studies. Taken together, these results strongly suggest that at least four AtABCG transporters are involved in controlling seed germination.

Figure 1: Seeds of atabcg31, atabcg30 and atabcg40 germinate faster than those of the corresponding wild type (WT). (a–d) Seed germination on ½ MS medium without stratification. atabcg31 (a), atabcg30 (c) and atabcg40 (d) mutant seeds germinated faster than the WT, whereas atabcg25 mutant seeds (b) did not. WT seeds collected at the same time were used as the control. Photographs were taken at the start of incubation (0 h) and 48 h later. A representative result out of three independent experiments giving rise to similar results is shown. Each experiment consisted of two or three replicates. Scale bar, 1.25 mm (a), 1 mm (b–d). (e,f) Germination rate of WT seeds compared with that of atabcg mutant seeds on ½ MS medium. Non-stratified (e) or stratified (f) seeds were sowed on ½ MS medium, and germinated seeds were determined based on radicle extrusion. Stratification consisted of pretreatment of seeds for 2 days at 4 °C in the dark. In all experiments, fresh seeds were used, and the sowed seeds were incubated under 16-h light and 8-h dark conditions at 22 °C. Data are mean±s.e.m. of three independent experiments with four technical replicates (N=3, n=4 × 100 seeds). HAS, hours after sowing. Full size image

AtABCGs are expressed either in the embryo or endosperm

To further explore whether these AtABCG proteins indeed influence germination by regulating ABA transport within the seed on imbibition, we analysed the transcript levels of the corresponding genes in the endosperms and embryos of mature wild-type seeds. Tissue-specific quantitative reverse transcription–PCR (qRT–PCR) revealed that AtABCG31 was not expressed in dissected embryos, but was expressed at high levels in the endosperm (Fig. 2a). AtABCG25 was mainly expressed in the endosperm and, to a lesser extent, in the embryo (Fig. 2b). This result is consistent with previous in silico data31. In contrast, AtABCG30 and AtABCG40 transcripts were strongly expressed in dissected embryos, but only weakly in the endosperm (Fig. 2d,e). To validate our approach, we performed qRT–PCR with two genes known to be expressed either in the endosperm (AtEPR1, an extensin-like gene involved in seed germination)46 or the dissected embryo (AtABI4, encoding an ABA-response transcription factor)31. Consistent with previous reports, AtEPR1 transcripts were detected solely in the endosperm, whereas AtABI4 transcripts were detected nearly exclusively in the embryo (Fig. 2c,f). Our analysis of multiple lines of promoter–β-glucuronidase-expressing plants (Fig. 2g and Supplementary Fig. 2a) confirmed the qRT–PCR results; the AtABCG31 promoter was active only in the endosperm layer, and β-glucuronidase activity driven by the AtABCG30 and AtABCG40 promoters was observed in dissected embryos, but not in the endosperm. Together, these results show that AtABCG31 and AtABCG25 expression is specific to the endosperm, where their protein products may act as ABA efflux transporters. In contrast, AtABCG30 and AtABCG40 expression is specific to the embryo, where their protein products may function as ABA influx transporters. To function as transporters that facilitate ABA flux from the endosperm to the embryo, these four transporters are expected to be localized to the plasma membrane. AtABCG25 and AtABCG40 were previously shown to localize to the plasma membrane39,40,47. Here we show that AtABCG31 and AtABCG30 are also localized to the plasma membrane of Arabidopsis mesophyll protoplasts and tobacco epidermis cells when transiently expressed under the control of the 35S promoter (Fig. 2h and Supplementary Fig. 2b). Co-expression of green fluorescent protein (GFP)-fused AtABCG31 or AtABCG30 with PM-rK (ref. 48), a plasma membrane marker protein (an mCherry fusion of the plasma membrane aquaporin, AtPIP2A), showed complete co-localization of the two ABCG proteins with PM-rK. Plasma membrane localization of AtABCG31 was recently also shown by Choi et al.49.

Figure 2: AtABCG31 and AtABCG25 are expressed mainly in the endosperm, whereas AtABCG30 and AtABCG40 are expressed specifically in the embryo. (a–f) q-PCR analysis of AtABCG31 (a); AtABCG25 (b); AtEPR1, a gene specifically expressed in the endosperm (c); AtABCG40 (d); AtABCG30 (e); and AtABI4, a gene specifically expressed in the embryo (f). q-PCR was performed using total RNA extracted from dissected embryos or endosperms, or whole seeds of the Col-0 wild type. Data were normalized using AtUbiquitin11. E, dissected embryos; En, dissected endosperms; WS, whole seeds. Data are mean±s.e.m. of three independent experiments (N=3, n=2). (**P<0.01, ***P<0.005; (a,b,c) compared with the embryo (E) and (d,e,f) compared with the endosperm (En) by Student’s t-test). (g) AtABCG31 promoter–GUS (pAtABCG31::uidA) reporter gene activity was detected only in the endosperm, and not in the embryo (left). AtABCG30 promoter–GUS (pAtABCG30::uidA) and AtABCG40 promoter–GUS (pAtABCG40::uidA) activities were detectable only in the embryo and not in the endosperm (middle and right). Embryos (upper panel) and endosperms (lower panel) were dissected from mature seeds and incubated in GUS solution for 30 min. Results representative of 8 (AtABCG30), 12 (AtABCG31) and 9 (AtABCG40) different T3 promoter–GUS lines. Scale bar, 0.8 mm. (h) Plasma membrane localization of AtABCG31 and AtABCG30 in Arabidopsis mesophyll protoplasts. Protoplasts were isolated from Arabidopsis leaves transformed with p35S::sGFP::AtABCG31 (upper panels) or p35S::AtABCG30::GFP (lower panels) and pd35S::AtPIP2A::mCherry (PM-rK) using polyethylene glycol transformation. The left panel shows the GFP fluorescence of ABCGs, the middle panel the red mCherry fluorescence of the plasma membrane marker PM-rK protein and the right panel shows the merged images of GFP, mCherry and bright-field. Scale bar, 10 μm. GUS, β-glucuronidase. Full size image

ABCG31 and ABCG25 repress embryonic growth

We next sought to determine whether the embryo or endosperm is responsible for the altered germination phenotype of mutant seeds by performing SCBAs as described by Lee et al.32,33. SALK insertion Arabidopsis lines are generated in the Col-0 ecotype, which displays relatively weak and short-term dormancy compared with ecotypes such as Ler or Cvi, for which the SCBA was initially used. As a result, a SCBA using freshly collected Col-0 material may result in less robust embryonic growth arrest results. To better test the role of AtABCG31 and AtABCG25 in controlling embryonic growth, we performed SCBAs in presence of paclobutrazol (PAC), a GA synthesis inhibitor. Lack of GA synthesis promotes ABA accumulation and efficiently blocks embryonic growth in SCBAs13,34,50. Indeed, in presence of PAC, seed coats of Col-0 prevented embryonic growth (Supplementary Fig. 3a), and expressed the germination repressor AtABI5 (Supplementary Fig. 3b)15, as effectively as those of Cvi. However, consistent with previous results, PAC did not affect the embryonic growth of dissected embryos or their ability to establish seedlings9 (Supplementary Fig. 3c). In this assay, we also used dissected embryos of the aba2-1 mutant, which is defective in ABA biosynthesis and consequently germinates precociously51. For quantitative analysis, we measured the angle between cotyledons and radicles as well as radicle length (Fig. 3a,b), as indicators of early embryonic growth. As shown in Fig. 3, seed coats (testa and endosperm) of the atabcg31 or atabcg25 single knockouts did not inhibit the growth or early seedling establishment of the aba2-1 embryo as effectively as did those of the wild type in the SCBA. Our findings for the atabcg25 seed coat are inconsistent with our observation that the germination rate of atabcg25 whole seeds is similar to that of the wild type (Fig. 1b,e and Supplementary Fig. 4a). This inconsistency may be due to a defect in atabcg25 embryos, which exhibit retarded embryonic growth even when dissected out from the surrounding tissues (Supplementary Fig. 4b). The retarded growth of embryos seems to cancel out the weak inhibitory effect of the endosperm on germination; thus, no aberrations in whole-seed germination are apparent. Reciprocal crossing of atabcg25 with atabcg31 yielded g31/g25-1 (atabcg31 pistil; atabcg25 pollen) and g31/g25-2 (atabcg31 pollen; atabcg25 pistil) double mutants, which lack both ABC transporters. The seed coats and endosperms of the double knockouts (g31/g25-1 or g31/g25-2) inhibited the growth and seedling establishment of aba2-1 embryos much less effectively than did those of the single knockouts or the wild type (Fig. 3c–e and Supplementary Fig. 7). These results suggest that atabcg31 and atabcg25 endosperms release reduced amounts of germination inhibitor. In contrast, the seed coat bedding of the other candidate ABC transporter mutants, atabcg30 and atabcg40, did not differ from those of the wild type in terms of their effect on the development of aba2-1 mutant embryos (Supplementary Fig. 5a, and Fig. 3c,d), indicating that the amount of inhibitor molecules released from the seed coat beddings of wild-type, atabcg30 and atabcg40 plants was similar.

Figure 3: Seed coats of atabcg31 and atabcg25 repressed the embryonic growth of aba2-1 less effectively than did those of the wild type (WT). (a,b) Diagram showing the quantification of the angle between the radicle and the cotyledon and the radicle length, respectively. (a) The angle between the radicle and cotyledon was measured using Axio. (b) Radicle length was measured using Axio. (c–e) Embryos dissected from aba2-1 mutant seeds were placed on a layer of seed coat beddings dissected from the WT (Col-0), atabcg31, atabcg25, atabcg31 atabcg25 (g31/g25-1, g31/g25-2), and atabcg40. The legend in the graph indicates the genotypes of the seeds from which the seed coat beddings were derived. The angle between the cotyledon and radicle (c) and the radicle length (d) of the aba2-1 embryos were measured as indicators of embryonic growth. (c,d) Data are means±s.e.m. of n=108–112 obtained from three independent experiments. Each experiment was performed in duplicate. Each replicate (one SCB) consisted of 20 embryos (N=3, n=2 × 20 embryos). (e) Photographs taken 4 h (top) and 120 h (bottom) after imbibition. Scale bar, 1.25 mm. HAT, hours after transfer. Results shown are representative of three independent experiments (see Supplementary Fig. 7). Each experiment was performed in duplicate. Similar results were obtained in all experiments. Full size image

atabcg30 and atabcg40 embryos are impaired in ABA uptake

We next sought to determine whether AtABCG30 and AtABCG40 are necessary for embryonic uptake of endospermic ABA. For this purpose, we tested whether atabcg30 and atabcg40 mutants are defective in the embryonic uptake of ABA. We evaluated the growth of atabcg30 (g30), atabcg40 (g40) and g30/g40 embryos in SCBAs using seed coats isolated from the highly dormant Cvi ecotype, which were previously shown to efficiently block wild-type embryonic growth in SCBAs32. While most embryos of Col-0 wild-type seeds did not grow until 85 h after imbibition (Supplementary Fig. 5a), the embryos dissected from the seeds of atabcg30 and atabcg40 and the corresponding double-knockout plants (g30/g40) grew and developed to plantlets (Supplementary Fig. 5a and Fig. 4a–c). Thus, these observations indicate that the mutations in AtABCG30 and AtABCG40 genes could not efficiently uptake endospermic ABA released by Cvi seed coats relative to wild-type embryos. We further investigated this possibility by monitoring the development of dissected atabcg30 and atabcg40 embryos in the absence or presence of exogenous ABA. Under control conditions, no difference in the development of embryos was observed between the wild-type and mutant embryos (Supplementary Fig. 5b); however, in the presence of ABA, the development of embryos dissected from atabcg30, atabcg40 and g30/g40 double-mutant seeds was inhibited to a lesser extent than those dissected from the wild type (Fig. 4d–f). These observations further indicate that atabcg30 and atabcg40 embryos do not take up the ABA released from the seed coat bedding of the Cvi ecotype as effectively as does the wild type.

Figure 4: Dissected embryos of atabcg30, atabcg40 and atabcg30 atabcg40 are less sensitive than the wild type (WT) to endosperm-dependent or exogenous ABA-dependent germination repression. (a–c) Seed coat bedding assay, using embryos dissected from WT (Col-0) or atabcg mutant seeds placed on a layer of seed coat beddings (endosperm and testa) dissected from dormant Cvi seeds (Cvi (D)). (b,c) Data are means±s.e.m. of n=105–109 obtained from three independent experiments. Each experiment was performed in duplicate. Each replicate (one SCB) consisted of 20 embryos (N=3, n=2 × 20 embryos). (d–f) Embryos dissected from WT (Col-0) and atabcg mutant seeds placed on ½ MS medium supplemented with 0.1 μM ABA. The angle between the cotyledon and radicle (b,e) and the radicle length (c,f) were measured as indicators of embryonic growth for each genotype of embryo. (e,f) Data are means±s.e.m. of n=108 obtained from three independent experiments. Each experiment was performed in triplicate. Each replicate consisted of 12 embryos (N=3, n=3 × 12 embryos). In a, photographs were taken at 4 h (top) and 96 h (bottom) after imbibition. In d, photographs were taken 4 h (top) and 166 h (bottom) after imbibition. Scale bar, 1 mm. (a,d) A representative result out of three independent experiments is shown (six (a) or nine (d) different samples). In all experiments, similar results were obtained. Full size image

Four AtABCGs mediate ABA transport in seeds

To directly test whether AtABCG31 and AtABCG25 release ABA from the tissues surrounding the embryo, we removed embryos from the seeds and compared the remaining tissues for secreted and retained endogenous ABA levels. As shown in Fig. 5a,b (with PAC) and Supplementary Fig. 6c,d (without PAC), the tissues surrounding the embryos of atabcg31 and atabcg25 single and double mutants secreted less and retained more ABA. On the basis of these results and previous studies that found that endospermic ABA inhibits germination25,29,30,31,32,33, we conclude that AtABCG31 and AtABCG25 contribute to the secretion of ABA from the endosperm. It is interesting that the total ABA content did not differ between the wild type and atabcg31 or atabcg25 (Supplementary Fig. 6e), further indicating the importance of endospermic ABA release to control embryonic growth.

Figure 5: Four AtABCGs mediate ABA transport in Arabidopsis seeds. (a,b) The endosperms and testa of atabcg mutants release less ABA than do those of the wild type (WT). Endosperms attached to testa were dissected from 500 WT, atabcg31, atabcg25 and atabcg31 atabcg25 (g31/g25-1, g31/g25-2) seeds 24 h after imbibition on medium containing 10 μM PAC. The tissues were then incubated on ½ MS liquid medium for 24 h before ABA content was assayed using an ELISA. The aqueous medium was collected for assay of secreted ABA (a), and the remaining tissues were washed three times and extracted to assay the retained ABA (b). (c) atabcg30, atabcg40 and two different lines of double mutant embryos (g30/g40-1 and g30/g40-2) accumulated less ABA than the corresponding WT. Accumulation of 3H-ABA by embryos dissected from WT and mutant seeds during a 30-min period. Embryos were incubated in a solution of 12.5 nM 3H-ABA (1.63 Tbq mmol−1) and 17.5 pM 14C-glycerol (5.40 GBq mmol−1) at pH 6.5. The radioactivity uptake was normalized to the 14C-glycerol d.p.m. (disintegrations per min) value. (d) Time-dependent loading assay of 3H-ABA to yeast cells expressing AtABCG31 or AtABCG30, or transformed with the empty vector (EV). Yeast cells were incubated in SG-URA medium containing 50 nM 3H-ABA (7.4 kBq, 1.63 Tba mmol−1) at pH 6.5. Data are means±s.e.m. of n=12 from three independent experiments (N=3, n=4). (e) Four AtABCGs participate in the delivery of ABA from the endosperm to the embryo in Arabidopsis seeds. AtABCG31 and AtABCG25 are expressed in the endosperm and secrete ABA from the endosperm to the embryo. AtABCG30 and AtABCG40 are ABA uptake transporters located in the embryo. To inhibit embryo germination, de novo biosynthesized ABA is transported from the endosperm to the embryo by the four transporters. Solid arrows indicate the direction of AtABCG-mediated ABA transportation. Data are means±s.e.m. of n=6 (a,b) or n=4 (c) from three or four independent experiments. (*P<0.05; **P<0.01 compared with the WT by Student’s t-test and ##P<0.01 compared with the single mutants by Student’s t-test). Full size image

Next, we incubated embryos dissected from the wild type, the atabcg30 and atabcg40 single mutants, and the corresponding double mutants with radiolabelled ABA and determined the amount of radioactivity taken up after 30 min at room temperature. Both mutants took up significantly less ABA than the wild type, and the double-knockout mutants took up less than the single knockouts (Fig. 5c). In contrast, embryos incubated in the presence of radiolabelled ABA on ice did not differ in their ABA contents (Supplementary Fig. 6a). Therefore, the difference in ABA uptake shown in Fig. 5c was mediated by an active transport mechanism and not by simple diffusion. Taken together, these observations further strengthen and lend direct support to the notion that AtABCG30 and AtABCG40 function as endospermic ABA importers. While ABA transport activity has been reported for AtABCG25 and AtABCG40 in heterologous systems39,40, it has not been reported for AtABCG30 and AtABCG31. Therefore, to obtain additional evidence that these transporters also catalyse ABA fluxes, we expressed these AtABCGs in yeast and performed ABA transport experiments using the yeast system. Yeast expressing AtABCG31 took up lower amounts of ABA than yeast transformed with an empty vector, while yeast expressing AtABCG30, which was localized to the plasma membrane (Supplementary Fig. 6f), accumulated far more radiolabelled ABA (Fig. 5d). These results are in line with those obtained with embryos and seed coats from the corresponding mutants, indicating that AtABCG31 acts as an exporter, while AtABCG30 catalyses the import of ABA.