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. Author manuscript; available in PMC: 2025 Apr 4.
Published in final edited form as: Plant J. 2025 Mar 1;121(6):e70096. doi: 10.1111/tpj.70096

ABA importers ABCG17 and ABCG18 redundantly regulate seed size in Arabidopsis

Yuqin Zhang 1,2,✉, Moran Anfang 1, James H Rowe 3, Annalisa Rizza 3, Zhuorong Li 2, Ning Su 2,4, Hamutal Bar 1, Laurence Charrier 5, Markus Geisler 5, Alexander M Jones 3, Eilon Shani 1,✉
PMCID: PMC7617562  EMSID: EMS203671  PMID: 40106270

Abstract

The stress hormone abscisic acid (ABA) plays a crucial role in mediating plant responses to the environment and regulating plant development. In this study, we demonstrate that two ABA importers, ABCG17 and ABCG18, control seed size by regulating the ABA levels transported into the embryo. Double knockdown of ABCG17 and ABCG18 resulted in lower ABA accumulation in the embryo, wider siliques, and increased overall seed size. Leaf phloem-specific ABA induction in the aba2-1 background showed that ABA could move from the vasculature to control seed size. ABCG17 and ABCG18 are expressed in leaves, and the reproductive organs septum, and valves but not in the developing seeds, suggesting that ABCG17 and ABCG18 affect seed size maternally. Together, the results shed light on the molecular mechanisms by which ABA is transported to the embryo to determine seed size.

Introduction

The plant hormone abscisic acid (ABA) is a stress-related signaling molecule1,2,3,4. In addition, ABA participates in multiple stages of plant growth and development, including seed development5,6,7, dormancy8, maturation, and germination9,10,11. Current models suggest that there are two ABA accumulation peaks during Arabidopsis seed development8,11,12. The first ABA maximum occurs during early seed maturation at around 9 days after pollination (DAP).

It is thought that this ABA accumulation is mainly driven by maternal tissues8,10. The second ABA accumulation peak occurs at 12 DAP, and high levels of ABA are maintained until the late stages of seed development (21 DAP)9,13,14. This maxima affects seed dormancy during the desiccation phase of seed development, and ABA is likely produced by the embryo during this phase9. There are two considerable drops in ABA levels during seed maturation. The first takes place around 4 DAP, at the initiation of endosperm cellularization, and is thought to result in larger Arabidopsis seeds11. The second ABA drop takes place between 9 and 12 DAP, between the end of the embryo growth stage and the beginning of the seed desiccation stage11.

Seed size is an essential trait that influences plant growth and development, as well as the yield of crops15,16. Several genes and pathways have been identified as regulators of seed size in Arabidopsis including the ubiquitin-proteasome system, hormones, and several transcription factors15,17,18,19. The ubiquitin-proteasome system is a major regulator of protein degradation in eukaryotic cells. In Arabidopsis, mutations in genes encoding components of the ubiquitin-proteasome pathway have been shown to affect seed size. For example, mutations in the RING-type E3 ligase gene, BIG BROTHER, result in abnormally large seeds due to the accumulation of cell wall proteins in the endosperm20. G-protein-mediated and mitogen-activated protein kinase-mediated signaling also regulate grain size21,22,23.

Like ABA, the plant hormones gibberellins (GAs) play important roles in seed size determination. GAs promote seed growth, whereas ABA can promote or inhibit growth, depending on its concertation24,25. The balance between these two hormones is, therefore, critical for seed size regulation26,27. While the direct effect of GA on seed size is not entirely clear, mutations in genes involved in ABA synthesis, such as ABA DEFICIENT 2 (ABA2), lead to larger seeds11,27,28,29. In addition, the expression of ABI5 (ABA-INSENSITIVE5) is reduced in aba2-1 plants, and the abi5 mutant exhibits larger seeds11.

ABA function is regulated at multiple levels including biosynthesis, metabolism, perception and signal transduction, and transport. Since 2010, several ABA transporters from different families have been characterized, shedding light on mechanisms of ABA homeostasis, transport, and redistribution. The first two identified ABA transporters were Arabidopsis ATP-binding cassette (ABC) family proteins ABCG25 and ABCG4030,31. ABCG25 is a plasma membrane-localized ABA transporter involved in intercellular ABA signaling. It is mainly expressed in the vasculature and controls stomatal movement. Overexpression of ABCG25 leads to higher leaf temperature, suggesting that ABA accumulation in the guard cells causes stomatal closure and reduces transpiration. ABCG40 is also a plasma membrane-localized ABA transporter. It is broadly expressed in roots and guard cells of leaves. ABCG40 functions as an ABA importer and regulates stomatal closure31. Kang et al. showed that in Arabidopsis ABCG25 and ABCG31 function as ABA exporters that move ABA out of the endosperm and that ABCG30 and ABCG40 cooperate to import ABA into the embryo, promoting seed dormancy32.

Additional ABA transporter genes have been identified in Medicago truncatula (MtABCG20)33, in wheat (LR34res)34, in rice (OsPM1)35, in Arabidopsis (genes encoding the nitrate transporter/peptide transporter (NPF) proteins)36,37,38, and in tomato (the NPF-encoding SlAIT1.1)39. However, none of these transporters have been shown to modulate seed size or long-distance ABA transport. ABCG17 and ABCG18 were recently characterized as ABA importers that regulate ABA long-distance transport and ABA homeostasis in guard cells and roots40. ABCG17 and ABCG18, localized on the plasma membrane, are mainly expressed in the leaf mesophyll cells, where they promote ABA uptake to tune stomatal closure. Upon abiotic stresses, including high salinity, drought stress, and ABA treatment, both ABCG17 and ABCG18 are transcriptionally repressed, which results in more free apoplastic ABA, leading to increased ABA movement to guard cells to balance stomatal closure and to the root to modulate lateral root emergence40.

The first evidence that long-distance ABA transport is required for the regulation of seed size in plants came from the recent identification and characterization of OsDG1 in rice41. OsDG1 is a multidrug and toxic compound extrusion (MATE) type transporter, which has been shown to promote ABA efflux41. The osdg1 mutant has grain-filling defects caused by noticeably reduced starch content in caryopses. Although ABA is synthesized in leaves of both wild-type and osdg1 mutant seedlings, only wild-type caryopses accumulate leaf-derived ABA, which activates starch synthesis genes41. This process is enhanced at above-normal temperatures to impose an impact on seed development.

In Arabidopsis, ABA is required to set seed size in plants5,8,11,15, but the complete map of ABA synthesis sites, and the biological importance of ABA movement during seed development has not been established. Therefore, it remains unclear if the entire ABA pool is synthesized directly in the embryo, if ABA moves only a few cells from the seed coat, or if ABA is translocated in a long-distance manner to the seeds, or a combination of all three above.

We previously provided evidence that ABCG17 and ABCG18 are ABA importers that redundantly regulate ABA long-distance transport necessary for control of stomatal closure and lateral root emergence40. Here, we show that ABCG17 and ABCG18 also regulate seed size. The double abcg17/18 knockdown (artificial microRNA targeting ABCG17 and ABCG18 transcript at the same time, mir17,18) leads to reduced ABA accumulation in the embryo and increased seed size. At the reproductive tissues, ABCG17 and ABCG18 are mainly expressed in the valves of the embryo. In addition, we showed that phloem-specific ABA induction influences seed size and that cell type-specific phloem activation of ABCG17 and ABCG18 results in larger seeds. These results support the hypothesis that ABCG17 and ABCG18 redundantly govern ABA transport to control seed size.

Results

ABCG17 and ABCG18 double knockdown leads to larger seeds

In a previous study, we demonstrated that ABCG17 and ABCG18 are two PM-localized ABA importers40. Since ABCG17 and ABCG18 are genetically linked, and obtaining a double mutant through T-DNA crossings is not feasible, the ABCG17 and ABCG18 double mutant was generated using an amiRNA-ABCG17/ABCG18 driven by 35S promoter (mir17,18). In addition to a physiologically related guard cell closure phenotype, the double-knockdown mir17,18 had significantly larger seeds than wild-type (WT) plants. Seeds of mir17,18 were wider and longer compared to WT (Fig. 1A-C). To test if the larger seed phenotype is caused by the double knockdown of ABCG17 and ABCG18, or by the loss of one of the proteins, we compared the single mutants (abcg17-1, abcg18-1, abcg18-2) to mir17,18 and WT. Although mir17,18 seeds were significantly larger, the seed size of all single mutants was similar to WT (Fig. 1D-E). mir17 showed a slight difference in seed width, with no significant difference in seed length (Sup. Fig. 1A). Previous studies showed that ABA is involved in seed size determination11,15,42. We, therefore, hypothesized that ABCG17 and ABCG18 control seed size by mediating ABA translocation and accumulation levels during seed development.

Fig. 1. ABCG17 and ABCG18 redundantly regulate seed size.

Fig. 1

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A, Light microscopy image of seeds from WT and mir17,18 plants. Scale bar = 200 μm. B-C, Average (±SD) seed B) widths and C) lengths of the indicated genotypes. n ≥ 102; P value determined by Student’s t test. D-E, Average (±SD) seed D) lengths and E) widths of the indicated genotypes. n ≥ 80; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test. F, Images of seeds from the indicated lines. Com is used to indicate the complementation lines pABCG17:ABCG17 or pABCG18:ABCG18. Scale bar = 200 μm. G, Average (±SD) seed widths for WT and indicated complementation lines. n ≥ 14, P < 0.05, one-way ANOVA with Student’s t test. Com stands for two independent complementation lines in the indicated background. H, Average (±SD) seed yield per plant for WT and indicated lines. n ≥ 6; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test. I, Images of carpels of 35-day-old WT and mir17,18 plants grown in soil under normal conditions. Scale bar = 0.5 mm. J-K, Average (±SD) carpel J) widths and K) lengths of the indicated genotypes. n = 12 plants; P values determined by Student’s t test. L, Images of siliques of the indicated genotypes. Scale bar = 1 cm. M-N, Average (±SD) silique M) widths and N) lengths of the indicated genotypes. mir17,18 is abcg17 and abcg18 double-knockdown amiRNA line; mir17,g18 is mir17 (amiRNA-ABCG17) transformed into the background of abcg18-1 T-DNA insertion line. n ≥ 13; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test.

To validate that seed-size phenotypes result from the double knockdown of ABCG17 and ABCG18, we tested the seed-size phenotype in two independent knockdown lines. mir17,g18 is the mir17 (amiRNA-ABCG17) transformed into the background of the abcg18-1 T-DNA insertion line. mir18,g17 is the mir18 (amiRNA-ABCG18) transformed into the background of the abcg17-1 T-DNA insertion line. Both lines were previously described40. amiRNA transformation into T-DNA insertion line is needed since ABCG17 and ABCG18 are genetically linked. Seeds of both mutant lines were significantly wider than seeds from WT plants (Fig. 1F-G). In addition, we used native promoters that drive ABCG17 or ABCG18 to test for rescue of a T-DNA insertion mutant40. Except for one ABCG18 line (18Com-2) that showed intermediate rescue, all other lines showed significant rescue of seed-size phenotypes (Fig. 1F-G), confirming that ABCG17 and ABCG18 are both necessary for the regulation of seed size. Furthermore, we found that while the seed number per silique was reduced in the mir17,18 double mutant (Sup. Fig. 1B-C), the seed mass per plant was significantly higher in the double-knockdown combinations compared to the WT (Fig. 1H).

We also evaluated carpel widths in the double-knockdown lines. Interestingly, mir17,18 lines showed thicker carpel widths but no significant difference in carpel lengths compared to WT carpels (Fig. 1I-K). Since the Arabidopsis silique is formed by the fusion of two carpels2, we evaluated silique widths and lengths and found that mir17,18 and mir17,g18 double-mutant plants had wider siliques but no significant difference in silique length compared to WT (Fig. 1L-N). These data indicate that ABCG17 and ABCG18 redundantly balance seed and carpel development.

ABCG17 and ABCG18 are expressed in valves and funiculus

We next characterized the expression patterns of the two ABCGs in siliques at 4 DAP using β-glucuronidase (GUS) reporter lines. In pABCG17:GUS and pABCG18:GUS lines, imaging of GUS indicated that ABCG17 and ABCG18 were primarily expressed in the valves, but not in the ovules (Fig. 2A). Analyses of YFP signal in cross-sections of siliques of pABCG17:NLS-YFP and pABCG18:NLS-YFP plants at 4 DAP were consistent with results obtained from the GUS lines with valves (Fig. 2B, Sup. Fig. 2A). No expression was detected in the developing seeds (i.e., embryo or endosperm). Notably, it is possible that expression in different tissues is below our detection level. Data from the eFP browser indicate that ABCG18 is expressed in testa during early seed developmental stages (no expression indicated for ABCG17). The Arabidopsis RNA-seq database (ARS) that integrates publicly available Arabidopsis RNA-seq library data43 showed a high similarity to our GUS and YFP reporters, with undetectable expression in the developing seeds (Sup. Fig. 2B).

Fig. 2. ABCG17 and ABCG18 are primarily expressed in valves during the early stage of seed development but not in the embryo or endosperm.

Fig. 2

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A, Images of pABCG17:GUS (pG17:GUS) and pABCG18:GUS (pG18:GUS) reporters in siliques stained for GUS activity (blue) at 4 DAP. Scale bars = 50 μm. B, Images of pABCG17:NLS-YFP (pG17:NLS-YFP) and pABCG18:NLS-YFP (pG18:NLS-YFP) reporters in siliques at 4 DAP. YFP signal (green) is detected in valves. Chlorophyll autofluorescence in red. Scale bar = 10 μm. White arrows indicate the signal in the valves.

The results indicate that ABCG17 and ABCG18 adjust seed size, while we cannot detect the expression of these genes in the developing seeds. We, therefore, speculated that ABCG17 and ABCG18 control ABA movement from the maternal region of the plant to the developing seeds. To assess this hypothesis, we tested whether the ABCG17, ABCG18 double-knockdown seed-size phenotype is influenced by maternal genotype. Seed size was examined in F1 reciprocal paternal/maternal crosses between WT and mir17,18. The larger seed size was observed when mir17,18 functioned paternally, but seed size was intermediate when mir17,18 functioned maternally (i.e., from the zygote) (Fig. 3A-C). The maternal effect of ABCG17 and ABCG18 on seed size is consistent with the observed expression of ABCG17 and ABCG18 in maternal tissues and suggests that ABCG17 and ABCG18 affect seeds from maternal tissues. Notably, a smaller but significant paternal effect was quantified (mean seed size 95% of WT), suggesting a partial contribution of ABCG17 and ABCG18 from the zygote itself.

Fig. 3. Seed size is affected by both maternal and zygotic ABCG17 and ABCG18 activity.

Fig. 3

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A, Images of mature seeds reciprocally crossed with mir17,18 and WT plants. Scale bar = 200 μm. B-C, Average (±SD) seeds B) lengths and C) widths of WT and mir17,18 reciprocally crossed lines. n ≥ 18; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test. ♀ stands for female and ♂ stands for male.

ABCG17 and ABCG18 double-knockdown leads to lower ABA levels in the embryo

In order to test how ABA accumulation is affected at the spatial level by ABCG17 and ABCG18 knockdown, we utilized the ABA reporter pRAB18:GFP44. The pRAB18:GFP reporter is not expressed in the seeds (i.e., embryo or endosperm). However, we found that ABCG17 and ABCG18 double knockdown resulted in enhanced pRAB18:GFP signal from the valves compared to WT (Sup. Fig. 3). This supports our hypothesis that ABCG17 and ABCG18 mediate ABA transport into seeds probably from the values.

To better understand ABA levels in the developing seeds, we used the FRET-based ABACUS2-400n and ABACUS2-100n ABA reporters, which enables quantitative, high-resolution analysis of ABA levels in plants45. Imaging the silique (4 days after pollination) showed high ABA levels in the outer tissues of the developing seeds, the valves, funiculus, septum and seed coat in ABCG17 and ABCG18 double-knockdown compared to WT (Fig. 4A-B). On the contrary, there was a significant reduction in ABA levels in the ABCG17 and ABCG18 double-knockdown embryos compared to WT embryos (Fig. 4C-E). The low levels of ABA in the mutant embryo, together with the larger seed size in the ABCG17 and ABCG18 double-knockdown lines are in agreement with previous reports showing that the ABA biosynthesis mutant aba2-1 and the ABA signaling mutant abi5 develop larger seed11. Altogether, the data suggest that ABCG17 and ABCG18 reduce seed size by regulating ABA levels from maternal tissues into the embryo.

Fig. 4. ABA levels are lower in abcg17,18 double knockdown embryos than in WT, but higher in valves, septum, funiculi and seed coat.

Fig. 4

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A, Maximum Z- projection of emission ratios of developing nlsABACUS2-100n mir17,18 siliques. Siliques were staged for 4 DAP from F1 plants (ABACUS2 as the male), then hand dissected to reveal the inner valve tissues, septum, funiculi and seeds. Scale bar indicates 50 μm. B, Emission ratios of developing inner valve, septum, funiculi and seed coat, as represented in A. The FRETENATOR ROI Labeller was used to label tissues and points indicate the mean emission ratio of each tissue type within a Z-stack. n = 3; two-way ANOVA (Tissue: p = 0.034, genotype: p < 0.0001. Interaction: p = 0.137) with Sidak’s test, **: P < 0.01, ****: P < 0.0001. C, Z-sum projection of nlsABACUS2-400n fluorescence (green) and autofluorescence (magenta) of F1 nlsABACUS2-400n X Col-0 or F1 nlsABACUS2-400n X mir17,18 seeds at 4 DAP (ABACUS2 as the male). Right: Emission ratios of embryos cropped from images to the left. Scale bars = 20 μm. D, Emission ratios of cropped early globular embryos, as represented in C. n ≥ 5; P value determined by Student’s t test. E, Emission ratios of cropped early (blue) and late (red) globular embryos. Points indicate the median emission ratio of individual embryo Z-stacks. n ≥ 3; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test.

Ectopic production of ABA in the phloem increases seed size

Our data suggest that ABCG17 and ABCG18 redundantly promote the movement of ABA from maternal tissues into the embryo. To test if ABCG17 and ABCG18 can affect ABA transport from phloem to seeds, we generated pSUC2:XVE:ABCG17 and pSUC2:XVE:ABCG18 lines, in which expression of the transporter is driven by a phloem-specific pSUC2:XVE promoter. The XVE activator is strictly regulated by estradiol, with limited activity in the absence of an inducer. We could not detect SUC2 expression in the developing seed (Sup. Fig. 4), suggesting that it is a valuable tool for this experiment. pSUC2:XVE:ABCG17 and pSUC2:XVE:ABCG18 plants were shorter than WT plants (Sup. Fig. 5) and produced larger seeds compared to WT and their respective controls (Fig. 5A-C). These results imply that the expression of either ABCG17 or ABCG18 manipulates ABA levels and affects leaf size.

Fig. 5. Phloem-born ABA regulates seed size.

Fig. 5

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A, Images of seeds from WT plants and from the indicated genotypes that express ABCG17 or ABCG18 only in phloem, in the background of pRAB18:GFP reporter. Scale bar = 200 μm. B-C, Average (±SD) of seeds B) length and C) width of the indicated genotypes. n ≥ 18; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test. D, Images of seed length of the WT (Col-0), aba2-1 mutant and pSUC2:XVE:ABA2 aba2-1 (pSUC2:XVE:ABA2 plants inducibly express ABA2 specifically in phloem companion cells, in the aba2-1 mutant background). The three genotypes were treated with the following treatments: mock-treated (Control), β-estradiol (E2) applied specifically to rosette leaves; and β-estradiol (E2) applied to the whole plant. Scale bar = 250 μm. β-estradiol (E2) concentration = 5 μM, plants were sprayed 3 times a week until the seeds were harvested. E, Quantification of the images shown in (D). Average (±SD) of seed length from β-estradiol (E2) treatment only to the leaves; whole plant, or mock-treated (Control) plants of the indicated genotypes. Estradiol concentration = 5 μM. n ≥ 24; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t test.

To further test the role of phloem-born ABA in affecting seed size, we generated pSUC2:XVE:ABA2 aba2-1 lines, in which ABA2 expression is driven by a phloem-specific pSUC2:XVE promoter (an estradiol-induced system) in the aba2-1 mutant background. Importantly, no SUC2 expression was detected in the developing seed (Sup. Fig. 4). We found that, without the application of estradiol, pSUC2:XVE:ABA2 aba2-1 displayed seed elongation phenotype, mirroring the phenotype observed in aba2-1 mutants. However, estradiol treatment only to the rosette leaves, or to the entire plant, suppressed the aba2-1 phenotype to WT levels (Fig. 5D-E). Therefore, phloem-specific ABA synthesis is sufficient to complement the aba2-1 mutant seed phenotype, suggesting that ABA can move from the phloem to the developing seeds.

ABCG1 is an ABA-induced plasma membrane-localized transporter

ABCG17 or ABCG18 were originally identified in an amiRNA screen40. In the amiRNA-1228 line, expression of ABCG17, ABCG18, and ABCG1 are reduced (Fig. 6A) as shown previously40. The seeds of the amiRNA-1228 mutant are significantly larger than those of WT plants (Fig. 6B). ABCG1 has not been reported to be involved in ABA transport or activity. To test if ABCG1 is expressed in the seed, we generated pABCG1:GUS lines and discovered that ABCG1 is weakly expressed in developing seeds (Sup. Fig. 6A). We also generated a pABCG1:NLS-YFP line, and its analysis confirmed that ABCG1 is minimally expressed in developing seeds (Sup. Fig. 6B). We further tested the ABCG1 transcriptional response to ABA treatment; both pABCG1:GUS and pABCG1:NLS-YFP lines showed an enhanced response to ABA treatment in both shoot and root (Fig. 6C-D). To determine where ABCG1 localizes at the subcellular level, we cloned the coding sequence of ABCG1 tagged with the YFP coding sequence at the N-terminus driven by the 35S promoter. Confocal microscopy of the root meristem of the p35S:YFP-ABCG1 transgenic line indicated that ABCG1 is localized to the plasma membrane (Fig. 6E).

Fig. 6. ABCG1 is transcriptionally induced by ABA and localized to the plasma membrane.

Fig. 6

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A, Phylogenetic clade of ABCG17, ABCG18, and their close Arabidopsis paralogues. B, Average (±SD) of length and width of seeds from WT (blue) and amiRNA1228 line. n ≥ 40; P was determined by one-way ANOVA with Student’s t test. C, Images of GUS staining for pABCG1:GUS (pG1:GUS) 5-day-old seedlings grown with or without 10 μM ABA. Scale bars = 1 mm. D, Representative images of pABCG1:NLS-YFP (pG1:NLS-YFP) in roots treated or not with 10 μM ABA for 7 hours. Scale bars = 10 μm. E, ABCG1 subcellular localization in p35S:YFP-ABCG1 (YFP-G1) stable transgenic Arabidopsis lines. Yellow indicates YFP-ABCG1 fluorescence, and red indicates propidium iodide (PI). Fluorescence was imaged in the root meristem epidermis layer. Scale bars = 5 μm. F-G, Relative efflux of F) [3H]ABA and G) [14C]IAA from tobacco protoplasts, expressing ABCG1 or ABCG17, relative to initial efflux. n ≥ 4, P values determined by Welch’s t test. H, Averages (±SD) of seed length and seed width of abcg1 mutant. n ≥ 30; P values determined by Student’s t-test. I, Images of seeds from WT, two triple knockdown lines resulting from mir18 transformation into abcg1,17 double mutant. Scale bar = 250 μm. J-K, Average (±SD) of length and width of seeds from indicated genotypes. n ≥ 26; different letters represent significant differences at P < 0.05, one-way ANOVA with Student’s t-test.

To test if ABCG1 can transport ABA, a biochemical transport assay with radiolabeled ABA ([3H]ABA) was performed in tobacco plants that overexpress ABCG1. Unlike the positive control ABCG17, ABCG1 did not show an export activity that was significantly different from the control. Further, ABCG1 did not transport a radioactively labeled hormone from the auxin class ([14C]IAA) (Fig. 6F-G). While the results may suggest that ABCG1 is not an ABA transporter, it could be that the protein is not functional under this experimental setup (e.g., codon usage, miss-localization in tobacco protoplasts, or that the YFP fusion alters its activity). Notably, ABCG1 was shown to be involved in the transport of longer-chain aliphatic monomers from the cytoplasm to the apoplastic space during root suberin formation46, a process that is induced by ABA38,46, 47,48.

To evaluate the contribution of ABCG1 to seed-size phenotype, we examined the abcg1 mutant; seeds were similar in length and width to WT seeds (Fig. 6H). In addition, when we transformed mir18 into the double-mutant abcg1,17 background to generate the triple mutant m18,g17,g1, we found that ABCG1 did not alter seed size compared to the double mutant (Fig. 6I-K). While it could be that ABCG1 is not directly involved in seed size development, it is possible that its activity is masked by additional unknown genetic factors or physical constraints. Together, these data indicate that ABCG1 is a plasma membrane protein that is transcriptionally upregulated by ABA treatment.

Discussion

Our previous study showed that ABCG17 and ABCG18 are two plasma membrane-localized ABA importers that redundantly modulate stomatal closure and lateral root initiation40. Here we further characterized the functions of these two importers and found that the double-knockdown lines had larger seeds compared to the WT and single abcg17 and abcg18 mutants, suggesting the two ABCG ABA transporters function redundantly to modulate seed development and seed size.

Given that the double knockdown lines showed higher ABA accumulation in the valves, septum, funiculus and seed coat, but reduced ABA content in the embryo (as shown using nlsABACUS2), and that ABCG17 and ABCG18 are ABA importers mainly expressed in the valves of 4 DAP siliques, we hypothesized that the two ABCG proteins are redundantly indispensable for ABA accumulation in the zygote while the seeds are in early stage of development. We speculate that ABCG17 and ABCG18 act from the valves, which reduced ABA accumulation in the developing seeds of the ABCG17 and ABCG18 double mutant leading to a delay in endosperm cellularization, resulting in larger seeds.

At present, the exact mechanisms governing the accumulation of abscisic acid (ABA) in plant valves remain unclear. Specifically, it is uncertain whether ABA in the valves is synthesized locally or transported from the vasculature of photosynthetic leaves, or if both processes contribute to its presence. ABCG17 and ABCG18, two important ABC transporters, are known to be expressed in the mesophyll cells40, where they function to import ABA into these cells. This import process serves to regulate ABA availability, limiting its concentration in the guard cells and roots. In the ABCG17 ABCG18 double mutant, elevated ABA levels are observed in the guard cells and roots, leading to enhanced stomatal closure and a suppression of later root emergence. Interestingly, while these transporters appear to control ABA levels in guard cells and roots, the same double mutant shows lower ABA levels in the embryo, suggesting that the transporters play a pivotal role in modulating ABA distribution within the plant. This raises the possibility that ABA does not simply passively flow through the phloem to accumulate in the seeds but instead is actively regulated and gated by the activities of ABCG17 and ABCG18. However, the contribution of these transporters in the valves remains an open question—specifically, whether their expression in these tissues has a direct impact on seed size. Is the ABA level in the seed governed by transporter activity within the rosette leaves, or does it involve regulation in the reproductive organs? Moreover, the role of additional transporters in ABA transport within the seeds has yet to be fully elucidated. It is still unclear how the transport of ABA in the seed is coordinated to maintain homeostasis between the zygote and endosperm, two key components of seed development. Further research into the interactions between these transporters and other potential ABA transporters will be critical to understanding the intricate regulation of ABA in seed development and its broader role in plant physiology.

Plant ABCG transporters are renowned for their versatility, particularly through a phenomenon known as “multispecificity.” This term refers to the ability of these transporters to selectively translocate a limited number of substrates, which are often chemically diverse. Unlike the polyspecificity observed in organisms such as yeast, plant ABCGs exhibit a more targeted and refined specificity49. For instance, Nicotiana tabacum’s ABCG1/PDR1 plays a role in pathogen defense by selectively translocating cyclic diterpenes like sclareol, manool, and cembrane, while excluding other monoterpenes such as eucalyptol50. Similarly, Arabidopsis ABCG37/PDR9 demonstrates multispecificity by exporting both the auxin precursor indole-3-butyric acid (IBA) and scopoletin, a compound involved in iron uptake51, 52,53. Additionally, Medicago truncatula’s ABCG46 exemplifies multispecificity by transporting structurally distinct precursors of the phytoalexin medicarpin, including p-coumaric acid and liquiritigenin54. These examples underscore the critical role of multispecificity in regulating plant growth, defense, and metabolism, ultimately shaping the plant’s dynamic interaction with its environment. In this instance, ABCG1 has been shown to participate in the transport of long-chain aliphatic monomers, a key process in regulating root suberin formation. Here, we demonstrate that ABCG1 is not directly involved in seed size or abscisic acid (ABA) transport, further contributing to the ongoing discussion surrounding the multispecificity of the ABCG family. Similar specificity was shown for the ABA importer ABCG40 and the ABA exporter ABCG25, with both exhibiting specificity towards ABA enantiomers30,31,55.

It will be important to investigate if the activity of rice DG1, which facilitates ABA long-distance transport to control rice seed development in a temperature-dependent manner41, is maintained in Arabidopsis and other dicots. This might be a challenging task since the MATE transporter family is large, with complex redundancy between family members56,57. If DG1 activity is indeed conserved, studying the relationships between the two ABA transport mechanisms will be interesting. These two activities could complement each other to stream ABA from the leaves to the developing seeds. Alternatively, they might function in response to different environmental stimuli with DG1 mediating high-temperature responsive ABA transport41 and ABCG17 and ABCG18 meditating ABA transport in response to abiotic stress40. Interestingly, it was recently reported that the rice ABA biosynthesis mutant nced3 (Osnced3), produces smaller grains while the OsNCED3 overexpression lines generated larger grains58, opposite to Arabidopsis ABA2, suggesting for low mechanistic conservation or complex regulation.

Recently, three NPF transporters, NPF2.12, NPF2.13, and NPF2.14, were found to regulate GA and ABA translocation in Arabidopsis to promote endodermal root suberization38. Though these three transporters are very close in the phylogenetic tree, NPF2.12 and NPF2.13 are localized on the plasma membrane and function as ABA importers, whereas NPF2.14 is localized on the tonoplast and functions as an ABA exporter in oocytes. Further, NPF2.12 and NPF2.13 are required for long-distance GA precursor GA12 shoot-to-root translocation38. This raises the question of whether the ABA intermediates and conjugated forms (e.g., Abscisic acid glucosyl ester) are those that are transported long and short distances59.

Previous studies have shown that diverse plant hormones, including, auxin, ABA, cytokinin, brassinosteroid, and GA, play critical roles in regulating seed size 21,5,60,61,62,63,64. It was also reported that ABA biosynthesis and signal transduction regulate seed development10, as well as DG1-mediated activity of ABA transport to balance rice grain filling41. However, the spatial activity map within the seeds that orchestrate the zygote and endosperm ABA homeostasis and communication is not completely clear. By carrying out in situ hybridization and GUS staining experiments, Cheng et al. found that a key enzyme in ABA biosynthesis pathway, ABA2, is expressed in both embryo and endosperm, suggesting that ABA may be synthesized directly in these tissues11,19. However, we demonstrated here that maternal ABA is transported to the embryo and determines seed size.

How does ABA regulate seed size? An earlier study showed that there is a correlation between the timing of endosperm cellularization and final seed size, with delayed cellularization resulting in larger seeds11. Further, ABA negatively modulates endosperm proliferation5,11, 65,66,67,68. Our study showed reduced ABA accumulation in the embryo in the ABCG17 and ABCG18 double-knockdown plants. It will be interesting to investigate if there is a delay in endosperm cellularization in this mutant. At this point, the relationship between the ABA levels in the embryo and seed size is not clear. Does ABA directly synchronize cell division and expansion in the endosperm, or rather it affects seed size indirectly by orchestrating nutrient sugar allocation is not fully understood. It will be important to understand in the future the complete ABA spatiotemporal map during seed development and reveal the direct mechanisms downstream to ABA perception that control seed size.

Materials and methods

Plant materials and growth conditions

All Arabidopsis thaliana lines used in this work are in Col-0 background (Columbia ecotype from Salk Institute). For assays on plates, sterilized seeds were plated on 16 × 16 cm square Petri dishes or 8.5-cm round Petri dishes with growth media containing 0.5× Murashige-Skoog (MS) medium, 1% sucrose, and 0.8% plant agar, pH 5.7. The seeds were stratified for 2-3 days at 4 °C and then transferred to growth chambers (Percival, CU41L5) at 21 °C and 100 μE m−2 S−1 light intensity under long-day conditions (16 h light/8 h dark). For seed propagation, transformation, crossing, or soil pot assays, seeds were sown into wet soil, and the plants were grown in growth rooms under long-day conditions (16 h light/8 h dark) at 21 °C. Sequencing data for Arabidopsis genes used in this study can be found in the Arabidopsis Genome Initiative database under the following accession numbers: ABCG1 (AT2G39350), ABCG17 (AT3G55100), and ABCG18 (AT3G55110). pSUC2:YFP was obtained from Gregory Vert Lab69.

Agrobacterium transformation

The GV3101 electrocompetent Agrobacterium tumefaciens strain was incubated on ice with ~100 ng of plasmid for 2 min and electroporated using a MicroPulser (Bio-Rad Laboratories; 2.2 kV, 5.9 ms). Immediately after electroporation, 600 μl LB medium was added, and the samples were shaken for 2 h at 28 °C. The Agrobacteria were then plated on selective LB agar plates containing the relevant antibiotics for 2 days at 28 °C.

Arabidopsis transformation

An Agrobacterium colony was chosen and verified by colony PCR and sequencing before growth in 150 ml LB medium containing 25 μg/ml gentamycin and 50 μg/ml rifampicin plus construct specific antibiotic for 2 days at 28 °C. Agrobacteria were collected by centrifuging for 15 min at 1790 g (4000 rpm). The supernatant was discarded, and the pellet was resuspended in 60 ml 5% sucrose and 0.05% Silwet L-77. Arabidopsis flowers were then submerged in the bacterial solution for around 5 min. After this, plants were kept in the dark overnight and then grown until siliques ripen and dried. T1 seeds were collected in bulk and sown on MS media containing the appropriate antibiotics for transformant plant selection. Resistant plants were transferred to soil and grown until maturity for seed collection.

Genotyping

T-DNA insertion lines for single mutants ordered from Gabi Kat (www.gabi-kat.de) and The Arabidopsis Information Resource (www.arabidopsis.org/) are listed in Table S1. Primers for the T-DNA insertion mutant genotyping were designed using the T-DNA Primer Design Tool powered by Genome Express Browser Server (http://signal.salk.edu/tdnaprimers.2.html). Homozygous mutants were characterized by PCR carried out with primers listed in Table S2.

Cloning

mir17,18; mir17,g18 and mir18,g17 lines, were previously described40. ABCG1, ABCG17, and ABCG18 coding regions were amplified using Phusion High-fidelity Polymerase (New England Biolabs) from Col-0 complementary DNA (cDNA) using primers listed in Table S3 (cloning of ABCG17 and ABCG18 were previously reported40). Promoters of ABCG1, ABCG17, and ABCG18 were amplified from Col-0 DNA using Phusion High-fidelity Polymerase (New England Biolabs) using primers listed in Table S3 (cloning of ABCG17 and ABCG18 promoters was previously reported40). Promoters of ABCG1, ABCG17, and ABCG18 are 1759, 1276, and 1580 base pairs long, respectively, including the 5′ untranslated regions. ABCG1, ABCG17, and ABCG18 coding regions as well as their promoter fragments were cloned into pENTR/D-TOPO (Invitrogen K2400), verified by sequencing, and subsequently cloned into binary destination vectors using LR Gateway reaction (Invitrogen 11791). p35S:YFP-ABCG1, p35S:YFP-ABCG17, and p35S:YFP-ABCG18 were generated using the pH7WGY2 vector and were selected using spectinomycin in Escherichia coli and hygromycin in plants. p35S:ABCG1, p35S:ABCG17, and p35S:ABCG18 were generated using the pH2GW7 vector and selected using spectinomycin in E. coli and hygromycin in plants. pABCG1:NLS-YFP, pABCG17:NLS-YFP, and pABCG18:NLS-YFP were generated using R1-R2:NLS-YFP in the pART27 vector and selected using spectinomycin in E. coli and kanamycin in plants. pABCG1:GUS, pABCG17:GUS, and pABCG18:GUS were generated using the pWGB3 vector and were selected using kanamycin and hygromycin in E. coli and hygromycin in plants. Using primers containing the recognition sequence for the restriction enzyme AscI, pABCG17 and pABCG18 were amplified and ligated into ABCG17 CDS and ABCG18 CDS at the AscI site. The LR Gateway reaction was carried out with the binary vector pGWB1. pABCG17:ABCG17 and pABCG18:ABCG18 were transformed into mir18,g17 and mir17,g18-1, respectively, for phenotype complementation assays. To generate pSUC2:XVE:ABA2:NosT, a multisite Gateway reaction (LRII+) was carried out: pSUC2:XVE (in p1p4r position) with ABA2 CDS (in pENTR) and NosT terminator (in 2R3e position) was cloned into pB7m34GW. The WMD3 website was used to design amiRNAs (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) (Table S4). The amiRNA sequences were synthesized by Syntezza Bioscience Ltd. and were cloned into the pUC57 vector with Gateway system borders and then into the pH2GW7 destination vector using the Gateway system.

Plant genetics

All transgenic lines generated from the destination vectors were transformed into Col-0 with the exception of pSUC2:XVE:ABA2CDS:NosT, which was transformed into the aba2-1 mutant. T1 seeds were collected and selected on 1/2 MS plates containing appropriate antibiotics or on soil with basta spraying. At least 10 independent lines for each construct were generated. Characterization of two representative homozygous lines for each construct are reported.

Seed-size measurements

Images of the seeds were taken using a light microscope. The seed length and seed width were quantified using Fiji software.

Estradiol treatments

β-estradiol (E2758, Sigma Aldrich) was dissolved in ethanol at 10 mM and stored at −20 °C. 5 μm β-estradiol as the final working concentration with 0.01-0.02% Tween 80 was used in the β-estradiol treatment assays. 10 days-old seedlings were treated with estradiol (5 μm) after transplanting to the soil. Estradiol was applied 2 times a week until siliques matured (turned yellow). The experiment included three groups: 1. Control (mock-treated plants); 2. Estradiol treatment only on the leaves, (no flowers and siliques); and 3. Estradiol treatment to the entire plant.

GUS staining and histology

Histochemical detection of GUS activity was carried out using 5-bromo-4-chloro-3-indolyl-D-glucuronide as a substrate as previously described70. Samples were placed on slides with glass coverslips and imaged with a Zeiss binocular microscope. Histology of GUS-stained samples was performed using protocol described previously71.

Radioactive ABA translocation and transport assays

[3H]ABA (ART2192; 1 mCi/ml and 10 Ci/mmol) and [14C]IAA (ARC0160; 0.1 mCi/ml and 55 mCi/mmol) export from tobacco (Nicotiana benthamiana) mesophyll protoplasts were analyzed as described72. Tobacco mesophyll protoplasts were prepared 4 days after agrobacterium-mediated transfection with p35S:YFP-ABCG1 or empty vector as a control. Relative export from protoplasts was calculated from exported radioactivity into the supernatant as follows: Percent export = (radioactivity in the supernatant at time t = x min) − (radioactivity in the supernatant at time t = 0) × (100)/(radioactivity in the supernatant at t = 0 min). Values presented are means from eight ([3H]ABA) and four ([14C]IAA) independent transfections.

FRET confocal imaging

UBQ10pro::nlsABACUS2-400n (line7) pollen was used to pollinate emasculated Col-0 or mir17,18 flowers. At 4 days after crossing, siliques were embedded in 5% agarose (w/v), and sectioned with a Leica VT1200S vibratome (Leica Biosystems, section width: 100 μm). Samples were mounted in sterile deionized water and imaged immediately. An upright SP8-Fliman was used for nlsABACUS2-400n imaging. All images were acquired as Z-stacks in 16 bit mode, with a 20× dry 0.70 HC PLAN APO dry objective. Typically, sequential scanning was used with the following laser/detector settings: Sequence 1 was 442 excitation 50%, HYD1 460-500 nm, 200 gain and HYD2 525-550 nm, 200 gain. Sequence 2 was 514 excitation 30%, HYD2 525-550 nm, 200 gain and PMT2 650-750 nm, gain 650. Other parameters were offset 0; scan speed 400; line accumulation 2-4; bidirectional X on; pinhole 1 airy unit; Z-step size equal to the optical section thickness; zoom 2.45; resolution 800 x 800 pixels.

Biosensor image analysis

Embryos were cropped with Fiji and then analyzed with FRETENATOR: Segment and ratio (v2.0 alpha, https://github.com/JimageJ/FRETENATOR2) to quantify emission ratio as described previously73,74. Local label-based background subtraction was used to remove autofluorescence from images.

Statistical analysis

Multiple comparisons were performed by using one-way ANOVA with the least significant difference post hoc test in SPSS 19.0. Two-tailed Student’s t tests were used for graphs with only two groups. Statistical significance was determined at P < 0.05 unless otherwise stated.

Supplementary Material

Supplementary Data

Acknowledgments

Funding

This work was supported by grants from the Israel Science Foundation (2378/19 and 1462/24 to E.S.), the Human Frontier Science Program (HFSP-LIY000540/2020 to E.S.), the European Research Council (757683-RobustHormoneTrans and 101118769-HYDROSENSING to E.S.), the Swiss National Funds (project 31003A_165877 and 310030_197563 to MG), the BR funds of Chinese Academy of Sciences (118900M089 to Y.Z.), the Gatsby Charitable Foundation (to A.M.J.) and UK Biotechnology and Biological Sciences Research Council (BB/P018572/1 to A.M.J.).

Footnotes

Competing interests: The authors declare that they have no competing interests.

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