Arabidopsis ABA INSENSITIVE4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm

Abstract

Regulation of seed germination requires coordinate action by the embryo and surrounding endosperm. We used Arabidopsis thaliana to establish the relative roles of embryo and endosperm in the control of seed germination and seedling establishment. We previously showed that endospermic oil reserves are used postgerminatively via gluconeogenesis to fuel seedling establishment and that lipid breakdown is repressed by abscisic acid (ABA) in embryo but not endosperm tissues. Here, we use RNA amplification to describe the transcriptome of the endosperm and compare the hormone responses of endosperm and embryo tissues. We show that the endosperm responds to both ABA and gibberellin but that ABA in particular regulates nuclear but not plastid-encoded photosynthetic gene expression in the embryo. We also show that ABA INSENSITIVE4 (ABI4) expression is confined to the embryo, accounts for the major differences in embryo response to ABA, and defines a role for ABI4 as a repressor of lipid breakdown. Furthermore, ABI5 expression in the endosperm defines a second region of altered ABA signaling in the micropylar endosperm cap. Finally, embryo and endosperm ABA signaling mutants demonstrate the spatial specificity of ABA action in seed germination. We conclude that the single cell endosperm layer plays an active role in the regulation of seed germination in Arabidopsis.

Figure 1.

Comparison of the Embryo and Endosperm Transcriptome 24 h after Transfer to 22°C. (A) Number of detected expressed genes in embryo, endosperm, or both tissues. (B) Plot of Z-score transformed embryo versus endosperm expression data shows similar expression of most genes in both tissues. (C) and (D) Transmission electron micrograph of representative embryo and endosperm cells 1 d after transfer to 22°C showing cells densely packed with ribosomes in the embryo (C) compared with the endosperm (D). Numbers at the bottom left of the panels indicate the mean ±se of ribosomes per μm² of cytoplasm observable in a 100-μm-thin section. CW, cell wall; RB, ribosomes; LB, lipid body; PX, peroxisome. Bars = 1 μM.

Figure 2.

Transcriptomic Response of Embryo and Endosperm Tissues to ABA. (A) and (B) Number of probe sets found to be downregulated or upregulated by ABA in the embryo or endosperm by SAM analysis. (C) and (D) Regulation of the plastid transcriptome by ABA in the embryo (C) and endosperm (D).

Figure 3.

Expression of ABI3, ABI4, and ABI5 in the Embryo and Endosperm 1 d after Transfer to 22°C. (A) Detection by real-time RT-PCR. Data represent mean and sd of three replicate determinations. (B) GUS expression from the ABI3, ABI4, and ABI5 promoters in dissected embryos or endosperm/seed coats in control (CON) or ABA- or PAC-treated seeds. (C) to (H) Sections to show endosperm expression of the ABI:GUS fusions in ABA-treated seeds. (C) and (D) ABI3:GUS. (E) and (F) ABI4:GUS. Inset shows toluidene blue–stained ABI4:GUS section confirming the presence of the endosperm cell layer barely visible in (F) due to lack of expression. (G) and (H) ABI5:GUS. (I) Illustration of the expression pattern of ABI3, ABI4, and ABI5 in seeds.

Figure 4.

Eicosenoic Acid Levels in Wild Type, abi4-1, and abi5-1 Embryos 5 d after Imbibition in the Presence of 20 μM ABA. Bars represent the mean and sd of four independent determinations.

Figure 5.

The Endosperm Is Required for the ABA Regulation of Seed Germination. (A) Real-time RT-PCR to show the expression of rbohD and rbohF in germinating seeds. (B) The rbohd single mutant germination shows decreased sensitivity to ABA. (C) and (D) abi4-1 mutant seed 5 d after transfer to 22°C growing in the presence of 10 μM ABA (C) and 10 μM PAC (D). Bars = 100 μM.