LEAFY COTYLEDON1 expression in the endosperm enables embryo maturation in Arabidopsis

Abstract

The endosperm provides nutrients and growth regulators to the embryo during seed development. LEAFY COTYLEDON1 (LEC1) has long been known to be essential for embryo maturation. LEC1 is expressed in both the embryo and the endosperm; however, the functional relevance of the endosperm-expressed LEC1 for seed development is unclear. Here, we provide genetic and transgenic evidence demonstrating that endosperm-expressed LEC1 is necessary and sufficient for embryo maturation. We show that endosperm-synthesized LEC1 is capable of orchestrating full seed maturation in the absence of embryo-expressed LEC1. Inversely, without LEC1 expression in the endosperm, embryo development arrests even in the presence of functional LEC1 alleles in the embryo. We further reveal that LEC1 expression in the endosperm begins at the zygote stage and the LEC1 protein is then trafficked to the embryo to activate processes of seed maturation. Our findings thus establish a key role for endosperm in regulating embryo development.

Fig. 1. Loss-of-function of LEC1 in the endosperm disrupts seed development.

a Cartoon showing a fis2-6⁻ lec1⁻ female gamete crossing with a cdka;1⁻ LEC1⁺ pollen to generate a small seed with lec1^−/− endosperm and LEC1^+/− embryo. Red dot, mutant lec1⁻ allele; black dot, wild type LEC1⁺ allele. Em embryo, Endo endosperm. b Green mature siliques collected from different genetic crosses. lec1 seeds display purple color. Arrows represent aborted seeds (red, fis2⁻ phenotype; black, cdka;1⁻ phenotype); asterisks indicate small seeds generated from the fis2-6⁻ × cdka;1⁻ bypassing cross. Five siliques from each of the five crosses were examined. Scale bar: 500 μm. c Percentage of various progeny produced from the bypassing genetic crosses. CDKA;1^+/+ indicate the normal sized wild type seeds, while cdka;1^+/− indicate the small seeds. NA not aborted seeds, A aborted seeds, n number of seeds scored. d–k Images of the CDKA;1^+/+ (normal size) and the cdka;1^+/− (small) seeds with different genotypes produced from the bypassing genetic crosses. Five seeds from each genotype were examined. The crosses of fis2-6^+/− × cdka;1^+/− produce d type normal seeds and h type small seeds. The crosses of fis2-6^+/− × cdka;1^+/− lec1-1^−/− produce e type normal seeds and i type small seeds. The crosses of fis2-6^+/− lec1-1^−/− × cdka;1^+/− produce f type normal seeds and j type small seeds, while the crosses of fis2-6^+/− lec1-1^−/− × cdka;1^+/− lec1-1^−/− produce g type normal size lec1 seeds and k type small seeds. l An example of aborted seeds produced by fis2-6⁻. Five seeds were examined. m An example of aborted seeds produced by cdka; 1⁻. Five seeds were examined. Scale bar: 100 μm.

Fig. 2. Fully developed haploid seeds with lec1 embryos and LEC1 endosperms.

a Cartoon showing the genetic cross using the SeedGFP-HI line as female parent and lec1-1 as male parent to induce haploid progeny. Red dot: mutant lec1⁻ allele; black dot: wild type LEC1⁺ allele; green dot: weak centromeres CENH3-GFP from the HI parent; white dot: wild type centromeres. Em embryo, Endo endosperm. Note: the At2S3:GFP reporter in the SeedGFP-HI line is not shown in the cartoon. b Typical siliques containing GFP florescent seeds produced from the SeedGFP-HI × wild type (WT) or SeedGFP-HI × lec1-1 crosses. Approximately 30 siliques were produced from each type of the genetic crosses. Red arrows represent aborted seeds; black arrows indicate diploid seeds; white arrows indicate haploid seeds. Scale bar: 1000 μm. c Diploid and haploid seeds hand-sorted by their GFP signal patterns: seeds with uniform GFP signals (diploid) and seeds with mottled GFP fluorescence (haploid). At least four seeds from each genotype were examined for each replicate. This experiment was repeated three times independently. Scale bar: 500 μm. d Hand-dissected embryos from the mature diploid and haploid seeds produced from the crosses. Seed GFP florescence were detected under UV lights. Three embryos from each genotype were examined. Scale bar: 100 μm. e Detection of GFP signals in the centromere of root tip cells under confocal microscope. Insets: magnified areas of root cells indicated. Cell walls stained with propidium iodide (PI) are shown in red. Confocal images are shown as the merged channel of GFP and PI. Three embryos of each seed type were examined. This experiment was repeated three times independently. WT and lec1-1 embryos were used as control. Scale bar: 50 μm.

Fig. 3. Gain-of-function of LEC1 in the endosperm rescues lec1-3 mutant seed phenotype.

a Green mature siliques collected from T1 plants with different transgenes in the lec1-3 background. Siliques of T1 plants PLL-1 (pLEC1::LEC1-GFP lec1-3^−/−), PPL-1 (pPHE::LEC1-GFP lec1-3^−/−), and PZL-1 (pZOU::LEC1-GFP lec1-3^−/−) contain two types of seeds: [WT] seeds and [lec1-3] seeds. PPA-1 (pPHE::ABI3-GFP lec1-3^−/−), PPg-1 (pPHE::GFP lec1-3^−/−), and PZA-1 (pZOU::ABI3-GFP lec1-3^−/−) only produce defective seeds (lec1-3). [WT] indicates WT phenotype, [lec1-3] indicates lec1-3 phenotype. At least five independent individual transgenic lines were examined for each construct transformation. Images of a typical embryo from each line are shown on the right next to the images of the siliques. WT (Ler-0) and lec1-3 are used as control. N normal seeds, A abnormal seeds (lec1-3), n number of seeds scored. Em embryo. Scale bar: 500 μm. b Germination of seeds with different transgenic backgrounds at day 7. Fifty seeds were used for one biological germination test. This experiment was repeated three times independently. c–m Phenotypes of mature seeds from each of the transgenic backgrounds as indicated. Ten seeds from each genotype were examined. Scale bar: 300 μm. n SDS-PAGE gel image showing the 2S and 12S storage proteins in seeds from each of the transgenic backgrounds. This experiment was repeated three times independently.

Fig. 4. Endosperm-expressed LEC1 enters embryo to regulate seed maturation genes.

a–u Localization patterns of GFP signals in PLL, PPL, and PPg seeds at various seed development stages from zygote to maturation as indicated. Five seeds or embryos at each stage from each line were examined. Embryos (a–f) are outlined in white dash lines for clarity. Amber stars indicate endosperm nuclei and white stars indicate embryo nuclei. GFP signals are shown in green. Cell walls stained with PI are shown in red. Shown here are merged confocal images from GFP and PI channels. Scale bars: 20 μm (a–l); 50 μm (m–o); 100 μm (p–u). v Relative expression of GFP in the whole seeds of PLL, PPL, PPg, and WT at linear stage. w Relative expression of GFP in the embryos (em) of PLL, PPL, PPg, and WT seeds at linear stage. x Relative expression of LEC2, FUS3 and ABI3 in the embryos (em) of PLL, PPL, PPg seeds at linear stage. v–x a to b indicate statistical difference with one-way ANOVA followed by the post-hoc Tukey multiple comparison tests (p < 0.05). The CACS gene was used as an internal control. Values are mean ±  standard error of three biological replicates.

Fig. 5. A model depicting the regulation of seed maturation by the mobile transcription factor LEC1.

In early stages of seed development, the onset of LEC1 expression occurs in the endosperm, followed by mobilization of the LEC1 protein to the embryo, likely via the plasmodesmata (PD) in the suspensor cells. After entering the embryo, the endosperm-synthesized LEC1 overcomes the repressive chromatin constraints to trigger the de-repression of seed maturation-related transcription factor genes including LEC2, ABI3, FUS3, and LEC1 itself (L.A.F.L.). Subsequently, the L.A.F.L. transcription factors activate the expression of seed storage reserve synthesis genes, enabling normal seed maturation. In the lec1 mutant seeds, the L.A.F.L. network remains repressed due to lack of LEC1 expression in both the endosperm and the embryo, thus leading to dramatically reduced accumulation of seed storage reserves and consequently the formation of desiccation-intolerant purple seeds. Notes: endosperm (Endo), embryo (Em), and suspensor (Sp) are colored with light amber, light green, and light blue, respectively; amber circles represent endosperm nuclei; dark green circles indicate embryo nuclei; orange and gray circles represent wild type and mutant LEC1 proteins, respectively; black lines represent genomic DNA and the orange/black rectangular bars indicate the wild type and mutant LEC1 genes, respectively; purple circles represent histones; red stars represent repressive histone modifications, such as H3K27me3; and bright green squares represent seed storage reserves (SSPs: seed storage proteins).