The Armadillo repeat gene ZAK IXIK promotes Arabidopsis early embryo and endosperm development through a distinctive gametophytic maternal effect

Abstract

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin-dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of miniseed3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways.

Figure 1.

Ds Insertion Locations in zix Alleles and Protein Domains in ZIX. Unnamed shaded box, exon; open box, untranslated region; line, intron; named shaded box, protein domain; and ARM, Armadillo repeat. Bar = 100 nt.

Figure 2.

Ovule and Seed Phenotypes of zix before Fertilization and 3 DAP. (A) Wild-type mature embryo sac 2 d after emasculation. (B) zix embryo sac 2 d after emasculation. (C) and (D) Wild-type seeds at the eight-cell embryo and early globular stages 3 DAP. (E) and (F) zix seeds at the elongated zygote and one-cell embryo stages 3 DAP. (G) and (H) Wild-type seeds at the elongated zygote and one-cell embryo stages 1.5 DAP. Bars = 50 µm. [See online article for color version of this figure.]

Figure 3.

ZIX Expression in Mature Gametophytes and Early Seeds. In situ hybridization of ZIX RNAs in pollen ([A] and [B]), mature embryo sac ([C] and [D]), fertilized seeds at the zygotic stage ([E] and [F]), one-cell embryo stage ([G] and [H]), and eight-cell embryo stage ([I] and [J]). as, antisense probe; s, sense probe. Arrows point to the egg cell ([C] and [D]) or the zygote ([E] and [F]). Bars = 40 µm.

Figure 4.

ZIX Promoter Activities in Gametophytes and Early Seeds. Prefertilization ovules or seeds at 1 to 3 DAP of ZIX:GUS lines from reciprocal crosses with wild-type plants were stained with GUS solution and imaged with DIC microscopy. cc, central cell; ec, egg cell; sy, synergids. Arrows with a star point to the early zygote ([F] and [G]); normal arrows point to the elongated zygote (H) or one-cell embryo ([I] and [N]). Bars = 4 µm in the inset in (A) and 20 µm in all others. (A) Functional megaspore; inset: pollen. (B) Two-nucleate embryo sac. (C) Four-nucleate embryo sac. (D) Eight-nucleate/seven-cell embryo sac. (E) Four-cell embryo sac. (F) to (J) Seeds from ZIX:GUS × wild-type crosses. (K) to (O) Seeds from wild-type × ZIX:GUS crosses. (F), (G), (K), and (L) Early zygote. (H) and (M) Elongated zygote. (I) and (N) One-cell embryo. (J) and (O) Two-cell embryo.

Figure 5.

Allele-Specific ZIX Transcript Levels in Fertilized Seeds. (A) Endogenous ZIX transcripts were amplified by RT-PCR and the PCR products digested with DraI and separated by agarose gel electrophoresis. The Col allele was smaller than the Ler allele after DraI digestion. Numbers refer to days after pollination. Genomic Ler and Col ZIX alleles were included in the assay as controls for genomic DNA contamination in the cDNA pool. cC, cDNA Col allele; cL, cDNA Ler allele; gC, genomic Col allele; gL, genomic Ler allele; M, DNA ladder. (B) Relative ZIX expression levels in fertilized seeds of reciprocal crosses shown in (A) measured by qPCR and normalized by ACT11 expression. Means of expression levels were calculated from three technical replicates and two biological replicates. Error bars indicate se. Numbers refer to days after pollination. The primers used in these qPCRs generate products that span an exon-intron junction and hybridize specifically to ZIX cDNA (see Methods). [See online article for color version of this figure.]

Figure 6.

Effects of Various Parental Mutant Backgrounds on Parent-of-Origin–Dependent ZIX Expression in Early Seeds. (A) Endogenous ZIX transcript levels, as determined by RT-PCR, in early seeds fertilized by pollen from hypomethylated mutants (met1, ddm1, cmt3, and kyp). Numbers refer to days after pollination. cC, cDNA Col allele; cL, cDNA Ler allele; gC, genomic Col allele; gL, genomic Ler allele; M, DNA ladder; mt, mutant pollen. (B) to (E) Promoter activities of paternal ZIX:GUS alleles in the mea (B) and fie (C) maternal background and of maternal ZIX:GUS alleles in the dme ([D] and [E]) maternal background. Bars = 20 µm.

Figure 7.

ZIX-GFP Protein Localization in Gametophytes, Early Seeds, and Roots. (A) to (J) Overlays of DIC and GFP pseudo-colored wide-field microscopy images. Red is background autofluorescence. Red arrows point to the early zygote ([F] and [G]), elongated zygote (H), or one-cell embryo (I). (A) Functional megaspore. (B) Two-nucleate embryo sac. (C) Four-nucleate embryo sac. (D) Eight-nucleate/seven-cell embryo sac. (E) Four-cell embryo sac. cc, central cell; ec, egg cell; sy, synergids. Inset in (E): pollen grain. sp, sperm; vc, vegetative cell. (F) and (G) Early zygote. (H) Elongated zygote. (I) One-cell embryo. (J) Two-cell embryo. (K) and (L) Whole-mount RNA in situ hybridization images of ZIX transcripts in roots. as, antisense probe; s, sense probe. (M) to (R) Overlays of DIC, FM-64 (red), and GFP pseudo-colored confocal laser scanning microscopy images. (M) Primary roots. (N) Lateral root initiation zone. (O) Emerging lateral roots. (P) Close-up of root cells. N, nucleus; n, nucleolus. (Q) Early seed with four-endosperm nuclei. (R) Isolated two-cell (top panel) and one-cell embryos (bottom panel). Yellow arrows point to GFP-ZIX in the nuclei. Bars = 20 µm in ovule, seeds, and embryo images, 40 µm in root images, and 5 µm in (P).

Figure 8.

Effects of Maternal zix Allele on the Expression of Other Genes in Early Seeds 2 DAP. (A) Overlay of DIC and GFP pseudo-colored image. (B) to (F) GFP pseudo-colored images (red, yellow, and pink are background autofluorescence). (G) to (J) DIC images. wt, the wild type. (A) and (B) Maternal and paternal ORC2:ORC2-GFP alleles in zix seeds. (C) and (D) Maternal and paternal CDKA1;1:CDKA1;1-YFP alleles in zix seeds. (E) and (F) Maternal and paternal AGL62:AGL62-GFP alleles in zix seeds. (G) and (H) Paternal MINI3:GUS allele in wild-type and zix seeds. (I) and (J) Maternal MINI3:GUS allele in wild-type and zix seeds. Arrows point to the GFP expression in the zygote or one-cell embryo. Bars = 20 µm.