Global Regulation of Embryonic Patterning in Arabidopsis by MicroRNAs

Abstract

The development of the embryo in Arabidopsis (Arabidopsis thaliana) involves a carefully controlled set of cell divisions and cell fate decisions that lead to a mature embryo containing shoot and root meristems and all basic tissue types. Over the last 20 years, a number of transcriptional regulators of embryonic patterning have been described, but little is known about the role of posttranscriptional regulators such as microRNAs (miRNAs). Previous work has centered on the study of null or very weak alleles of miRNA biosynthetic genes, but these mutants either arrest early in embryogenesis or have wild-type-looking embryos. Here, we significantly extend those analyses by characterizing embryos mutant for a strong hypomorphic allele of DICER-LIKE1 (dcl1-15). Our data demonstrate that miRNAs are required for the patterning of most regions of the embryo, with the exception of the protoderm. In mutant embryos with the most severe morphological defects, the majority of tissue identities are lost. Different levels of miRNAs appear to be required to specify cell fates in various regions of the embryo. The suspensor needs the lowest levels, followed by the root apical meristem and hypocotyl, cotyledons, and shoot apical meristem. Furthermore, we show that erecta acts as a suppressor of dcl1-15, a novel role for this signaling pathway in embryos. Our results also indicate that the regulation of the messenger RNA levels of miRNA targets involves not just the action of miRNAs but has a significant transcriptional component as well.

Figure 1.

Axes and tissues. This late heart stage embryo shows the embryonic axes (left side) and tissues (right side) mentioned in the text.

Figure 2.

Phenotypes of dcl1 embryos and seedlings. A to D, Early globular (A and B) and heart (C and D) stage wild-type (WT) and dcl1-15 embryos. The arrow in B points to the split hypophysis. E to K, Mature stage embryos showing the wild type (E) and the six different classes of dcl1 phenotypes. All embryos are dcl1-15/dcl1-15 er/er except for F (dcl1-9/dcl1-15 er/er) and K (dcl1-5/dcl1-5 ER/ER). The arrow in J points to a xylem element. The arrow in K points to the overproliferated and persistent suspensor (the suspensor has degenerated in all the other classes and the wild type). L and M, Twenty-day-old wild-type (L) and dcl1-9/dcl1-15 er/er (M) seedlings, photographed at the same magnification. Bar = 25 µm (A–D) and 50 µm (E–K).

Figure 3.

Distribution of dcl1 phenotypic classes in different genetic backgrounds. WT, Wild type.

Figure 4.

Expression of reporters for the protoderm and suspensor. A and B, Heart stage embryos expressing pSUC3:GFP in the suspensor and derivatives of the hypophysis in both the wild type (WT) and mutant. C to F, Early heart stage embryos expressing pAtML1:H2B-YFP. The reporter is expressed in the wild type throughout the protoderm but excluded from the root pole (arrow in C) and the suspensor. In some mutants, pAtML1:H2B-YFP is excluded from the root pole (arrow) and suspensor (D). In others, it is expressed in the root pole (arrow) but not the suspensor (E) or it expands into the top of the suspensor (arrow in F). Bar = 25 µm for all.

Figure 5.

Expression of auxin-related reporters. A to E, Expression of DR5rev:GFP. A and B, In transition stage wild-type (WT) embryos, the reporter is expressed in the derivatives of the hypophysis, while expression is displaced apically in the mutant. C to E, In bent cotyledon stage wild-type embryos, DR5rev:GFP is strongly expressed in the root pole, the tip of the cotyledons, the SAM where new primordia will emerge, and at lower levels in the provasculature. In less severe mutants, reporter expression is similar to that in the wild type (D), but in severe mutants, it is reduced to a central dot (arrow) and patchy root pole and epidermal expression (E). F to J, Expression of pPIN1:PIN1-GFP. F and G, In wild-type early heart stage embryos, the reporter is expressed in the tip of the cotyledons and in the provasculature, while in some mutants, it is expressed throughout the embryo. H to J, In wild-type bent cotyledon stage embryos, pPIN1:PIN1-GFP is expressed in the provasculature and in future leaf primordia (H). In less severe mutants, the reporter is still expressed in the putative provasculature (I), while in more severe mutants, it is reduced to a dot in the center (arrow in J). In C to J, magenta represents chlorophyll autofluorescence. dcl1-15 phenotypic classes are as follows: class 2 (D and I) and class 5 (E and J). Bar = 25 µm (A, B, F, and G), 50 µm (E and J), and 100 µm (C, D, H, and I).

Figure 6.

Expression patterns of reporter genes for the meristems. A to D, Reporters for the SAM. E to H, Reporters for the RAM. A and B, Late heart stage embryos expressing pSTM:GUS in the central zone of the SAM in the wild type (WT) but not in the mutant. C and D, Early bent cotyledon stage embryos expressing the enhancer trap M0233 in the boundary between the SAM and the cotyledons in the wild type but not in the mutant. Magenta indicates chlorophyll autofluorescence. E and F, Late heart stage embryos expressing pWOX5:GFP in the QC in the wild type but not in the mutant. G and H, Bent cotyledon stage embryos expressing pPIN4:GUS in the QC and surrounding initials in both the wild type and the mutant. dcl1-15 phenotypic classes are as follows: class 5 (B and F), class 4 (D), and class 2 (H). Bar = 25 µm (A, B, E, and F) and 50 µm (C, D, G, and H).

Figure 7.

Early expression patterns for reporters for the central-peripheral domains. A to D and F to I, Reporters for the central and adaxial domains. E and J, Reporter for the peripheral domain. A and B, Early heart stage embryos expressing pSCR:GFP in the QC in the wild type (WT) and slightly displaced toward the center in the mutant. C and D, Heart stage embryos expressing pZLL:ZLL-YFP in the provasculature and adaxial side of the cotyledons in the wild type and all over the embryo in the mutant. E and J, Late heart stage embryos expressing pKAN1:GUS in peripheral tissues of the hypocotyl in the wild type and over most of the embryo in the mutant. F and G, Early heart stage embryos with the phb-6 gene trap expressed in the provasculature and adaxial side of the cotyledons in the wild type and reduced to the position of the provasculature in the mutant. H and I, Late globular stage embryos with the rev-9 enhancer trap expressed in the apical and central domains in the wild type and reduced to part of the apical domain in the mutant. Bar = 25 µm for all.

Figure 8.

Late expression patterns for reporters for the central-peripheral domains. A to G, Reporters for the central and adaxial domains. H to L, Reporters for the peripheral and abaxial domains. A to D, Bent cotyledon stage embryos expressing pSCR:GFP in the endodermis and QC in the wild type (WT). The expression domain is progressively reduced in the mutants as phenotypes increase in severity (B and C) to a dot in the center of the embryos (D). E to G, Early bent cotyledon stage embryos with the rev-9 enhancer trap expressed in the provasculature and adaxial side of the cotyledons in the wild type. Expression is progressively reduced in the mutants, depending on severity (F and G). H to J, Bent cotyledon stage embryos expressing pKAN1:GUS in peripheral tissues of the basal hypocotyl in the wild type. Expression is absent (I) or reduced (J) in the mutant. K and L, Bent cotyledon stage embryos expressing the E2023 enhancer trap in the periphery of the upper hypocotyl and the abaxial side of the cotyledons in the wild type. Very reduced expression is seen in the basal hypocotyl of the mutant (L). Magenta indicates chlorophyll autofluorescence. dcl1-15 phenotypic classes are as follows: class 2 (B and F), class 3 (C), class 5 (D, G, and L), class 4 (I and J). Bar = 100 µm (A) and 50 µm (B–L).