The maize transcription factor myb-related protein-1 is a key regulator of the differentiation of transfer cells

Abstract

Transfer cells are highly modified plant cells specialized in the transport of solutes. They differentiate at many plant exchange surfaces, including phloem loading and unloading zones such as those present in the sink organs and seeds. In maize (Zea mays) seeds, transfer cells are located at the base of the endosperm. It is currently unknown how apical-basal polarity is established or why the peripheral cells at the base of the endosperm differentiate into transfer instead of aleurone cells. Here, we show that in epidermal cells committed to develop into aleurone cells, the ectopic expression of the transfer cell-specific transcriptional activator Myb-Related Protein-1 (MRP-1) is sufficient to temporarily transform them into transfer cells. These transformed cells acquire distinct transfer cell features, such as cell wall ingrowths and an elongated shape. In addition, they express a number of MRP-1 target genes presumably involved in defense. We also show that the expression of MRP-1 is needed to maintain the transfer cell phenotype. Later in development, an observed reduction in the ectopic expression of MRP-1 was followed by the reversion of the transformed cells, which then acquire aleurone cell features.

Figure 1.

Ectopic Expression of MRP-1. (A) GUS staining of a ProAL9:GUS transgenic kernel at 11 DAP. The aleurone shows positive GUS staining. (B) and (C) In situ hybridization experiments in 12-DAP nontransgenic (B) or ProAL9:MRP-1 transgenic (C) sibling kernels, using a MRP-1 antisense riboprobe. The hybridization signal (white spots) was detected in the BETL in both cases (large arrows) and in one side of the aleurone in (C) (arrowheads). (D) and (E) Magnification of the areas boxed in (C), containing the abgerminal epithelial cells (D) or the BETL cells (E). The image in (D) has been rotated 90° clockwise. Al, aleurone; Em, embryo; En endosperm; Pd, pedicel. Bars = 1 mm in (A) to (C) and 50 μm (D) and (E). (B) and (C) are dark-field micrographs; (D) and (E) combine dark field and epifluorescence. [See online article for color version of this figure.]

Figure 2.

The Ectopic Expression of MRP-1 Induces TC-Like Modifications. (A) to (C) Images of the abgerminal side of nontransgenic endosperms at 12 (A) or 10 ([B] and [C]) DAP. (D) to (I) Images of the abgerminal side of transgenic endosperms at 12 (D) or 10 DAP ([E] to [I]). (Enclosed in a common frame.) (J) to (N) Images of the BETL at 12 (J) or 10 DAP ([K] to [N]). (A), (D), and (J) Wax-embedded material sectioned at 8 μm and stained with calcofluor white. CWIs appear light blue with calcofluor white staining (some indicated by arrowheads in [D]). Transformed cells (those containing CWIs) appear in (D), elongated along the germinal-abgerminal axis; compare these with the cubic cells at the aleurone layer (Al) or the rounded starchy endosperm cells (SE). The germinal-abgerminal (G-AB) or apico-basal (AP-B) polarity is indicated for figures (A), (D), and (J). The image in (J) has been rotated 90° for comparison with the EETL. However, note that the images in (K) and (L) are shown in a natural orientation, with the placento-chalaza at the bottom part of the micrograph. (B), (E), (K), and (L) Semithin sections (0.5 μm) of LR white–embedded material (10 DAP) stained with toluidine blue. Cell wall secondary growth is stained pale blue in the transformed cells (E) and TCs ([K] and [L]). (C), (F) to (I), (M), and (N) Ultrathin sections (0.05 μm) of LR white–embedded material stained with lead nitrate and uranyl acetate. Images were taken using TEM. The corresponding areas of the cells shown in these images are framed in red in (B), (E), and (L). P, pericarp side; PCH, placento-chalaza; CW, cell wall; M, mitochondria, SM, synthesis machinery associated with the internal side of the CWI. Bars = 50 μm in (A), (D), and (J), 25 μm in (B), (E), (K), and (L), and the indicated values in the TEM images. Materials analyzed in (A), (D), and (J) were derived from the transgenic line EER-3b. Materials analyzed in (B), (C), (E) to (I), and (K) to (N) were derived from the transgenic line EER-2a.

Figure 3.

The Ectopic Expression of MRP-1 Induces the Expression of TC-Specific Genes. (A) Immunolocalization of the BETL-2 protein (brown color, greyish in areas that accumulate less protein) in both the BETL and the EETL in a 12-DAP transgenic seed. Inset, higher (×5) magnification of cells expressing BETL-2 at the top of the endosperm (framed). (B) Confocal microscopy immunodetection of BETL-2 in the BETL and pedicel (Pd). (C) Confocal microscopy immunodetection of BETL-2 in the EETL and pericarp (P). (D) Negative result for the immunodetection of BETL-2 in the abgerminal aleurone layer (AL) of a nontransgenic kernel at 12 DAP; the rounded cells below the aleurone are starchy endosperm cells (Se). BETL-2 is produced in the TCs and EETL (arrows) but accumulates in the adjacent maternal tissue (arrowheads). P indicates the pericarp side. Bars = 1 mm in (A), 100 μm in (B), and 50 μm in (C) and (D).

Figure 4.

Expression Analyses of BETL Markers at the EETL (A) Diagram showing a sagittal section of a maize kernel at ∼15 DAP. En, starchy endosperm; Em, embryo; Al, aleurone; ESR, embryo surrounding region; Pd, pedicel (maternal tissue). (B) and (C) Real-time RT-PCR expression analyses of MRP-1 and six TC-specific genes (BETL-1, BETL-2, BETL-9, BETL-10, TCRR-1, and INCW-2), an embryo surrounding region marker (ESR-6), and the aleurone marker AL-9. Solid red and blue bars, upper (Top) and lower (Bottom) halves of a transgenic seed, respectively (dissected as shown in [A]); striped red and blue bars, upper (Top) or lower (Bottom) halves of a seed not expressing (B) or expressing low levels (C) of MRP-1. Materials analyzed were derived from the transgenic lines EER-3b and EER-2a, respectively. Values are means + sd of three technical replicates. Asterisks denote that the transcript level was below the technique sensitivity.

Figure 5.

The EETL Cells Express Reduced Levels of Aleurone Markers. (A) to (C) Expression of a TC-specific marker (BETL-9) in a 12-DAP transgenic kernel, as determined by in situ hybridization. (B) and (C) Higher-magnification images of the EETL (B) and the BETL (C) from (A). Note in both cases the expression of the TC marker in two or three layers of elongated cells. (D) and (E) Expression of an aleurone-specific marker (AL-9) in 12-DAP transgenic (D) and nontransgenic (E) sibling kernels. The section in (D) was obtained from the same kernel shown in (A). Em, embryo. Bars = 1 mm in (A), (D), and (E) and 50 μm (B) and (C).

Figure 6.

The TC-Like Features Observed in Epithelial Cells at the EETL by 12 DAP Are Almost Absent in 16- to 17-DAP Kernels. (A) and (B) In situ hybridization analyses of a 17-DAP transgenic kernel, using probes against a TC marker (BETL-9; [A]) or an aleurone marker (AL-9; [B]). Cells at the abgerminal (AB) side of the endosperm do not express the TC marker (cf. [A] to Figure 5A), while they express the aleurone marker normally. (C) Immunolocalization of the BETL-2 protein (brown color) in the same transgenic kernel as in (A) and (B). Al-L, aleurone-like cells; EC, enlarged cells. (D) Immunolocalization of the BETL-2 protein (negative result) in a nontransgenic kernel. Al, aleurone; P, pericarp. (E) Lower-magnification image for comparison of Aleurone-like cells (Al-L) and a patch of enlarged cells (EC). (F), (H), and (I) Semithin sections (0.5 μm) of LR white–embedded material (16 DAP) stained with toluidine blue. (F) A normal aleurone layer (Al) and several starchy endosperm cells (SE) in a nontransgenic kernel. (H) Enlarged cells showing a massive CWI (arrow). (I) A complete enlarged cell showing a thicker cell wall at the pericarp-facing side, discrete cell wall masses at a lateral side (arrows), and short stretches of cell wall thickenings (CWT). (G), (J), (K), and (L) Ultrathin sections (0.05 μm) of LR white–embedded material stained with lead nitrate and uranyl acetate. Images were taken using TEM. The corresponding areas of the cells shown in these images are framed in red in (F) and (I). Arrows in (J) and (K) indicate the position of small CWIs. CW, cell walls. Materials analyzed in (A) to (E) were derived from the transgenic line EER-3b. Materials analyzed in (F) to (L) were derived from the transgenic line EER-2a. Bars = 1 mm in (A) and (B), 100 μm in (E), 50 μm in (C) and (D), 25 μm in (F), 20 μm in (H) and (I), and the indicated value in the TEM images.