Establishment of cereal endosperm expression domains: identification and properties of a maize transfer cell-specific transcription factor, ZmMRP-1

Abstract

In maize, cells at the base of the endosperm are transformed into transfer cells that facilitate nutrient uptake by the developing seed. ZmMRP-1 is the first transfer cell-specific transcriptional activator to be identified. The protein it encodes contains nuclear localization signals and a MYB-related DNA binding domain. A single gene copy is present in maize, mapping to a locus on chromosome 8. ZmMRP-1 is first expressed soon after fertilization, when the endosperm is still a multinuclear coenocyte. The transcript accumulates in the basal nucleocytoplasmic domain that gives rise to transfer cells after cellularization. The transcript can be detected throughout transfer cell development, but it is not found in mature cells. ZmMRP-1 strongly transactivates the promoters of two unrelated transfer cell-specific genes. The properties of ZmMRP-1 are consistent with it being a determinant of transfer cell-specific expression. Possible roles for ZmMRP-1 in the regulation of endosperm and transfer cell differentiation are discussed.

Figure 1.

ZmMRP-1 Contains a MYB-Related Domain. (A) Nucleotide sequence of the ZmMRP-1 cDNA and deduced amino acid sequence of the protein. The nucleotide sequence obtained by RACE is shown in italic type. The putative MYB-like DNA binding domain is shaded. Arrows above the sequence mark the positions of different primers used during the characterization studies (see text). An arrowhead marks the position of the single intron of the gene. The stop codon is marked with an asterisk. (B) Amino acid alignment of the putative DNA binding domain of ZmMRP-1 and those from MCB1, LeMYB1, MybSt1, LHY, CCA1, and the maize R2-R3 MYB gene C1. Amino acids identical in at least four sequences are shaded.

Figure 5.

RT-PCR Analysis of the Expression of ZmMRP-1. Total RNA samples (1 μg) from coleoptiles (C), leaves (L), roots (R), silk (S), tassels (T), unpollinated flowers (U), kernels at 3, 8, 17, and 29 DAP, no RNA (0), and genomic DNA (DNA). (A) ZmMRP-1–specific primers. (B) Primers designed to amplify a ubiquitous, weakly expressed R2-R3 MYB gene. In both cases, PCR primers were designed to span a genomic sequence containing an intron. The arrow in (A) indicates the ZmMRP-1 band derived from cDNA. Size markers are indicated at left.

Figure 2.

RNA Gel Blot Analysis of ZmMRP-1 Expression. mRNA samples (2 μg) from different maize tissues were hybridized with a ZmMRP-1 probe (A) or a ubiquitin probe (B). T, upper part of 10-DAP developing kernels; B, lower part of 10-DAP developing kernels; U, unpollinated flowers; R, roots; C, coleoptiles; S, silk; Ts, tassels; G, germinating kernels (detached from coleoptiles and roots).

Figure 3.

RNA Gel Blot Analysis of ZmMRP-1 Expression during Seed Development. mRNA samples (2 μg) from maize kernels at different developmental stages (DAP indicated at top) were hybridized with a ZmMRP-1 probe (A) or a ubiquitin probe (B).

Figure 4.

In Situ Hybridization Analysis of the Expression of ZmMRP-1. Sagittal sections of maize kernels at various developmental stages were hybridized with antisense ([A], [B], [C], [D], [E], [G], and [I]) or sense ([F] and [H]) RNA probes derived from the 3′ end of the ZmMRP-1 cDNA. (A) and (B) Less than 3-DAP kernel. (B) shows the same section as (A) but at higher magnification and after counterstaining with DAPI to show the nuclei. (C) and (D) 3 DAP. (E) and (F) 5 DAP. (G) and (H) 11 DAP. (I) 16 DAP. (J) Development of a maize kernel. The endosperm, as a coenocyte at 1 and 3 DAP or cellular after 5 DAP, is shown yellow. The embryo-surrounding region is shown green, and the embryo is shown blue. Drawings are not to scale. Red dots indicate nuclei where ZmMRP-1 is transcribed. Em, embryo; En, endosperm; ESR, embryo-surrounding region; PC, placentochalaza; Ph, phloem terminals at the pedicel; TC, transfer cells. Unlabeled arrows point to positive hybridization signals (bright silver grains) at the base of the endosperms. Bars = 100 μm in (A) to (D) and 500 μm in (E), (G), and (I). (A) to (I) are dark-field photographs; sections were counterstained with calcofluor to visualize the cytoplasm and, in the case of (B) to (D), with DAPI to visualize nuclei.

Figure 6.

Nuclear Localization of ZmMRP-1. Tobacco protoplasts were transformed with the GFP coding sequence under the control of the 35S promoter (A), a translational fusion between ZmMRP-1 and GFP (B), and carrier DNA alone (C). Protoplasts were photographed under UV light. Green signal, GFP fluorescence; red signal, chloroplast autofluorescence. N, nuclei.

Figure 7.

ZmMRP-1 Transactivates BETL-Specific Promoters in Tobacco Protoplasts. (A) Transactivation of BETL promoters in tobacco protoplasts. Tobacco protoplasts were cotransfected with the promoter–GUS reporter plasmid indicated in the key at bottom, plus either a control plasmid containing the ubiquitin promoter and the NOS terminator (−) or the effector plasmid ubiquitin promoter ZmMRP-1 coding sequence NOS terminator (+). The scale on the y axis is logarithmic; the mean obtained for each experiment is shown above the corresponding column. (B) Deletion analyses of the BETL-2 promoter. GUS activity measurements are the means of four independent experiments. The error bars show the average scatter from the four experiments. GUS activity is expressed as μmol 7-hydroxy-4-methylcoumarin mg⁻¹ protein min⁻¹. Green, REP construct; dark blue, full-length promoter; light blue, DEL construct; magenta, no DNA added. The effector plasmid used is indicated below each group of bars. O2, Opaque-2; O2(AD), activation domain from O2. Error bars indicate ±sd.

Figure 8.

Transactivation of the BETL-1 Promoter by ZmMRP-1 in Transgenic Plants. Transgenic maize leaves (the structures of the transgenes are shown above the leaves) were bombarded with a ubiquitin promoter–GUS construct (A), a ubiquitin promoter–ZmMRP-1 construct (B), or a ubiquitin promoter construct (C). In all cases, the NOS terminator (T) was used. After 24 hr of incubation, leaves were stained for GUS (indigo spots). PROM, promoter; Ubi, ubiquitin.