Identification of cis-regulatory sequences that activate transcription in the suspensor of plant embryos

Abstract

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the scarlet runner bean (Phaseolus coccineus) G564 gene to understand how genes are activated specifically within the suspensor during early embryo development. Previously, we showed that the G564 upstream region has a block of tandem repeats, which contain a conserved 10-bp motif (GAAAAG(C)/(T)GAA), and that deletion of these repeats results in a loss of suspensor transcription. Here, we use gain-of-function (GOF) experiments with transgenic globular-stage tobacco embryos to show that only 1 of the 5 tandem repeats is required to drive suspensor-specific transcription. Fine-scale deletion and scanning mutagenesis experiments with 1 tandem repeat uncovered a 54-bp region that contains all of the sequences required to activate transcription in the suspensor, including the 10-bp motif (GAAAAGCGAA) and a similar 10-bp-like motif (GAAAAACGAA). Site-directed mutagenesis and GOF experiments indicated that both the 10-bp and 10-bp-like motifs are necessary, but not sufficient to activate transcription in the suspensor, and that a sequence (TTGGT) between the 10-bp and the 10-bp-like motifs is also necessary for suspensor transcription. Together, these data identify sequences that are required to activate transcription in the suspensor of a plant embryo after fertilization.

Fig. 1.

SRB G564 gene and transcriptional activity in tobacco and Arabidopsis. (A) Comparison of SRB and Arabidopsis seeds. (B) Localization of G564 mRNA in a SRB globular-stage embryo. In situ image was taken from Weterings et al. (4). (C–E) GUS activity in transgenic tobacco embryos carrying the G564 (−921 to +56)/GUS (C and D) and the G564 (−662 to +56)/GUS (E) chimeric genes. (F and G) GUS activity in transgenic Arabidopsis embryos carrying the G564 (−921 to +56)/GUS chimeric gene. The photograph (G) was taken from Le et al. (3). Preglobular-stage (C and F), globular-stage (D), transition-stage (G), and mature (E) embryos were hand dissected from seeds and tested for GUS activity. GUS assay incubation time was 8 h (C–E) and 2 h (F and G). (H) Conceptual representation of the G564 gene and upstream region. Blue and yellow boxes represent exons and 150-bp tandem repeats, respectively. Red arrows indicate a 10-bp motif (5′-GAAAAG^C/_TGAA-3′) identified to be conserved in the upstream regions of SRB G564 and C541 genes (4). Orange arrows indicate a sequence similar to the 10-bp motif (5′-GAAAAA^C/_T^G/_AAA-3′), designated as the 10-bp-like motif. Blue arrow indicates a 5′-deletion at −921 that showed GUS activity in the suspensor of transgenic tobacco embryos (C and D). Black arrow indicates a 5′-deletion at −662 that showed a weak GUS activity in the suspensor of transgenic tobacco embryos (Fig. 5A). The 5′-deletion results were taken from Weterings et al. (4) and data presented here (Fig. 5A). Numbers indicate positions relative to the transcription start site (+1). (I) Nucleotide sequence alignment of the 5 150-bp tandem repeats. Nucleotides conserved across all 5 repeats are indicated by asterisks. The 10-bp-like and 10-bp motifs, and region 2 (5′-TTGGT-3′) in the fourth repeat are shown in orange, red, and pink, respectively. Light blue brackets include the 54-bp regions analyzed further (Fig. 4A). The number to the left of aligned sequences indicates positions of the repeats. Gaps were introduced for optimal alignment. At, Arabidopsis; ax, axis; c, cotyledon; ep, embryo proper; s, suspensor. (Scale bars: A, 1 cm; B–G, 50 μm.)

Fig. 2.

One 150-bp tandem repeat is sufficient for suspensor transcription. Suspensor GUS activity in transgenic tobacco embryos containing different GOF constructs. Names and conceptual representations of GOF constructs to the left of each embryo. Numbers indicate positions relative to the G564 transcription start site (+1). Yellow boxes indicate 150-bp tandem repeats. Orange and red arrows indicate the 10-bp-like and 10-bp motifs, respectively; 35S/GUS indicates the CaMV 35S minimal promoter/GUS gene (7). Black arrows indicate fragments inserted in the opposite direction to the transcription start site. The GOF4 construct contains 2 fragments (−1208 to −1057, and −1902 to −1879). Numbers in the Lines column indicate the number of individual transformants displaying suspensor GUS activity over total number of transformants analyzed. Photographs of GOF1, GOF2, and GOF4 were taken after 16-h GUS incubation. Photographs of GOF3, GOF5 to GOF8, and 35S Negative were taken after 24-h GUS incubation. (Scale bar: 50 μm.)

Fig. 3.

The 10-bp-like and 10-bp motifs are required for suspensor transcription. Suspensor GUS activity in transgenic tobacco embryos containing 15-bp scanning mutatgenesis constructs (A), constructs with GOF fragments focusing on the −880 to −827 region (B), and the GOF12 constructs with mutagenesis in the 10-bp-like and 10-bp motifs (C). Blue blocks indicate the 15-bp mutation sequence (5′-GGCCGCGGGGGGCCC-3′). Black crosses on the 10-bp-like and 10-bp motifs indicate mutagenesis of these motifs (see SI Materials and Methods); +++ in the Expression column indicates that suspensor GUS activity was strong and detected by 2-h incubation in the majority of GUS-positive lines; −/+ in the Expression column indicates that suspensor GUS activity was weak and not detected by 2-h incubation in the majority of GUS-positive lines; − in the Expression column indicates no detectable suspensor GUS activity. Numbers in the Emb. column indicate the number of embryos displaying suspensor GUS activity by 24-h incubation over total number of analyzed embryos of GUS-positive lines. Other figure details are the same as those outlined in Fig. 2. Higher magnification photos of these results are shown in Figs. S1 and S2. Emb., embryos; N/A, not applicable. Photographs were taken after 24-h GUS incubation. (Scale bar: 50 μm.)

Fig. 4.

A region between the 10-bp-like and 10-bp motifs is also required for suspensor transcription. (A) Alignment of 54-bp tandem repeat regions that were tested for GUS activity. Asterisks indicate conserved nucleotides across 4 repeats. Nucleotides shown in orange, red, and pink indicate the 10-bp-like, 10-bp, and region 2 motifs, respectively. Nucleotides shown in larger and bold fonts indicate nucleotides that are conserved in GUS-positive repeats. (B) Nucleotide sequences of the 54-bp fragment and summary of the 15-bp scanning mutagenesis experiment (Fig. 3A). Green lines display borders of the 15-bp mutation constructs (M3 to M6), as shown in Fig. 3A. Regions 1 and 2 indicate 5-bp sequences containing a nucleotide G at −878 and −852, respectively. (C) Suspensor GUS activity in embryos containing the 54-bp sequence (−880 to −827) with G to A mutations at −878 (M13) and −852 (M14). Other figure details are the same as those outlined in Figs. 2 and 3. Photographs were taken after 24-h GUS incubation. (Scale bar: 50 μm.)

Fig. 5.

The 10-bp motif and degenerate 10-bp motifs combined with the TTGGT sequence can program suspensor transcription. (A) Suspensor GUS activity in transgenic tobacco embryos containing the −662 G564 deletion (D-662), the −629 G564 deletion (D-629), D-662 constructs with 1 (GOF14) and 2 (GOF13) copies of the 10-bp motif, and the GOF14 construct with a 45-bp mutation in the region at −662 to −618 (M15). Higher magnifications are shown in Fig. S3. (B) Suspensor GUS activity in transgenic tobacco embryos containing the CaMV 35S minimal promoter/GUS constructs with 2 (GOF15) and 5 (GOF16) copies of the 10-bp motif. (C) Sequences and results of suspensor GUS activity in transgenic tobacco embryos containing the D-662 constructs with 2 copies of sequences similar to the 10-bp motif present in 5′-flanking regions of suspensor-active genes. (D) Consensus 10-bp motif sequence generated from 17 sequences similar to the 10-bp motif shown in C and the 10-bp motif (5′-GAAAAGCGAA-3′) using WebLogo (28). Green boxes indicate 45-bp replacement mutations. Numbers in the Position column indicate positions of 10-bp sequences relative to their transcription start sites (+1). Asterisks indicate 10-bp sequences found in the opposite direction to the transcription start site. Other figure details are the same as those outlined in Figs. 2 and 3. Emb., Embryos; Expr., Expression; N/A, not applicable. Photographs were taken after 24-h GUS incubation. (Scale bar: 50 μm.)