Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm

Abstract

The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

Keywords: endocycle; seed development.

Fig. 1.

Generation of RBR1DS1 construct and RBR1/3-specific antibodies. (A) Schematic alignment of maize RBR amino acid sequences relative to that of human RB, which displays blocks of related sequences as thick bars highlighted by different shading. The conserved “A” and “B” pocket domains are shown as thick boxes. The sense and antisense regions selected to engineer the RBR1DS1 construct are indicated by green and red arrows, respectively. The regions selected for raising antibodies are indicated by broken boxes. (B) SDS/PAGE of recombinant proteins. Affinity-purified polypeptides corresponding to GST-RBR1P, GST-RBR3N, GST, GST-RBR3P, and GST-RBR1N are indicated by arrowheads. (C) Affinity-purified antibodies recognize specific recombinant polypeptides. Antibodies against GST-RBR3N (termed α-RBR3N), GST-RBR1N (α-RBR1N), and GST-RBR1P (α-RBR1P) specifically recognize the corresponding proteins. The ∼50-kDa band detected by α-RBR1P presumably represents an incomplete RBR1P polypeptide. Each lane contained 10 ng of protein. Primary antibodies were used at a 1:500 dilution. (D) Identification of RBR1 protein in cellular extracts. The ∼96-kDa band corresponding to RBR1 (arrowhead) was identified in 9-DAP endosperm extracts using two independent antibodies targeted to different regions of the proteins: α-RBR1P and α-RBR1N. Fifty micrograms of protein extract was analyzed in each lane. Primary antibodies were used at a dilution of 1:250.

Fig. 2.

Effects of RBR1 down-regulation on downstream gene expression. (A) Expression levels of RBR1-4, MCM2-7, and PCNA RNAs in developing RBR1DS1 endosperm relative to wild type, as measured by quantitative RT-PCR. A horizontal red line indicates the reference expression level of one unit in control samples. Error bars indicate SEMs. Each bar represents the mean of three to six replicates. RNA expression levels for MCM4, MCM5, and MCM7, which exceeded the scale of the histogram, are shown at the top. (B) Immunoblot analysis showing that RBR1DS1 endosperm has no detectable RBR1 protein and displays up-regulation of RBR3 and MCM3 proteins. (Upper) Presence or absence of the RBR1DS1 transgene (as scored by PCR) in 16-DAP transgenic (+) and wild-type (-) endosperm, respectively. RBR1 protein in wild-type endosperm extracts was detected using two different antibodies (α-RBR1P and α-RBR1N). Actin protein was detected as a loading control. RBR1 and up-regulated polypeptides are indicated by arrowheads. Both transcript and protein analyses were carried out on half endosperms dissected from wild-type and RBR1DS1 kernels from segregating ears.

Fig. 3.

Endoreduplication is stimulated in RBR1DS1 transgenic endosperm. (A) Mean ploidy levels in RBR1DS1 (red bars) and wild-type (black bars) endosperms at five developmental stages between 10 and 22 DAP. Relative transgenic ploidy levels are expressed as fold changes in C values. (B) Distribution of endosperm nuclei (expressed as a percentage of the total number of nuclei) among different ploidy classes in 16-DAP RBR1DS1 (red bars) and wild-type (black bars) endosperms. (C) Distribution of nuclear DNA (expressed as a percentage of the total amount of DNA) among different ploidy classes in 16-DAP RBR1DS1 (red bars) and wild-type (black bars) endosperms. Significance levels of pair-wise differences between control and transgenic RBR1DS1 values as determined by Student t test: *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate SEMs. All analyses were carried out on dissected endosperms from genotyped kernels segregating on the same ears. Each bar represents the mean of 5–12 samples.

Fig. 4.

PCD is enhanced in RBR1DS1 endosperm. (A) Average amounts of low-molecular-weight (<1,000 bp) DNA in transgenic RBR1DS1 endosperm relative to wild type in two mature ears. Error bars indicate SDs. Differences between wild-type and transgenic endosperms for each ear are significant as determined by Student t test: *P < 0.05; **P < 0.01. Each bar represents the mean of four to six samples. (B) Immunoblot analysis showing the relative distribution of cytochrome c, the α-subunit of mitochondrial F₁-ATPase, and actin in subcellular fractions from 19-DAP endosperm enriched for mitochondria (M) and cytosol (C). All analyses were carried out on dissected endosperms from genotyped kernels segregating on the same ears.

Fig. 5.

Reduced cell size in RBR1DS1 endosperm. (A) Cell areas were measured in three endosperm zones (indicated A–C) of segregating wild-type and RBR1DS1 kernels. (Inset) Cell contours and numbering. Scale bar = 1 mm. (B) Plots showing average cell areas in both wild-type and RBR1DS1 endosperms in the three zones measured at 2-d intervals between 10 and 18 DAP. Error bars indicate SEMs. Each datapoint represents the mean of 600–1,503 measurements. Complete analysis is shown in Fig. S4B and Table S4.

Fig. 6.

Genetic interaction between CDKA;1 and RBR1. (A) Western blot analysis (Top and Top Middle) of CDKA;1 and CDKA1DN protein expression in 19-DAP endosperms of the four genotypes (wild type, CDKA1DN, RBR1DS1, and CDKA1DN/RBR1DS1) derived from a CDKA1DN × RBR1DS1 cross. Antibodies against CDKA;1 (α-CDKA;1) recognize both the endogenous and the mutant CDKA1DN proteins. CDKA1DN protein is specifically recognized by antibodies against human influenza hemagglutinin epitope tag (α-HA), which was engineered in its sequence (21). Antibodies against actin were used as gel loading control. (Bottom) Phosphorylation of histone H1 substrate by p13^Suc1-adsorbed kinase activity in the four genotypes. (B) Expression levels of RBR1 target transcripts in 19-DAP endosperms of the four genotypes described in A. Bars represent the mean of three biological replicates each made from four, pooled endosperm RNAs. (C) Mean ploidy levels in 19-DAP endosperms of the four genotypes described in A. Significance levels of pair-wise comparisons with CDKA1DN according to Student t test: *P < 0.05; **P < 0.001. (D) RNA expression levels of the DNA METHYLTRANSFERASE 1 homolog, DMT101, in the four genotypes described in A. Datapoint composition as described in B. All analyses were carried out on dissected endosperms from genotyped kernels segregating on the same ear. Error bars indicate SEMs.

Fig. 7.

The CDKA;1/RBR1 pathway controls different aspects of maize endosperm development. In this schematic model, the CDKA;1/RBR1 pathway is shown to regulate gene expression, endoreduplication, cell proliferation, cell death, and cell size during the development of maize endosperm. CDKA;1 controls endoreduplication through RBR1 but does not impact RBR1-dependent gene expression. RBR1 represses CDK activity. Broken lines indicate hypothetical relationships. Details in main text.