A dominant negative mutant of cyclin-dependent kinase A reduces endoreduplication but not cell size or gene expression in maize endosperm - PubMed (original) (raw)

. 2004 Jul;16(7):1854-69.

doi: 10.1105/tpc.022178. Epub 2004 Jun 18.

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A dominant negative mutant of cyclin-dependent kinase A reduces endoreduplication but not cell size or gene expression in maize endosperm

João T Leiva-Neto et al. Plant Cell. 2004 Jul.

Abstract

Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD gamma-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis.

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Figures

Figure 1.

Figure 1.

Detection of CDKA-DN Expression in Developing Transgenic Maize Endosperms. Endosperm protein extracts (40 μg/lane) from 8- to 18-DAP kernels were separated by 12.5% SDS-PAGE and blotted onto nitrocellulose. (A) Immunoblotting with monoclonal anti-HA antibody detected a 36-kD protein corresponding to CDKA-DN. (B) Immunoblotting with polyclonal anti-CDKA antibody detected CDKA-DN and a 34-kD protein corresponding to the endogenous CDKA. (C) CDKA transcripts were detected in 16-DAP endosperm by semiquantitative RT-PCR using gene-specific primers to amplify the transgene (CDKA-DN), both the endogenous and transgenic CDKA (CDKA + CDKA-DN), and actin, which was used as loading control. RT-PCR products from individual CDKA-DN endosperms are shown in the first two lanes (CDKA-DN), and those from nontransgenic controls are in the last two lanes (control).

Figure 2.

Figure 2.

Assay of Histone H1 Phosphorylation by CDK Immunoprecipitates and p13suc1 Pulldowns from Nontransgenic Control, CDKA-WT, and CDKA-DN Transgenic Endosperms. The presence or absence of transgenic HA-tagged CDKA in the assay is indicated in the top panels of (A) to (D); lanes corresponding to transgenic and control endosperms are designated. (A) Histone H1 phosphorylation by HA-immunoprecipitate of 12-DAP CDKA-WT and nontransgenic control endosperms. (B) Histone H1 phosphorylation by p13suc1-pulldown of 12-DAP CDKA-WT and nontransgenic control endosperms. (C) Histone H1 phosphorylation by HA-immunoprecipitate of 12-DAP CDKA-DN and nontransgenic control endosperms. (D) Histone H1 phosphorylation by p13suc1-pulldown of 12-DAP CDKA-DN and nontransgenic control endosperms.

Figure 3.

Figure 3.

Flow Cytometric Analysis of Nuclei from Developing Transgenic CDKA-DN and Nontransgenic Control Endosperms. Nuclei were isolated in PARTEC buffer and analyzed by flow cytometry as described in Methods. (A) to (E) Endosperms from 8- to 18-DAP CDKA-DN kernels. (F) to (J) Endosperms from 8- to 18-DAP nontransgenic control kernels. C-value is indicated for each nuclear peak.

Figure 4.

Figure 4.

Analysis of Flow Cytometric Data Describing Nuclear C-Values for Developing CDKA-DN and Nontransgenic Control Endosperms. (A) The mean C-value was calculated for each stage of endosperm development shown in Figure 3. (B) Calculation of the percent of nuclei in each C-value class in CDKA-DN and nontransgenic control endosperm at 18 DAP. (C) Calculation of the proportion of total DNA contributed by nuclei in each C-value class in CDKA-DN and nontransgenic control endosperm at 18 DAP. The error bars show the standard deviation of the measurements.

Figure 5.

Figure 5.

DAPI-Stained Longitudinal Sections of 14-DAP CDKA-DN and Nontransgenic Control Endosperms. Paraffin-embedded kernels were sectioned near the site of silk attachment and stained with DAPI as described in Methods. CDKA-DN (A) and nontransgenic (B) control kernels are shown. Arrows identify nuclei of contrasting size in comparable cells of the central starchy endosperm region. The inset in (A) illustrates the location of the aleurone (Al), starchy endosperm (SE), embryo surrounding region (ESR), basal transfer layer (BTL), and embryo (E). Bar = 0.5 mm.

Figure 6.

Figure 6.

Comparison of Cell Sizes in CDKA-DN and Nontransgenic Control Endosperms. Comparison of cell sizes in CDKA-DN ([A] and [C]) and nontransgenic control ([B] and [D]) endosperms at 13 ([A] and [B]) and 19 DAP ([C] and [D]). Paraffin-embedded kernel sections were double stained with Calcofluor-white and DAPI; the procedure for identification of the region selected for cell size measurements is described in Methods. The inset in (A) identifies the location of the aleurone (Al), starchy endosperm (SE), embryo surrounding region (ESR), basal transfer layer (BTL), and embryo (E). Bar = 0.5 mm for (A) and (B); bar = 1.0 mm for (C) and (D).

Figure 7.

Figure 7.

Accumulation of Zein Storage Proteins in Mature CDKA-DN and Nontransgenic Control Endosperms. (A) A BC2 ear segregating for CDKA-DN and nontransgenic kernels. (B) Identification of CDKA-DN and nontransgenic kernels based on immunodetection of the HA-epitope tag. After photographing the kernels, endosperm protein was extracted and separated by 12.5% SDS-PAGE; immunoblotting was the same as described in Figure 1. (C) Analysis of zein storage proteins in transgenic CDKA-DN and control kernels shown in (B). After SDS-PAGE, the gel was stained with Coomassie blue. (D) ELISA of 27-kD γ-zein in CDKA-DN and control endosperms shown in (B). Zein proteins were extracted and immobilized for ELISA analysis as described in Methods.

Figure 8.

Figure 8.

RNA Gel Blot Analysis of RNA Transcripts in 20-DAP CDKA-DN and Nontransgenic Control Endosperms. Ten micrograms of total RNA was separated electrophoretically in 1.5% agarose and blotted to nylon membranes as described in Methods. RNA samples from CDKA-DN (first two lanes) and nontransgenic control endosperms (last two lanes) are designated. After hybridization, the nylon filters were exposed to a PhosphorImager screen (A), and the radioactivity was measured by PhosphorImager analysis. The mean radioactive values for three independent blotting experiments are shown in (B).

Figure 9.

Figure 9.

Levels of Nuclear Transcripts in CDKA-DN and Nontransgenic Control Endosperms. (A) Illustration of an autoradiograph obtained after hybridization of RNAs obtained by in vitro run-on transcription with CDKA-DN (left) and nontransgenic control nuclei (right) 20 DAP. λ DNA was used as a background control; slot blots corresponding to 19- and 22-kD α-zeins and 27-kD γ-zeins, waxy, sucrose synthase (SuSy), glyceraldehyde phosphate dehydrogenase (GAPDH), and actin are indicated. (B) Mean values of hybridization intensities calculated for three independent run-on transcription assays with 18-DAP CDKA-DN and nontransgenic control endosperm nuclei. The error bars indicate the standard deviation of the measurements.

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