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. 2014 Feb;239(2):493-509.
doi: 10.1007/s00425-013-1990-1. Epub 2013 Nov 16.

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization

Ricardo A Dante  1 , Paolo A Sabelli, Hong N Nguyen, João T Leiva-Neto, Yumin Tao, Keith S Lowe, George J Hoerster, William J Gordon-Kamm, Rudolf Jung, Brian A Larkins
Affiliations

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization

Ricardo A Dante et al. Planta. 2014 Feb.

Abstract

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin-proteasome pathway.

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Figures

Fig. 1
Fig. 1
Cyclin expression levels during maize endosperm development. a Cyclin and actin RNA levels during endosperm development. RT-PCR was performed on total RNA isolated from endosperm at different developmental stages (indicated by DAP) with cyclin- or ACT1-specific primers. Reaction products were separated by electrophoresis and exposed to a Phosphorscreen. b Cyclin RNA levels relative to actin RNA in developing endosperms. Signal from amplicons from two independent experiments was quantified, and expression was normalized relative to 7 DAP, which is considered one expression unit. Error bars show standard error of the means. c Immunoblot analysis of cyclins during maize endosperm development. Total soluble protein extracted from endosperm at different developmental stages (indicated by DAP) was separated by SDS-PAGE and immunoblotted with affinity-purified antibodies as indicated on right. An immunoblot with actin antibody is shown as the loading control
Fig. 2
Fig. 2
CDK expression levels during maize endosperm development. a CDK and actin RNA levels during endosperm development. RT-PCR was performed on total RNA isolated from endosperm at different developmental stages (indicated by DAP) with CDK- or ACT1-specific primers. Reaction products were separated by electrophoresis and exposed to a Phosphorscreen. b CDK RNA levels relative to actin RNA in developing endosperms. Signal from amplicons from two independent experiments was quantified and expression was normalized relative to 7 DAP, which is considered one expression unit. Error bars show standard error of the means. c Immunoblot analysis of CDKs present during maize endosperm development. Total soluble protein extracted from endosperm at different developmental stages (indicated by DAP) was separated by SDS-PAGE and immunoblotted with affinity-purified antibodies as indicated on the right. An immunoblot with actin antibody is shown as the loading control
Fig. 3
Fig. 3
Localization of cyclins in mitotic and endoreduplicating endosperm cells. Longitudinal sections of kernels at mitotic (a, c, e) and endoreduplication (b, d, f) stages of endosperm development were immunostained with CYCB1;3 (a, b), CYCD2;1 (c, d), and CYCD5 (e, f) antibodies (shown in green). DNA was stained with To-Pro 3-iodide® (shown in red). Developmental stages were 5 DAP (a), 7 DAP (c, e), 15 DAP (b), and 13 DAP (d, f). Arrowheads in b, c and d indicate nuclei with different intensities of cyclin labeling. Higher magnification insets illustrate subcellular localization patterns discussed in the text. al aleurone. Bars 50 μm
Fig. 4
Fig. 4
Expression of CDKB1;1 in wild-type (−) and RBR1DS1 (+) endosperms. a Quantitative RT-PCR analysis of CDKB1;1 transcript abundance was performed on 16-DAP endosperms. Error bars show standard errors of the means. b Immunoblot analysis was performed on individual, segregating RBR1DS1 and wild-type 16-DAP endosperms. Up-regulated CDKB1;1 in RBR1DS1 endosperms is indicated by arrowheads. Equal sample loading was verified via actin immunoblotting and reported by Sabelli et al. (2013)
Fig. 5
Fig. 5
Interaction of CDKA;1 and CDKA;3 with cyclins and assembly of active kinase complexes. Maize CDKAs and cyclins were co-expressed in Drosophila S2 cells and complexes were immunoprecipitated from extracts with the corresponding cyclin antibodies. Immunoprecipitates were analyzed by immunoblotting with a PSTAIR-motif antibody (upper panel in each figure) and histone H1 kinase assays (lower panel). a In vitro-synthesized CYCA1;1 added to extracts of S2 cells expressing CDKA;1 or CDKA;3. Co-expression in S2 cells of CDKA;1 or CDKA;3 with CYCB1;3 (b), CYCD2;1 (c) and CYCD5;1 (d)
Fig. 6
Fig. 6
Kinase activity associated with CYCA1, CYCB1;3, CYCD5, and CYCD2;1 in developing maize endosperm. Cyclins were immunoprecipitated from endosperm extracts with specific antibodies, and the activity of the associated kinase was measured by phosphorylation of histone H1 (CYCA1, CYCD5, and CYCD2) or GST-E2F1 (CYCB1;3) substrates. a Autoradiographic detection of histone H1 phosphorylated by CYCA1-, CYCD5-, and CYCD2;1-associated kinases. Equal exposures of control assays that contained IgG from pre-immune serum instead of cyclin antibodies are shown (pi). b Autoradiographic detection of GST-E2F1 phosphorylated by CYCB1;3-associated kinase. Equal exposures of control assays are shown (pi). c Relative kinase activity at 7, 11, and 15 DAP. Values are averages from two independent experiments and were normalized to those from the developmental stages displaying the highest kinase activity for each cyclin, which are considered as one expression unit. Error bars show standard deviations of the means
Fig. 7
Fig. 7
Cyclin stability in extracts from mitotic and endoreduplicating endosperm cells. Radio-labeled proteins synthesized in vitro were incubated with extracts from endosperm at different stages of development. Reaction products were separated by SDS-PAGE, and gels were exposed to X-ray films. Incubation times are indicated in minutes. a Stability of CYCB1;3 in 7-, 11-, and 15-DAP endosperm extracts. b Stability of CYCA1;1, CYCD5;1, CYCD2;1, 27-kDa γ-zein, and firefly luciferase in 7- and 15-DAP endosperm extracts. c Time-course analysis of cyclin degradation in 7-DAP endosperm extract and its inhibition by the proteasome inhibitor, MG-132; (+) and (−) symbols indicate presence or absence of MG-132, respectively
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