Complexes of D-type cyclins with CDKs during maize germination

Abstract

The importance of cell proliferation in plant growth and development has been well documented. The majority of studies on basic cell cycle mechanisms in plants have been at the level of gene expression and much less knowledge has accumulated in terms of protein interactions and activation. Two key proteins, cyclins and cyclin-dependent kinases (CDKs) are fundamental for cell cycle regulation and advancement. Our aim has been to understand the role of D-type cyclins and type A and B CDKs in the cell cycle taking place during a developmental process such as maize seed germination. Results indicate that three maize D-type cyclins—D2;2, D4;2, and D5;3—(G1-S cyclins by definition) bind and activate two different types of CDK—A and B1;1—in a differential way during germination. Whereas CDKA–D-type cyclin complexes are more active at early germination times than at later times, it was surprising to observe that CDKB1;1, a supposedly G2-M kinase, bound in a differential way to all D-type cyclins tested during germination. Binding to cyclin D2;2 was detectable at all germination times, forming a complex with kinase activity, whereas binding to D4;2 and D5;3 was more variable; in particular, D5;3 was only detected at late germination times. Results are discussed in terms of cell cycle advancement and its importance for seed germination.

Modulation of CycD3;1‐CDK complexes by phytohormones and sucrose during maize germination

“…A). The two bands at 34 and 32 kDa detected match the CDKA bands previously reported (Sánchez et al , Godínez‐Palma et al ). Using the anti‐CDKB1;1 antibody, ZmCycD3;1 was found bound to ZmCDKB1;1 at 0, 12 and 24 h of germination; a similar result was obtained when anti‐CycD3;1 was used to co‐immunoprecipitate ZmCDKB1;1 (Fig.…”

“…C, D). This is a similar pattern to the one shown by most other maize cyclins D studied during the germination process (Lara‐Núñez et al , Godínez‐Palma et al ).…”

“…Association of maize CycD3;1 with CDKA and CDKB1;1 during germination. Specific antibodies anti‐CDKA, anti‐CDKB1;1 (Godínez‐Palma et al ) and anti‐CycD3;1 were used for immunoprecipitation and identification of associated proteins in crude extracts from 0, 12 and 24 h of germinated axes. (A) Input, maize protein extract (PE) at 0, 12 and 24 h of germinated embryo axes; IP, antibody (anti‐CDKA, anti‐CDKB1;1 or anti‐CycD3;1) + protein A agarose + PE; C1, antibody + protein A agarose and C2, protein A agarose + PE.…”

“…Anti‐GST‐CycD3;1, anti‐GST‐CDKB1;1 and anti‐CDKA antibodies (previously reported by Godínez‐Palma et al ) were conjugated for 1 h with magnetized protein A agarose (1:10 dilution) at room temperature using binding buffer (50 m M NaCl, 20 m M Tris‐HCl, pH 7.5, 25% glycerol, 1.5 m M MgCl 2, 1 m M PMSF, 0.02% Triton X‐100). Protein from extracts (500 µg) was added and the mixture was incubated 4 h or overnight at 4°C in a Labquake Shaker Rotisserie.…”

“…Maize cycs D2;2, D4;2 and D5;3 differentially associate to CDKA and CDKB1;1 forming active complexes during germination (Godínez‐Palma et al ). Furthermore, addition of IAA or BA increases the kinase activity associated to cycs D4;2 and D5;3 during early germination, without modifying protein levels, suggesting that these phytohormones promote activation of these kinase complexes (Lara‐Núñez et al ).…”