The glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and hexokinase interact with cell cycle proteins in maize.

Cyclin/cyclin-dependent kinase (CDK) heterodimers have multiple phosphorylation targets and may alter the activity of these targets. Proteins from different metabolic processes are among the phosphorylation targets, that is, enzymes of central carbon metabolism. This work explores the interaction of Cyc/CDK complex members with the glycolytic enzymes hexokinase 7 (HXK7) and glyceraldehyde-3-phosphate dehydrogenase (GAP). Both enzymes interacted steadily with CycD2;2, CycB2;1 and CDKA;1 but not with CDKB1;1. However, Cyc/CDKB1;1 complexes phosphorylated both enzymes, decreasing their activities. Treatment with a CDK-specific inhibitor (RO-3306) or with lambda phosphatase after kinase assay restored total HXK7 activity, but not GAP activity. In enzymatic assays, increasing concentrations of CDKB1;1, but not of CycD2;2, CycB2;1 or CycD2;2/CDKB1;1 complex, decreased GAP activity. Cell cycle regulators may modulate carbon channeling in glycolysis by two different mechanisms: Cyc/CDK-mediated phosphorylation of targets (e.g., HXK7; canonical mechanism) or by direct and transient interaction of the metabolic enzyme (e.g., GAP) with CDKB1;1 without a Cyc partner (alternative mechanism).

Two cyclin Bs are differentially modulated by glucose and sucrose during maize germination.

Cell proliferation during seed germination is determinant for an appropriate seedling establishment. The present work aimed to evaluate the participation of two maize B-type Cyclins during germination and under the stimulus of two simple sugars: sucrose and glucose. We found out that the corresponding genes, ZmCycB1;2 and ZmCycB2;1, increased their expression at 24 h of germination, but only ZmCycB1;2 responded negatively to sugar type at the highest sugar concentration tested (120 mM). Also, CycB1;2 showed differential protein levels along germination in response to sugar, or its absence. Both CycBs interacted with CDKA;1 and CDKB1;1 by pull down assays. By an immunoprecipitation approach, it was found that each CycB associated with two CDKB isoforms (34 and 36 kDa). A higher proportion of CycB1;2-CDKB-36kDa was coincident to an increased kinase activity in the presence of sugar and particularly in glucose treatment at 36 h of imbibition. CycB1;2-CDKB activity increased in parallel to germination advance and this was dependent on sugar: glucose > sucrose > No sugar treatment. At RAM, CycB1;2 was more abundant in nuclei on Glucose at late germination; DNA-CycB1;2 colocalization was parallel to CycB1;2 inside the nucleus. Overall, results point out CycB1;2 as a player on promoting proliferation during germination by binding a specific CDKB isoform partner and changing its cellular localization to nuclei, co-localizing with DNA, being glucose a triggering signal.

Proliferating cell nuclear antigen associates to protein complexes containing cyclins/cyclin dependent kinases susceptible of inhibition by KRPs during maize germination.

The Proliferating Cell Nuclear Antigen, PCNA, has roles in both G1 and S phases of the cell cycle. Here we show that maize PCNA can be found in cells in structures of a trimer or a dimer of trimer, in complexes of high molecular mass that change in size as germination proceeds, co-eluting with cell cycle proteins as CycD3;1 and CDKs (A/B1;1). Using different methodological strategies, we show that PCNA actually interacts with CycD3;1, CDKA, CDKB1;1, KRP1;1 and KRP4;1, all of which contain PIP or PIP-like motifs. Anti-PCNA immunoprecipitates show kinase activity that is inhibited by KRP1;1 and KRP4;2, indicating the formation of quaternary complexes PCNA-CycD/CDKs-KRPs in which PCNA would act as a platform. This inhibitory effect seems to be differential during the germination process, more pronounced as germination advances, suggesting a complex regulatory mechanism in which PCNA could bind different sets of cyclins/CDKs, some more susceptible to inhibition by KRPs than others.