More is more (DNA and cells) with AELs

Vegetative growth of plants is accomplished by a combination of mitotic cell division and endoreduplication, a cell cycle process in which DNA replication occurs without cell division, leading to increased DNA content and cell expansion. The direct influence of cell number and cell size on overall plant development and productivity makes regulation of both the mitotic cell cycle and the endocycle important for crop yield. CYCLIN KINASE INHIBITORs (CKIs), such as KIP-RELATED PROTEINs (KRPs), regulate the transitions between GAP 1 and DNA Synthesis (G1-S) and GAP 2 and Mitosis (G2-M) phases of the cell cycle, placing members of the family at the key checkpoints for both meiotic cell cycles and endocycles. For instance, in Arabidopsis (Arabidopsis thaliana), overexpression of the ubiquitously expressed CKI KRP6 leads to fewer higher ploidy cells due to inhibition of the mitotic cell cycle (Guérinier et al., 2013; Zhou et al., 2002).

In this issue of Plant Physiology, Qu et al. demonstrate that the four members of the Arabidopsis EL1-LIKE family of casein kinases (AELs) redundantly regulate the cell cycle (Qu et al., 2021). Through a combination of scanning electron microscopy and flow cytometry, the authors described the pleiotropic phenotypes of two different triple AEL mutants (ael123 and ael124) that exhibit reduced fertility and both fewer and lower ploidy cells (Figure 1A). This suggests that AELs have a complex effect on both mitotic and endocycles, which can be partially explained by increased stability of KRP6 in ael123 and ael124.

Figure 1.

AEL casein kinases regulate the cell cycle. A, Scanning electron micrograph of leaf epithelial cells of wild type, ael123 and ael124 plants showing reduction of cell size and number in the mutant plants (scale bar = 20 μm). B, Model showing AELs phosphorylating KRP6 at S75 and S109, promoting interaction with E3-ligases F-box-like 17 (FBL17) and RING-H2 group F 1a (RHF1a), enhancing proteasome-mediated degradation of KRP6. Degradation of KRP6 releases its suppression of CDKA;1, promoting cell division. Adapted from  Qu et al. (2021).

The pleiotropic phenotypes of the AEL triple mutants led Qu and colleagues to hypothesize that both the observed cell number and ploidy defects could result from AEL phosphorylation of key factors regulating the cell cycle. By searching for candidates with predicted Casein kinase 1 phosphorylation sites among cell cycle proteins, they identified KRP6 and used yeast and in planta interaction assays to confirm protein–protein interaction with AEL1. Using mass spectrometry and Phos-tag gel electrophoresis, the authors demonstrated that AELs regulate KRP6 through direct phosphorylation at two Serines, Ser75 and Ser109, which they confirmed by expressing a nonphosphorylatable alanine substitution mutant in plants.

As with many cell-cycle proteins, the stability of KRPs is controlled by proteasome-mediated degradation (Genschik et al., 2014). Qu et al. showed that phosphorylation of KRP6 at Ser75 and Ser109 promotes interaction with E3 ubiquitin ligases, leading to proteasome-mediated degradation of KRP6. KRP6 suppresses the activity of CYCLIN DEPENDENT KINASE A;1 (CDKA;1), thus suppressing cell division (Inzé and De Veylder, 2006; Genschik et al., 2014). Degradation of KRP6 is a key step in release of the G1-S cell cycle checkpoint, and these findings place AELs upstream of this checkpoint release (Figure 1B; Genschik et al., 2014).

In addition to AELs, KRP6 is thought to be regulated through phosphorylation of key residues by a number of other kinases, including AMP-activated protein kinase, Cyclin-dependent kinase, and possibly through autophosphorylation (Guérinier et al., 2013). These results suggest that KRP6 could be at a nexus of cellular signaling pathways and the mitotic cell cycle, integrating diverse signals into decisions about cell cycle progression. Further studies could illuminate the upstream regulation of AELs.