Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.

Significance: Our work provides an insight into the complex network of basic helix–loop–helix (bHLH)/helix–loop–helix (HLH) transcription factors that regulates cell elongation. An unknown network motif (an incoherent feed-forward loop) has been discovered that was established by two negative regulators of brassinosteroid responses, namely the HLH transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1) and its unidentified homolog, IBH1-LIKE1 (IBL1). We also suggest that IBH1 and IBL1 coexist with PHYTOCHROME-INTERACTING FACTOR 4 (another key regulator of cell elongation) in transcriptional complexes.

Kip‐related proteins (KRPs) play a central role in the regulation of the cell cycle and differentiation through modulation of cyclin‐dependent kinase (CDK) functions. We have identified a CDK inhibitor gene from Medicago truncatula (Mt) by a yeast two‐hybrid screen. The KRPMt gene was expressed in all plant organs and cultured cells, and its transcripts accumulated after abscisic acid and NaCl treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST motif that is also detected in various Arabidopsis KRPs. In the yeast two‐hybrid test, the KRPMt protein interacted with CDK (Medsa;CDKA;1) and D‐type cyclins. However, in the pull‐down assays, B‐type CDK complexes were also detectable. Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in phosphorylation assays. The immunoprecipitated Medsa;CDKA;1/A;2 complex was strongly inhibited, whereas the mitotic Medsa;CDKB2;1 complex was the most sensitive to inhibition. Function of Medsa;CDKB1;1 complex was not inhibited by the KRPMt protein. The mitotic Medsa;CYCB2 and Medsa;CYCA2;1 complexes responded weakly to this inhibitor protein. Kinase complexes from G₂/M cells showed increased sensitivity towards the inhibitor compared with those isolated from G₁/S‐phase cells. In vitro phosphorylation of Medicago retinoblastoma‐related protein was also reduced in the presence of KRPMt. Phosphorylation of this inhibitor protein by the recombinant calmodulin‐like domain protein kinase (MsCPK3) resulted in enhanced inhibition of CDK function. The data presented emphasize the selective sensitivity of various cyclin‐dependent kinase complexes to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in cross‐talk between Ca²⁺ signalling and regulation of cell‐cycle progression in plants.