Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway

Abstract

Background: During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well supported by experimental data. At the same time there is only limited insight into the molecular events that provide linkage between the regulation of cell-cycle progression and hormonal and developmental control. Studies indicate that there are several alternative ways by which hormonal signalling networks can influence cell division parameters and establish functional links between regulatory pathways of cell-cycle progression and genes and protein complexes involved in organ development.

Scope: An overview is given here of key components in plant cell division control as acceptors of hormonal and developmental signals during organ formation and growth. Selected examples are presented to highlight the potential role of Ca(2+)-signalling, the complex actions of auxin and cytokinins, regulation by transcription factors and alteration of retinoblastoma-related proteins by phosphorylation.

Conclusions: Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase (CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and over-expression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1-ARGOS-ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation of the CYCD6;1 gene. Over-expression of the EBP1 gene (plant homologue of the ErbB-3 epidermal growth factor receptor-binding protein) increased biomass by auxin-dependent activation of both D- and B-type cyclins. The direct involvement of auxin-binding protein (ABP1) in the entry into the cell cycle and the regulation of leaf size and morphology is based on the transcriptional control of D-cyclins and retinoblastoma-related protein (RBR) interacting with inhibitory E2FC transcriptional factor. The central role of RBRs in cell-cycle progression is well documented by a variety of experimental approaches. Their function is phosphorylation-dependent and both RBR and phospho-RBR proteins are present in interphase and mitotic phase cells. Immunolocalization studies showed the presence of phospho-RBR protein in spots of interphase nuclei or granules in mitotic prophase cells. The Ca(2+)-dependent phosphorylation events can be accomplished by the calcium-dependent, calmodulin-independent or calmodulin-like domain protein kinases (CDPKs/CPKs) phosphorylating the CDK inhibitor protein (KRP). Dephosphorylation of the phospho-RBR protein by PP2A phosphatase is regulated by a Ca(2+)-binding subunit.

Fig. 1.

Schematic overview of potential links between calcium, hormonal and developmental signals and cell cycle regulators; note that the proposed scheme is not complete. Abbreviations: CK, cytokinin; E2F/DP, transcription factors; RBR, retinoblastoma-related protein; P, phospho-protein; CYC, cyclin; CDK, cyclin-dependent kinase; PP2A, phosphatase; SCR, SCARECROW; SHR, SHORT ROOT; SCF, SKP1 + CULLIN + F-box (SKP2); EBP1, plant homologue of epidermal growth factor-binding protein; SKP2, F-box protein; STM, SHOOT MERISTEMLESS; KRP, CDK inhibitor; CaM, calmodulin; CPK, calmodulin-like domain protein kinase; ABAP1, armadillo BTB Arabidopsis protein 1; TCP24, transcription factor; CDT1, DNA replication-licensing factor; ABP1, auxin-binding protein 1; ANT, aintegumenta; ARGOS, auxin-regulated gene in organ size; AXR1, RUB1-activating enzyme; ABA, abscisic acid; GL2, GLABRA (root hair); GEM, GL2 expression regulator; ACS5, 1-aminocyclo-propane-1-carboxil acid synthase. For detail see the text.

Fig. 2.

Auxin (2,4-D) activates while abscisic acid (ABA) depresses wheat histone H4 promoter function serving as a marker of S-phase cells in transgenic maize tissues. The GUS (β-glucuronidase) reporter gene indicates the promoter activity by blue staining.

Fig. 3.

Immunodetection of phospho-MsRBR protein in prophase alfalfa cells as nuclear granules by antibodies produced against the human phospho-pRb peptide (green labelling). These nuclear granules cannot be recognized in later mitotic phases as shown by anaphase cells. The red staining of DNA was carried out by DAPI (diamidino-2-phenylindole).

Fig. 4.

The transcript level from the rice retinoblastoma-related gene (RBR) influences the number of S-phase cells and biomass in transgenic rice cell cultures. Over-expression of the rice RBR1 gene reduces while down-regulation of this gene increases the frequency of DNA-synthesizing S-phase cells labelled with 5-ethyl-2′-deoxyuridine (EdU) as shown in yellow and green. The over-expressing culture (line 80) could not be cultured for a prolonged time.