RESUMO
The final shape and size of plant organs are determined by a network of genes that modulate cell proliferation and expansion. Among those, SCI1 (Stigma/style Cell-cycle Inhibitor 1) functions by inhibiting cell proliferation during pistil development. Alterations in SCI1 expression levels can lead to remarkable stigma/style size changes. Recently, we demonstrated that SCI1 starts to be expressed at the specification of the Nicotiana tabacum floral meristem and is expressed at all floral meristematic cells. To elucidate how SCI1 regulates cell proliferation, we screened a stigma/style cDNA library through the yeast two-hybrid (Y2H) system, using SCI1 as bait. Among the interaction partners, we identified the 14-3-3D protein of the Non-Epsilon group. The interaction between SCI1 and 14-3-3D was confirmed by pulldown and co-immunoprecipitation experiments. 14-3-3D forms homo- and heterodimers in the cytoplasm of plant cells and interacts with SCI1 in the nucleus, as demonstrated by Bimolecular Fluorescence Complementation (BiFC). Analyses of SCI1-GFP fluorescence through the cell-cycle progression revealed its presence in the nucleoli during interphase and prophase. At metaphase, SCI1-GFP fluorescence faded and was no longer detected at anaphase, reappearing at telophase. Upon treatment with the 26S proteasome inhibitor MG132, SCI1-GFP was stabilized during cell division. Site-directed mutagenesis of seven serines into alanines in the predicted 14-3-3 binding sites on the SCI1 sequence prevented its degradation during mitosis. Our results demonstrate that SCI1 degradation at the beginning of metaphase is dependent on the phosphorylation of serine residues and on the action of the 26S proteasome. We concluded that SCI1 stability/degradation is cell-cycle regulated, consistent with its role in fine-tuning cell proliferation.
RESUMO
BACKGROUND: DNA replication and transcription are dynamic processes regulating plant development that are dependent on the chromatin accessibility. Proteins belonging to the Agenet/Tudor domain family are known as histone modification "readers" and classified as chromatin remodeling proteins. Histone modifications and chromatin remodeling have profound effects on gene expression as well as on DNA replication, but how these processes are integrated has not been completely elucidated. It is clear that members of the Agenet/Tudor family are important regulators of development playing roles not well known in plants. METHODS: Bioinformatics and phylogenetic analyses of the Agenet/Tudor Family domain in the plant kingdom were carried out with sequences from available complete genomes databases. 3D structure predictions of Agenet/Tudor domains were calculated by I-TASSER server. Protein interactions were tested in two-hybrid, GST pulldown, semi-in vivo pulldown and Tandem Affinity Purification assays. Gene function was studied in a T-DNA insertion GABI-line. RESULTS: In the present work we analyzed the family of Agenet/Tudor domain proteins in the plant kingdom and we mapped the organization of this family throughout plant evolution. Furthermore, we characterized a member from Arabidopsis thaliana named AIP1 that harbors Agenet/Tudor and DUF724 domains. AIP1 interacts with ABAP1, a plant regulator of DNA replication licensing and gene transcription, with a plant histone modification "reader" (LHP1) and with non modified histones. AIP1 is expressed in reproductive tissues and its down-regulation delays flower development timing. Also, expression of ABAP1 and LHP1 target genes were repressed in flower buds of plants with reduced levels of AIP1. CONCLUSIONS: AIP1 is a novel Agenet/Tudor domain protein in plants that could act as a link between DNA replication, transcription and chromatin remodeling during flower development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas do Domínio Armadillo/genética , Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica de Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , DNA de Plantas/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: The orderly progression through mitosis is regulated by the Anaphase-Promoting Complex (APC), a large multiprotein E3 ubiquitin ligase that targets key cell-cycle regulators for destruction by the 26 S proteasome. The APC is composed of at least 11 subunits and associates with additional regulatory activators during mitosis and interphase cycles. Despite extensive research on APC and activator functions in the cell cycle, only a few components have been functionally characterized in plants. RESULTS: Here, we describe an in-depth search for APC subunits and activator genes in the Arabidopsis, rice and poplar genomes. Also, searches in other genomes that are not completely sequenced were performed. Phylogenetic analyses indicate that some APC subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. Expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. The absence of subunit APC7 from the genome of some green algae species and as well as from early metazoan lineages, could mean that APC7 is not required for APC function in unicellular organisms and it may be a result of duplication of another tetratricopeptide (TPR) subunit. Analyses of TPR evolution suggest that duplications of subunits started from the central domains. CONCLUSIONS: The increased complexity of the APC gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of APC regulation to cope with the sedentary life style and its associated environmental exposures.