Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition.

Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells.

Corrigendum: The GIP Gamma-Tubulin Complex-Associated Proteins are Involved in Nuclear Architecture in Arabidopsis Thaliana.

[This corrects the article DOI: 10.3389/fpls.2013.00480/full.].

The F-Box-Like Protein FBL17 Is a Regulator of DNA-Damage Response and Colocalizes with RETINOBLASTOMA RELATED1 at DNA Lesion Sites.

In Arabidopsis (Arabidopsis thaliana), the F-box protein F-BOX-LIKE17 (FBL17) was previously identified as an important cell-cycle regulatory protein. FBL17 is required for cell division during pollen development and for normal cell-cycle progression and endoreplication during the diploid sporophyte phase. FBL17 was reported to control the stability of the CYCLIN-DEPENDENT KINASE inhibitor KIP-RELATED PROTEIN (KRP), which may underlie the drastic reduction in cell division activity in both shoot and root apical meristems observed in fbl17 loss-of-function mutants. However, whether FBL17 has other substrates and functions besides degrading KRPs remains poorly understood. Here we show that mutation of FBL17 leads not only to misregulation of cell cycle genes, but also to a strong upregulation of genes involved in DNA damage and repair processes. This phenotype is associated with a higher frequency of DNA lesions in fbl17 and increased cell death in the root meristem, even in the absence of genotoxic stress. Notably, the constitutive activation of DNA damage response genes is largely SOG1-independent in fbl17 In addition, through analyses of root elongation, accumulation of cell death, and occurrence of γH2AX foci, we found that fbl17 mutants are hypersensitive to DNA double-strand break-induced genotoxic stress. Notably, we observed that the FBL17 protein is recruited at nuclear foci upon double-strand break induction and colocalizes with γH2AX, but only in the presence of RETINOBLASTOMA RELATED1. Altogether, our results highlight a role for FBL17 in DNA damage response, likely by ubiquitylating proteins involved in DNA-damage signaling or repair.

The Control of Arabidopsis thaliana Growth by Cell Proliferation and Endoreplication Requires the F-Box Protein FBL17.

A key step of the cell cycle is the entry into the DNA replication phase that typically commits cells to divide. However, little is known about the molecular mechanisms regulating this transition in plants. Here, we investigated the function of FBL17 (F BOX-LIKE17), an Arabidopsis thaliana F-box protein previously shown to govern the progression through the second mitosis during pollen development. Our work reveals that FBL17 function is not restricted to gametogenesis. FBL17 transcripts accumulate in both proliferating and postmitotic cell types of Arabidopsis plants. Loss of FBL17 function drastically reduces plant growth by altering cell division activity in both shoot and root apical meristems. In fbl17 mutant plants, DNA replication is severely impaired and endoreplication is fully suppressed. At the molecular level, lack of FBL17 increases the stability of the CDK (CYCLIN-DEPENDENT KINASE) inhibitor KIP-RELATED PROTEIN2 known to switch off CDKA;1 kinase activity. Despite the strong inhibition of cell proliferation in fbl17, some cells are still able to enter S phase and eventually to divide, but they exhibit a strong DNA damage response and often missegregate chromosomes. Altogether, these data indicate that the F-box protein FBL17 acts as a master cell cycle regulator during the diploid sporophyte phase of the plant.

The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana.

During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from γ-Tubulin Complexes (γ-TuCs) located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope (NE) are currently unknown. The γ-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

Microtubule nucleation and establishment of the mitotic spindle in vascular plant cells.

The microtubular cytoskeleton plays a major role in cellular organization and proliferation. The first step in construction of a microtubule is microtubule nucleation. Individual microtubules then participate in organization of more complex microtubule arrays. A strong body of evidence suggests that the underlying molecular mechanisms involve protein complexes that are conserved among eukaryotes. However, plant cell specificities, mainly characterized by the presence of a cell wall and the absence of centrosomes, must be taken into account to understand their mitotic processes. The goal of this review is to summarize and discuss current knowledge regarding the mechanisms involved in plant spindle assembly during early mitotic events. The functions of the proteins currently characterized at microtubule nucleation sites and involved in spindle assembly are considered during cell-cycle progression from G2 phase to metaphase.

The GCP3-interacting proteins GIP1 and GIP2 are required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability.

Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.