CDKB1;1 Forms a Functional Complex with CYCA2;3 to Suppress Endocycle Onset

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.     

The A-type cyclin CYCA2;3 is a key regulator of ploidy levels in Arabidopsis endoreduplication

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3-green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.     

The tobacco A-type cyclin, Nicta;CYCA3;2, at the nexus of cell division and differentiation

Although most of the components of the cell cycle machinery are conserved in all eukaryotes, plants differ strikingly from animals by the absence of a homolog of E-type cyclin, an important regulator involved in G1/S-checkpoint control in animals. By contrast, plants contain a complex range of A-type cyclins, with no fewer than 10 members in Arabidopsis. We previously identified the tobacco A-type cyclin Nicta;CYCA3;2 as an early G1/S-activated gene. Here, we show that antisense expression of Nicta;CYCA3;2 in tobacco plants induces defects in embryo formation and impairs callus formation from leaf explants. The green fluorescent protein (GFP)-Nicta;CYCA3;2 fusion protein was localized in the nucleoplasm. Transgenic tobacco plants overproducing GFP-Nicta;CYCA3;2 could not be regenerated from leaf disc transformation, whereas some transgenic Arabidopsis plants were obtained by the floral-dip transformation method. Arabidopsis plants that overproduce GFP-Nicta;CYCA3;2 showed reduced cell differentiation and endoreplication and a dramatically modified morphology. Calli regenerated from leaf explants of these transgenic Arabidopsis plants were defective in shoot and root regeneration. We propose that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E in animals, in the control of plant cell division and differentiation.     

Cytidinediphosphate diacylglycerol synthase—Mediated phosphatidic acid metabolism is crucial for early embryonic development of Arabidopsis

Embryonic development is a key developmental event in plant sexual reproduction; however, regulatory networks of plant early embryonic development, particularly the effects and functional mechanisms of phospholipid molecules are still unknown due to the limitation of sample collection and analysis. We innovatively applied the microspore-derived in vitro embryogenesis of Brassica napus and revealed the dynamics of phospholipid molecules, especially phosphatidic acid (PA, an important second messenger that plays an important role in plant growth, development, and stress responses), at different embryonic developmental stages by using a lipidomics approach. Further analysis of Arabidopsis mutants deficiency of CDS1 and CDS2 (cytidinediphosphate diacylglycerol synthase, key protein in PA metabolism) revealed the delayed embryonic development from the proembryo stage, indicating the crucial effect of CDS and PA metabolism in early embryonic development. Decreased auxin level and disturbed polar localization of auxin efflux carrier PIN1 implicate that CDS-mediated PA metabolism may regulate early embryogenesis through modulating auxin transport and distribution. These results demonstrate the dynamics and importance of phospholipid molecules during embryo development, and provide informative clues to elucidate the regulatory network of embryogenesis.     

Loss-of-Function Mutants and Overexpression Lines of the Arabidopsis Cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS Exhibit Different Defects in Prophase-I Meiocytes but Produce the Same Meiotic Products

In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products.     

Irx1 and Irx2 Are Coordinately Expressed and Regulated by Retinoic Acid,TGFβ and FGF Signaling during Chick Hindlimb Development

The Iroquois homeobox (Irx) genes play a crucial role in the regionalization and patterning of tissues and organs during metazoan development. The Irx1 and Irx2 gene expression pattern during hindlimb development has been investigated in different species, but its regulation during hindlimb morphogenesis has not been explored yet. The aim of this study was to evaluate the gene expression pattern of Irx1 and Irx2 as well as their regulation by important regulators of hindlimb development such as retinoic acid (RA), transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling during chick hindlimb development. Irx1 and Irx2 were coordinately expressed in the interdigital tissue, digital primordia, joints and in the boundary between cartilage and non-cartilage tissue. Down-regulation of Irx1 and Irx2 expression at the interdigital tissue coincided with the onset of cell death. RA was found to down-regulate their expression by a bone morphogenetic protein-independent mechanism before any evidence of cell death. Furthermore, TGFβ protein regulated Irx1 and Irx2 in a stage-dependent manner at the interdigital tissue, it inhibited their expression when it was administered to the interdigital tissue at developing stages before their normal down-regulation. TGFβ administered to the interdigital tissue at developing stages after normal down-regulation of Irx1 and Irx2 evidenced that expression of these genes marked the boundary between cartilage tissue and non-cartilage tissue. It was also found that at early stages of hindlimb development FGF signaling inhibited the expression of Irx2. In conclusion, the present study demonstrates that Irx1 and Irx2 are coordinately expressed and regulated during chick embryo hindlimb development as occurs in other species of vertebrates supporting the notion that the genomic architecture of Irx clusters is conserved in vertebrates.     

Conserved Non-Coding Regulatory Signatures in Arabidopsis Co-Expressed Gene Modules

Complex traits and other polygenic processes require coordinated gene expression. Co-expression networks model mRNA co-expression: the product of gene regulatory networks. To identify regulatory mechanisms underlying coordinated gene expression in a tissue-enriched context, ten Arabidopsis thaliana co-expression networks were constructed after manually sorting 4,566 RNA profiling datasets into aerial, flower, leaf, root, rosette, seedling, seed, shoot, whole plant, and global (all samples combined) groups. Collectively, the ten networks contained 30% of the measurable genes of Arabidopsis and were circumscribed into 5,491 modules. Modules were scrutinized for cis regulatory mechanisms putatively encoded in conserved non-coding sequences (CNSs) previously identified as remnants of a whole genome duplication event. We determined the non-random association of 1,361 unique CNSs to 1,904 co-expression network gene modules. Furthermore, the CNS elements were placed in the context of known gene regulatory networks (GRNs) by connecting 250 CNS motifs with known GRN cis elements. Our results provide support for a regulatory role of some CNS elements and suggest the functional consequences of CNS activation of co-expression in specific gene sets dispersed throughout the genome.     

OSD1 Promotes Meiotic Progression via APC/C Inhibition and Forms a Regulatory Network with TDM and CYCA1;2/TAM

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.