SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.     

Arabidopsis thaliana FIMBRIATA PETIOLES gene, controlling cell division and growth in floral organs

A new mutant, fimbriata petioles (fip), of Arabidopsis thaliana was obtained by chemical mutagenesis. The mutant is characterized by unusual anomalies of floral organs. Clusters of very large cells formed in the distal region of sepals and petals, which created fringed edges of these organs. An analysis of the morphology of the floral organs and leaves of the fip as 1 double mutant revealed a complementary interaction of the ASYMMETRIC LEAVES1 (AS1) and FIMBRIATA PETIOLES (FIP) genes. It was assumed that the FIP gene, together with the AS1 gene, controls cell proliferation, preventing their premature entry into endocycle.     

BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.     

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10⁻⁹ M of the gibberellin GA₄₊₇, whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA₄₊₇ has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA₄₊₇ than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process. Received: 27 February 1999 / Accepted: 21 May 1999     

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.     

Expression of CKS1At in Arabidopsis thaliana indicates a role for the protein in both the mitotic and the endoreduplication cycle

Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle. Received: 11 May 1998 / Accepted: 29 September 1998     

Misexpression of the cyclin-dependent kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death

A positive correlation between cell size and DNA content has been recognized in many plant cell types. Conversely, misexpression of a dominant-negative cyclin-dependent kinase (CDK) or CDK inhibitor proteins (ICK/KRPs) in Arabidopsis and tobacco leaves has revealed that cell growth can be uncoupled from cell cycle progression and DNA content. However, cell growth also appears to be controlled in a non-cell-autonomous manner by organ size, making it difficult in a ubiquitous expression assay to judge the cell-autonomous function of putative cell growth regulators. Here, we investigated the function of the CDK inhibitor ICK1/KRP1 on cell growth and differentiation independent of any compensatory influence of an organ context using Arabidopsis trichomes as a model system. By analyzing cell size with respect to DNA content, we dissected cell growth in a DNA-dependent and a DNA-independent process. We further found that ICK1/KRP1 misexpression interfered with differentiation and induced cell death, linking cell cycle progression, differentiation, and cell death in plants. The function of ICK1/KRP1 in planta was found to be dependent on a C-terminal domain and regulated negatively by an N-terminal domain. Finally, we identified CDKA;1 and a D-type cyclin as possible targets of ICK1/KRP1 expression in vivo.     

Functional identification of AtSKIP as a regulator of the cell cycle signaling pathway in Arabidopsis thaliana

Light is a key environmental cue controlling plant development, which involves meristemic activation by cell proliferation and differentiation. Here, we identify one gene, AtSKIP, associated with cell cycle-regulated root and leaf growth processes in Arabidopsis. The spatial pattern of β-glucuronidase (GUS) activity indicated that AtSKIP is expressed in the leaf primodia, root meristem region and root vascular system, and can be activated by light. Ectopic expression of AtSKIP resulted in enhanced leaf development but suppressed root elongation in Arabidopsis, whereas AtSKIP^DD seedlings displayed retarded leaf growth and normal root growth. Moreover, AtSKIP cells displayed enhanced sensitivity to a cytokinin in a callus induction assay, further demonstrated that AtSKIP expression altered endogenous cell cycle-regulated signaling in plants. Together, these data indicate that AtSKIP participates in cell cycle-mediated growth of leaf and root.     

The evolution of gene regulatory networks controlling Arabidopsis thaliana L. trichome development

Background

The variation in structure and function of gene regulatory networks (GRNs) participating in organisms development is a key for understanding species-specific evolutionary strategies. Even the tiniest modification of developmental GRN might result in a substantial change of a complex morphogenetic pattern. Great variety of trichomes and their accessibility makes them a useful model for studying the molecular processes of cell fate determination, cell cycle control and cellular morphogenesis. Nowadays, a large number of genes regulating the morphogenesis of A. thaliana trichomes are described. Here we aimed at a study the evolution of the GRN defining the trichome formation, and evaluation its importance in other developmental processes.

Results

In study of the evolution of trichomes formation GRN we combined classical phylogenetic analysis with information on the GRN topology and composition in major plants taxa. This approach allowed us to estimate both times of evolutionary emergence of the GRN components which are mainly proteins, and the relative rate of their molecular evolution. Various simplifications of protein structure (based on the position of amino acid residues in protein globula, secondary structure type, and structural disorder) allowed us to demonstrate the evolutionary associations between changes in protein globules and speciations/duplications events. We discussed their potential involvement in protein-protein interactions and GRN function.

Conclusions

We hypothesize that the divergence and/or the specialization of the trichome-forming GRN is linked to the emergence of plant taxa. Information about the structural targets of the protein evolution in the GRN may predict switching points in gene networks functioning in course of evolution. We also propose a list of candidate genes responsible for the development of trichomes in a wide range of plant species.

     

MIDGET connects COP1‐dependent development with endoreduplication in Arabidopsis thaliana

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post‐germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub‐nuclear accumulation of COP1. The MID protein is not degraded in a COP1‐dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1‐dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1‐dependent development with the regulation of endoreduplication.     

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.