The plant hormone ethylene regulates a wide range of developmental processes and the response of plants to stress and pathogens. Genetic studies in Arabidopsis led to a partial elucidation of the mechanisms of ethylene action. Ethylene signal transduction initiates with ethylene binding at a family of ethylene receptors and terminates in a transcription cascade involving the EIN3/EIL and ERF families of plant-specific transcription factors. Here, we identify two Arabidopsis F box proteins called EBF1 and EBF2 that interact physically with EIN3/EIL transcription factors. EBF1 overexpression results in plants insensitive to ethylene. In contrast, plants carrying the ebf1 and ebf2 mutations display a constitutive ethylene response and accumulate the EIN3 protein in the absence of the hormone. Our work places EBF1 and EBF2 within the genetic framework of the ethylene-response pathway and supports a model in which ethylene action depends on EIN3 protein stabilization.

Being sessile organisms, plants need rapid and finely tuned signaling pathways to adapt their growth and survival over their immediate and often adverse environment. Abscisic acid (ABA) is a plant hormone crucial for both biotic and abiotic stress responses. In this study, we highlight a function of six Arabidopsis MATH-BTB proteins in ABA signaling. MATH-BTB proteins act as substrate-binding adaptors for the Cullin3-based ubiquitin E3 ligase. Our genetic and biochemical experiments demonstrate that the MATH-BTB proteins directly interact with and target for proteasomal degradation the class I homeobox-leucine zipper (HD-ZIP) transcription factor ATHB6, which was previously identified as a negative regulator of ABA responses. Reducing CUL3(BPM) function leads to higher ATHB6 protein accumulation, reducing plant growth and fertility, and affects stomatal behavior and responses to ABA. We further demonstrate that ABA negatively regulates ATHB6 protein turnover, a situation reminiscent to ABI5, another transcription factor involved in ABA signaling.

In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis. We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-␤ -glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems. Our observations support a conserved function of the APC/C in plants but a different mode of regulation.

The SCF (for SKP1, Cullin/CDC53, F-box protein) ubiquitin ligase targets a number of cell cycle regulators, transcription factors, and other proteins for degradation in yeast and mammalian cells. Recent genetic studies demonstrate that plant F-box proteins are involved in auxin responses, jasmonate signaling, flower morphogenesis, photocontrol of circadian clocks, and leaf senescence, implying a large spectrum of functions for the SCF pathway in plant development. Here, we present a molecular and functional characterization of plant cullins. The Arabidopsis genome contains 11 cullin-related genes. Complementation assays revealed that AtCUL1 but not AtCUL4 can functionally complement the yeast cdc53 mutant. Arabidopsis mutants containing transfer DNA (T-DNA) insertions in the AtCUL1 gene were shown to display an arrest in early embryogenesis. Consistently, both the transcript and the protein of the AtCUL1 gene were found to accumulate in embryos. The AtCUL1 protein localized mainly in the nucleus but also weakly in the cytoplasm during interphase and colocalized with the mitotic spindle in metaphase. Our results demonstrate a critical role for the SCF ubiquitin ligase in Arabidopsis embryogenesis.

As part of the goal to generate a detailed transcript map for Arabidopsis thaliana, 1152 single run sequences (expressed sequence tags or ESTs) have been determined from cDNA clones taken at random in libraries prepared from different sources of plant material: developing siliques, etiolated seedlings, flower buds, and cultured cells. Eight hundred and ninety-five different genes could be identified, 32% of which showed significant similarity to existing sequences in Arabidopsis and an array of other organisms. These sequences in combination with their positioning on the Arabidopsis genetic map will not only constitute a new set of molecular markers for genome analysis in Arabidopsis but also provide a direct route for the in vivo analysis of their gene products. The sequences have been made available to the public databases.

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase ( CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G 2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable. INTRODUCTIONThe cell division cycle is a sequential process that permits the replication of the genome, the segregation of chromosomes to two daughter nuclei, and finally cytokinesis. Progression through the cell cycle involves many proteins, which are regulated both at the transcriptional and posttranslational levels. Today, it has become clear that the timed destruction of key proteins plays an essential role in cell cycle progression. The first protein shown to be subjected to cell cycle-specific proteolysis was cyclin B (Evans et al., 1983). For many other proteins, cell cycle-specific proteolysis has been reported: among them are the G 1 cyclins and the mitotic cyclins, the cyclin-dependent kinase inhibitors (CKIs), proteins involved in sister chromatid separation, and spindle components (reviewed in Peter and Herskowitz, 1994;Murray, 1995; King et al., 1996; Hoyt, 1997). The degradation of cell cycle regulator proteins is not just a way to break down a protein whose function is achieved at a specific step of the cell cycle, but it is also a way to directly control the cell cycle. Thus, the highly regulated proteolysis of CKIs allows DNA replication to begin, and degradation of proteins involved in sister chromatid separation is required at the onset of anaphase.Proteins subjected to degradation are marked with ubiquitin tags and subsequently are targeted to the degradative action of the 26S proteasome. The ubiquitin/26S proteasome proteolytic pathway is highly conserved in eukaryotes and is involved in many other important cellular functions aside from cell cycle progression (reviewed in Hochstrasser, 1995). Deg...

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.