Yasushi Yoshioka

GIGAS CELL1, a Novel Negative Regulator of the Anaphase-Promoting Complex/Cyclosome, Is Required for Proper Mitotic Progression and Cell Fate Determination in Arabidopsis

A novel plant-specific inhibitor of anaphase-promoting complex/cyclosome was identified from the gigas cell1 (gig1) mutation, which causes polyploidization of somatic cells due to ectopic endomitosis. This mutation also generated mixed-fate cells with both characters of guard cells and pavement cells, suggesting that GIG1 may have a role in cell fate determination, in addition to its role in proper mitotic progression. Increased cellular ploidy is widespread during developmental processes of multicellular organisms, especially in plants. Elevated ploidy levels are typically achieved either by endoreplication or endomitosis, which are often regarded as modified cell cycles that lack an M phase either entirely or partially. We identified GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION1 (OSD1) and established that mutation of this gene triggered ectopic endomitosis. On the other hand, it has been reported that a paralog of GIG1/OSD1, UV-INSENSITIVE4 (UVI4), negatively regulates endoreplication onset in Arabidopsis thaliana. We showed that GIG1/OSD1 and UVI4 encode novel plant-specific inhibitors of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. These proteins physically interact with APC/C activators, CDC20/FZY and CDH1/FZR, in yeast two-hybrid assays. Overexpression of CDC20.1 and CCS52B/FZR3 differentially promoted ectopic endomitosis in gig1/osd1 and premature occurrence of endoreplication in uvi4. Our data suggest that GIG1/OSD1 and UVI4 may prevent an unscheduled increase in cellular ploidy by preferentially inhibiting APC/CCDC20 and APC/CFZR, respectively. Generation of cells with a mixed identity in gig1/osd1 further suggested that the APC/C may have an unexpected role for cell fate determination in addition to its role for proper mitotic progression.

Arabidopsis Rad51B is important for double-strand DNA breaks repair in somatic cells.

Rad51 paralogs belong to the Rad52 epistasis group of proteins and are involved in homologous recombination (HR), especially the assembly and stabilization of Rad51, which is a homolog of RecA in eukaryotes. We previously cloned and characterized two RAD51 paralogous genes in Arabidopsis, named AtRAD51C and AtXRCC3, which are considered the counterparts of human RAD51C and XRCC3, respectively. Here we describe the identification of RAD51B homologue in Arabidopsis, AtRAD51B. We found a higher expression of AtRAD51B in flower buds and roots. Expression of AtRAD51B was induced by genotoxic stresses such as ionizing irradiation and treatment with a cross-linking reagent, cisplatin. Yeast two-hybrid analysis showed that AtRad51B interacted with AtRad51C. We also found and characterized T-DNA insertion mutant lines. The mutant lines were devoid of AtRAD51B expression, viable and fertile. The mutants were moderately sensitive to γ-ray and hypersensitive to cisplatin. Our results suggest that AtRAD51B gene product is involved in the repair of double-strand DNA breaks (DSBs) via HR

Nucleotide sequence of the promoter-distal region of thetra operon of plasmid R100, includingtraI (DNA helicase I) andtraD genes

The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.

Plant Cells Without Detectable Plastids are Generated in the crumpled leaf Mutant of Arabidopsis thaliana

Plastids are maintained in cells by proliferating prior to cell division and being partitioned to each daughter cell during cell division. It is unclear, however, whether cells without plastids are generated when plastid division is suppressed. The crumpled leaf (crl) mutant of Arabidopsis thaliana is a plastid division mutant that displays severe abnormalities in plastid division and plant development. We show that the crl mutant contains cells lacking detectable plastids; this situation probably results from an unequal partitioning of plastids to each daughter cell. Our results suggest that crl has a partial defect in plastid expansion, which is suggested to be important in the partitioning of plastids to daughter cells when plastid division is suppressed. The absence of cells without detectable plastids in the accumulation and replication of chloroplasts 6 (arc6) mutant, another plastid division mutant of A. thaliana having no significant defects in plant morphology, suggests that the generation of cells without detectable plastids is one of the causes of the developmental abnormalities seen in crl plants. We also demonstrate that plastids with trace or undetectable amounts of chlorophyll are generated from enlarged plastids by a non-binary fission mode of plastid replication in both crl and arc6.

Visualization of Plastids in Pollen Grains: Involvement of FtsZ1 in Pollen Plastid Division

Visualizing organelles in living cells is a powerful method to analyze their intrinsic mechanisms. Easy observation of chlorophyll facilitates the study of the underlying mechanisms in chloroplasts, but not in other plastid types. Here, we constructed a transgenic plant enabling visualization of plastids in pollen grains. Combination of a plastid-targeted fluorescent protein with a pollen-specific promoter allowed us to observe the precise number, size and morphology of plastids in pollen grains of the wild type and the ftsZ1 mutant, whose responsible gene plays a central role in chloroplast division. The transgenic material presented in this work is useful for studying the division mechanism of pollen plastids.

Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

The constitutive stress response induced by chloroplast dysfunction causes early differentiation via the activation of cell cycle inhibitors. The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

Interaction between Agrobacterium tumefaciens oncoprotein 6b and a tobacco nucleolar protein that is homologous to TNP1 encoded by a transposable element of Antirrhinum majus

When gene 6b on the T-DNA of Agrobacterium tumefaciens is transferred to plant cells, its expression causes plant hormone-independent division of cells in in vitro culture and abnormal cell growth, which induces various morphological defects in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b localizes to the nuclei, a requirement for the abnormal cell growth, and binds to a tobacco nuclear protein called NtSIP1 and histone H3. In addition, 6b has histone chaperone-like activity in vitro and affects the expression of various plant genes, including cell division-related genes and meristem-related class 1 KNOX homeobox genes, in transgenic Arabidopsis. Here, we report that 6b binds to a newly identified protein NtSIP2, whose amino acid sequence is predicted to be 30% identical and 51% similar to that of the TNP1 protein encoded by the transposon Tam1 of Antirrhinum majus. Immunolocalization analysis using anti-T7 antibodies showed nucleolar localization of most of the T7 epitope-tagged NtSIP2 proteins. A similar analysis with the T7-tagged 6b protein also showed subnucleolar as well as nuclear localization of the 6b protein. These results suggest the involvement of 6b along with NtSIP2 in certain molecular processes in the nucleolus as well as the nucleoplasm.

EMBRYO YELLOW gene, encoding a subunit of the conserved oligomeric Golgi complex, is required for appropriate cell expansion and meristem organization in Arabidopsis thaliana

We identified an embryo yellow (eye) mutation in Arabidopsis that leads to the abnormal coloration and morphology of embryos. The eye mutant formed bushy plants, with aberrant organization of the shoot apical meristem (SAM) and unexpanded leaves with irregular phyllotaxy. The epidermal cells of the eye mutant were much smaller than that of the wild‐type. Thus, EYE is required for expansion of cells and organs, and for formation of the organized SAM. Hydrophobic layers of epidermal cells were also disrupted, suggesting that EYE might be involved in the generation of the extra‐cellular matrix. The mutated gene encoded a protein that is homologous to Cog7, a subunit of the conserved oligomeric Golgi (COG) complex, which is required for the normal morphology and function of the Golgi appratus. The eye mutation caused mislocalization of a Golgi protein. In addition, the size of the Golgi apparatus was also altered. Thus, EYE might be involved in transport or retention of Golgi‐localized proteins and in maintenance of Golgi morphology. We propose that some Golgi‐localized proteins, distributions of which are controlled by EYE, play important roles in expansion of cells and organs, and in formation of the properly organized SAM in plants.

Characterization of the gene products produced in minicells by pSM1, a derivative of R100

SummaryAt least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo. pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca. 90 kb) and carries the R100 essential replication region as well as some non-essential functions. Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten oberserved polypeptides to specific coding regions of pSM1. Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100. The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here. This sequence contains an open reading frame,orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as theorf4 gene product. Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region. Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells.