Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate.

Upon floral induction, the primary shoot meristem of an Arabidopsis plant begins to produce flower meristems rather than leaf primordia on its flanks. Assignment of floral fate to lateral meristems is primarily due to the cooperative activity of the flower meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER. We present evidence here that AP1 expression in lateral meristems is activated by at least two independent pathways, one of which is regulated by LFY. In lfy mutants, the onset of AP1 expression is delayed, indicating that LFY is formally a positive regulator of AP1. We have found that AP1, in turn, can positively regulate LFY, because LFY is expressed prematurely in the converted floral meristems of plants constitutively expressing AP1. Shoot meristems maintain an identity distinct from that of flower meristems, in part through the action of genes such as TERMINAL FLOWER1 (TFL1), which bars AP1 and LFY expression from the influorescence shoot meristem. We show here that this negative regulation can be mutual because TFL1 expression is downregulated in plants constitutively expressing AP1. Therefore, the normally sharp phase transition between the production of leaves with associated shoots and formation of the flowers, which occurs upon floral induction, is promoted by positive feedback interactions between LFY and AP1, together with negative interactions of these two genes with TFL1.     

The Arabidopsis MERISTEM DISORGANIZATION 1 gene is required for the maintenance of stem cells through the reduction of DNA damage

In plants, stem cells reside in apical meristems, and provide the descendants required for post-embryonic growth and development throughout the life of a plant. To identify a novel factor required for the maintenance of stem cells, we isolated an Arabidopsis mutant, named meristem disorganization 1-1 (mdo1-1), that exhibits several developmental defects, such as abnormal phyllotaxy and plastochron, stem fasciation and retarded root growth. We found that the mutant plants fail to maintain stem cells, resulting in the differentiation or death of stem cells. The mutant plants also showed several phenotypes related to DNA damage, suggesting that the mutant cells are exposed constitutively to DNA damage even without external genotoxic stress. The growth defect and the hypersensitivity to DNA-damaging agents of mdo1-1 were enhanced significantly when combined with a lesion of the ATAXIA-TELANGIECTASIA MUTATED (ATM) gene, but not of the ATM/RAD3-RELATED (ATR) gene, suggesting that the function of the MDO1 gene is closely related to that of ATM kinase. The MDO1 gene encodes an unknown protein that is conserved in a wide variety of land plants. The results thus suggested that the MDO1 gene product is required for the maintenance of stem cells through a reduction in DNA damage.     

A Link among DNA Replication, Recombination, and Gene Expression Revealed by Genetic and Genomic Analysis of TEBICHI Gene of Arabidopsis thaliana

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed.     

Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.     

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999     

Pak1 and PIX regulate contact inhibition during epithelial wound healing

Wound healing in epithelia requires coordinated cell migration and proliferation regulated by signaling mechanisms that are poorly understood. Here we show that epithelial cells expressing constitutively active or kinase-dead mutants of the Rac/Cdc42 effector Pak1 fail to undergo growth arrest upon wound closure. Strikingly, this phenotype is only observed when the Pak1 kinase mutants are expressed in cells possessing a free lateral surface, i.e. one that is not engaged in contact with neighboring cells. The Pak1 kinase mutants perturb contact inhibition by a mechanism that depends on the Pak-interacting Rac-GEF PIX. In control cells, endogenous activated Pak and PIX translocate from focal complexes to cell-cell contacts during wound closure. This process is abrogated in cells expressing Pak1 kinase mutants. In contrast, Pak1 mutants rendered defective in PIX binding do not impede translocation of activated Pak and PIX, and exhibit normal wound healing. Thus, recruitment of activated Pak and PIX to cell-cell contacts is pivotal to transduction of growth-inhibitory signals from neighboring cells in epithelial wound healing.     

Histochemical analysis of root meristem activity in Arabidopsis thaliana using a cyclin:GUS (β-glucuronidase) marker line

We used a transgenic Arabidopsis line expressing a translational fusion between a mitotic cyclin and the reporter gene -glucuronidase (GUS) to investigate cell divisions in postembryonic root meristems. The fusion protein contains the cyclin destruction box (CDB) and this leads to a rapid degradation of the chimeric GUS-protein after mitosis. Hence, the staining pattern of the meristem marks dividing cells. We observed that upon germination the first cell divisions occur in epidermis cells at the junction with the hypocotyl. Moreover, the accelerated root growth on media supplemented with sucrose correlates with an increased number of dividing cells and an enlargement of the root meristematic zone. The conditional root expansion mutants pom pom1 and procuste1 (quill) suppress this sugar effect leading to a smaller meristematic zone. Simultaneous visualisation of the nucleus revealed that the CYCAT1:CDB:GUS expression is subcellularly localised around the nucleus. This particular staining starts at prophase and disappears after the completion of the new cell wall. In metaphase the staining invades the cytoplasm whereas in the telophase it concentrates again around the nucleus. This cell cycle-dependent distribution was used to characterise the two root specific cytokinesis mutants pleiade1 and hyade1. In both mutants, cells which fail to develop a complete cell wall during cytokinesis divide synchronously in further cell divisions leading to multinucleate cells. These experiments demonstrate the usefulness of the CYCAT1:CDB:GUS marker line for studying cell division of wild-type and mutants. Furthermore, this line can be used to analyse the influence of biotic and abiotic signals on the rate and spatial distribution of cell divisions.     

Shade avoidance 6 encodes an Arabidopsis flap endonuclease required for maintenance of genome integrity and development

Flap endonuclease-1 (FEN1) belongs to the Rad2 family of structure-specific nucleases. It is required for several DNA metabolic pathways, including DNA replication and DNA damage repair. Here, we have identified a shade avoidance mutant, sav6, which reduces the mRNA splicing efficiency of SAV6. We have demonstrated that SAV6 is an FEN1 homologue that shows double-flap endonuclease and gap-dependent endonuclease activity, but lacks exonuclease activity. sav6 mutants are hypersensitive to DNA damage induced by ultraviolet (UV)-C radiation and reagents that induce double-stranded DNA breaks, but exhibit normal responses to chemicals that block DNA replication. Signalling components that respond to DNA damage are constitutively activated in sav6 mutants. These data indicate that SAV6 is required for DNA damage repair and the maintenance of genome integrity. Mutant sav6 plants also show reduced root apical meristem (RAM) size and defective quiescent centre (QC) development. The expression of SMR7, a cell cycle regulatory gene, and ERF115 and PSK5, regulators of QC division, is increased in sav6 mutants. Their constitutive induction is likely due to the elevated DNA damage responses in sav6 and may lead to defects in the development of the RAM and QC. Therefore, SAV6 assures proper root development through maintenance of genome integrity.     

In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis

The aerial parts of the plant are generated by groups of rapidly dividing cells called shoot apical meristems. To analyze cell behavior in these structures, we developed a technique to visualize living shoot apical meristems using the confocal microscope. This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerizing microtubules) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems. Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation were inhibited, suggesting an important role for DNA synthesis in growth and patterning.     

Dual role of CDKs in DNA repair: to be, or not to be

The maintenance of genome integrity is essential for the regulation of cell proliferation and differentiation. DNA must be accurately duplicated and segregated to daughter cells at cell division, a process that is primarily regulated by cyclin-dependent kinases (CDKs). During cell growth, however, it is inevitable that DNA breaks will occur due to endogenous and exogenous stresses. Interestingly, there is increasing evidence that the catalytic activities of CDKs play critical roles in the DNA damage response, especially in the case of damage repaired by the homologous recombination (HR) pathway. In this review, we outline current knowledge of CDK regulation and its roles both in the unperturbed cell cycle and in DNA damage responses, and discuss the physiological roles of CDKs in HR repair.     

Expression of fasciation mutation in apical meristems of soybean, Glycine max (Leguminosae)

The phenotype of the apical meristem was used to examine the effect of fasciation mutation at the f locus in different genetic backgrounds in soybean Glycine max (L.) Merr. Comparisons of meristem development in fasciation mutant and wild type were conducted with scanning electron microscope (SEM) on isogenic lines BARC-11-11-ff and BARC-11-11-FF at postgermination and early vegetative stages. Studies of apical meristems of three independently originated fasciation mutants, PI 83945-4, PI 243541, and T173, were carried out at vegetative and early floral transition stages. Corolla Fasciation, the extreme mutant phenotype, was used for comparison of meristem development. Enlargement of the apical meristem and shortened plastochron were observed in the mutant lines 2 d after germination. Similar to Corolla Fasciation, in PI 83945-4, PI 243541, and T173, enlargement of the apical meristem was followed by growth along one axis at the V3 stage and establishment of a ridge-like meristem at the V4 stage. Influence of pedigree on the expression of the fasciation phenotype was demonstrated by different growth patterns (subangular vs. ridge-like) of the apical meristem in BARC-11-11-ff and PI 243541 with the same f gene. During transition of the apical meristem from vegetative to reproductive stage in all mutant lines further production of leaf primordia ceased. The developmental pattern of the apical meristems suggests that the f locus may have the same allele in fasciation mutants of independent origin in soybean.     

TIF1 activates the intra-S-phase checkpoint response in the diploid micronucleus and amitotic polyploid macronucleus of Tetrahymena

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.     

Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development

Chromatin assembly factor CAF-1 facilitates the formation of nucleosomes on newly replicated DNA in vitro. However, the role of CAF-1 in development is poorly understood because mutants are not available in most multicellular model organisms. Biochemical evidence suggests that FASCIATA1, FASCIATA2 and MSI1 form CAF-1 in Arabidopsis thaliana. Because fasciata mutants are viable, CAF-1 is not essential for cell division in plants. Arabidopsis CAF-1 mutants have defects in shoot apical meristems; in addition, CAF-1 is required to establish seedling architecture, leaf size and trichome differentiation. CAF-1 is needed to restrict branching of trichomes on rosette leaves. Increased trichome branching in CAF-1 mutants is not strictly correlated with increased nuclear DNA content. In addition, fas2 glabra3 double mutants show an additive genetic interaction, demonstrating that CAF-1 acts genetically parallel to the GLABRA3-containing, endoreduplication-coupled trichome branching pathway. However, CAF-1 is often needed to restrict endoreduplication, because seedlings of most CAF-1 mutants have increased ploidy. Notably, in the Landsberg erecta background, loss of CAF-1 does not affect ploidy, demonstrating that loss of CAF-1 can be compensated in some Arabidopsis accessions. These results reveal that the functions of FAS1, FAS2 and MSI1 are not restricted to meristems, but are also needed to control genome replication at multiple steps of development.     

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. Key message We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.     

ROR1/RPA2A, a putative replication protein A2, functions in epigenetic gene silencing and in regulation of meristem development in Arabidopsis

We screened for suppressors of repressor of silencing1 (ros1) using the silenced 35S promoter-neomycin phosphotransferase II (Pro(35S):NPTII) gene as a marker and identified two allelic mutants, ror1-1 and ror1-2 (for suppressor of ros1). Map-based cloning revealed that ROR1 encodes a 31-kD protein similar to DNA replication protein A2 (RPA2A). Mutations in ROR1 reactivate the silenced Pro(35S):NPTII gene but not RD29A promoter-luciferase in the ros1 mutant. DNA methylation in rDNA, centromeric DNA, and RD29A promoter regions is not affected by ror1. However, chromatin immunoprecipitation data suggest that histone H3 acetylation is increased and histone H3K9 dimethylation is decreased in the 35S promoter in the ror1 ros1 mutant compared with ros1. These results indicate that release of silenced Pro(35S):NPTII by ror1 mutations is independent of DNA methylation. ROR1/RPA2A is strongly expressed in shoot and root meristems. Mutations in ROR1/RPA2A affect cell division in meristems but not final cell sizes. Our work suggests important roles of ROR1/RPA2A in epigenetic gene silencing and in the regulation of plant development.     

Regulation of meristem organization and cell division by TSO1, an Arabidopsis gene with cysteine-rich repeats

In higher plants, meristem organization and cell division regulation are two fundamentally important and intimately related biological processes. Identifying and isolating regulatory genes in these processes is essential for understanding higher plant growth and development. We describe the molecular isolation and analyses of an Arabidopsis gene, TSO1, which regulates both of these processes. We previously showed that tso1 mutants displayed defects in cell division of floral meristem cells including partially formed cell walls, increased DNA content, and multinucleated cells (Liu, Z., Running, M. P. and Meyerowitz, E. M. (1997). Development 124, 665-672). Here, we characterize a second defect of tso1 in influorescence meristem development and show that the enlarged influorescence in tso1 mutants results from repeated division of one inflorescence meristem into two or more influorescence meristems. Using a map-based approach, we isolated the TSO1 gene and found that TSO1 encodes a protein with cysteine-rich repeats bearing similarity to Drosophila Enhancer of zeste and its plant homologs. In situ TSO1 mRNA expression pattern and the nuclear localization of TSO1-GFP are consistent with a regulatory role of TSO1 in floral meristem cell division and in influorescence meristem organization.     

Dominant-negative cAMP-responsive element-binding protein inhibits proliferating cell nuclear antigen and DNA repair,leading to increased cellular radiosensitivity

Selective inhibition of the epidermal growth factor receptor or mitogen-activated protein kinase (MAPK) results in radiosensitization of cancer cells. One potential mechanism involves cAMP-responsive element-binding protein, which is activated by radiation via the epidermal growth factor receptor/MAPK pathway and which regulates synthesis of proliferating cell nuclear antigen (PCNA), a protein involved in repair of ionizing radiation-induced DNA damage. To test for a role of CREB in cellular radiosensitivity, CHO cells were transfected with plasmids expressing dominant-negative CREB mutants (CR133 or KCREB), and various end-points were measured 48 h later. Basal levels of PCNA-CAT reporter construct activity were reduced by 60 and 40% following expression of CR133 and KCREB, respectively; similar decreases were observed in PCNA protein levels. Pulsed-field gel electrophoresis measurements showed that CR133 inhibited the repair of radiation-induced DNA double-strand breaks, and this effect was reversed by over-expression of PCNA; dominant-negative CREB also significantly inhibited split-dose recovery. Clonogenic assays were used to determine surviving fraction; the dose enhancement ratios for dominant-negative CREB-expressing cells compared with control (vector alone) were 1.5 and 1.3 for CR133 and KCREB, respectively. Importantly, co-transfection of mutant CREB and a construct constitutively expressing PCNA protein restored radiosensitivity of CHO cells back to wild-type levels. Moreover, cells expressing either CREB mutant showed no significant cell cycle redistribution. These data demonstrate that genetic disruption of CREB results in radiosensitization, and that this effect can be explained by a mechanism involving decreased PCNA expression and inhibition of DNA repair.