Search

Aluminum‐dependent stoppage of root growth requires the DNA damage response (DDR) pathway including the p53‐like transcription factor SUPPRESSOR OF GAMMA RADIATION 1 (SOG1), which promotes terminal differentiation of the root tip in response to Al dependent cell death. Transcriptomic analyses identified Al‐induced SOG1‐regulated targets as candidate mediators of this growth arrest. Analysis of these factors either as loss‐of‐function mutants or by overexpression in the als3‐1 background shows ERF115, which is a key transcription factor that in other scenarios is rate‐limiting for damaged stem cell replenishment, instead participates in transition from an actively growing root to one that has terminally differentiated in response to Al toxicity. This is supported by a loss‐of‐function erf115 mutant raising the threshold of Al required to promote terminal differentiation of Al hypersensitive als3‐1. Consistent with its key role in stoppage of root growth, a putative ERF115 barley ortholog is also upregulated following Al exposure, suggesting a conserved role for this ATR‐dependent pathway in Al response. In contrast to other DNA damage agents, these results show that ERF115 and likely related family members are important determinants of terminal differentiation of the root tip following Al exposure and central outputs of the SOG1‐mediated pathway in Al response. Summary Statement: Aluminum dependent stoppage of root growth in Arabidopsis thaliana requires a SUPPRESSOR OF GAMMA RADIATION 1 controlled transcriptional program that includes ERF115 and family members. Loss of ERF114 and ERF115 results in prevention of the severe response to Al normally seen in the Al hyperresponsive mutant als3‐1. [ABSTRACT FROM AUTHOR]

Summary: The establishment of root‐knot nematode (RKN; Meloidogyne spp.) induced galls in the plant host roots likely involves a wound‐induced regeneration response. Confocal imaging demonstrates physical stress or injury caused by RKN infection during parasitism in the model host Arabidopsis thaliana.The ERF115‐PAT1 heterodimeric transcription factor complex plays a recognized role in wound‐induced regeneration. ERF115 and PAT1 expression flanks injured gall cells likely driving mechanisms of wound healing, implying a local reactivation of cell division which is also hypothetically involved in gall genesis.Herein, functional investigation revealed that ectopic ERF115 expression resulted in premature induction of galls, and callus formation adjacent to the expanding female RKN was seen upon PAT1 upregulation. Smaller galls and less reproduction were observed in ERF115 and PAT1 knockouts. Investigation of components in the ERF115 network upon overexpression and knockdown by qRT‐PCR suggests it contributes to steer gall wound‐sensing and subsequent competence for tissue regeneration. High expression of CYCD6;1 was detected in galls, and WIND1 overexpression resulted in similar ERF115OE gall phenotypes, also showing faster gall induction.Along these lines, we show that the ERF115‐PAT1 complex likely coordinates stress signalling with tissue healing, keeping the gall functional until maturation and nematode reproduction. [ABSTRACT FROM AUTHOR]

SUMMARY: The genomic integrity of every organism is endangered by various intrinsic and extrinsic stresses. To maintain genomic integrity, a sophisticated DNA damage response (DDR) network is activated rapidly after DNA damage. Notably, the fundamental DDR mechanisms are conserved in eukaryotes. However, knowledge about many regulatory aspects of the plant DDR is still limited. Important, yet little understood, regulatory factors of the DDR are the long non‐coding RNAs (lncRNAs). In humans, 13 lncRNAs functioning in DDR have been characterized to date, whereas no such lncRNAs have been characterized in plants yet. By meta‐analysis, we identified the putative long intergenic non‐coding RNA induced by DNA damage (LINDA) that responds strongly to various DNA double‐strand break‐inducing treatments, but not to replication stress induced by mitomycin C. After DNA damage, LINDA is rapidly induced in an ATM‐ and SOG1‐dependent manner. Intriguingly, the transcriptional response of LINDA to DNA damage is similar to that of its flanking hypothetical protein‐encoding gene. Phylogenetic analysis of putative Brassicales and Malvales LINDA homologs indicates that LINDA lncRNAs originate from duplication of a flanking small protein‐encoding gene followed by pseudogenization. We demonstrate that LINDA is not only needed for the regulation of this flanking gene but also fine‐tuning of the DDR after the occurrence of DNA double‐strand breaks. Moreover, Δlinda mutant root stem cells are unable to recover from DNA damage, most likely due to hyper‐induced cell death. Significant Statement: We unraveled the functional relevance of the first putative lncRNA within the DNA damage response network of Arabidopsis thaliana. This putative lncRNA, termed LINDA, is an important part of the DNA damage response network, as it is needed for accurate regulation of cell death and cell cycle progression. [ABSTRACT FROM AUTHOR]

Summary: The processes that contribute to plant organ morphogenesis are spatial–temporally organized. Within the meristem, mitosis produces new cells that subsequently engage in cell expansion and differentiation programs. The latter is frequently accompanied by endoreplication, being an alternative cell cycle that replicates the DNA without nuclear division, causing a stepwise increase in somatic ploidy.Here, we show that the Arabidopsis SCL28 transcription factor promotes organ growth by modulating cell expansion dynamics in both root and leaf cells.Gene expression studies indicated that SCL28 regulates members of the SIAMESE/SIAMESE‐RELATED (SIM/SMR) family, encoding cyclin‐dependent kinase inhibitors with a role in promoting mitotic cell cycle (MCC) exit and endoreplication, both in response to developmental and environmental cues. Consistent with this role, mutants in SCL28 displayed reduced endoreplication, both in roots and leaves. We also found evidence indicating that SCL28 co‐expresses with and regulates genes related to the biogenesis, assembly, and remodeling of the cytoskeleton and cell wall.Our results suggest that SCL28 controls, not only cell proliferation as reported previously but also cell expansion and differentiation by promoting MCC exit and endoreplication and by modulating aspects of the biogenesis, assembly, and remodeling of the cytoskeleton and cell wall. [ABSTRACT FROM AUTHOR]

SUMMARY: Being sessile organisms, plants are ubiquitously exposed to stresses that can affect the DNA replication process or cause DNA damage. To cope with these problems, plants utilize DNA damage response (DDR) pathways, consisting of both highly conserved and plant‐specific elements. As a part of this DDR, cell cycle checkpoint control mechanisms either pause the cell cycle, to allow DNA repair, or lead cells into differentiation or programmed cell death, to prevent the transmission of DNA errors in the organism through mitosis or to its offspring via meiosis. The two major DDR cell cycle checkpoints control either the replication process or the G2/M transition. The latter is largely overseen by the plant‐specific SOG1 transcription factor, which drives the activity of cyclin‐dependent kinase inhibitors and MYB3R proteins, which are rate limiting for the G2/M transition. By contrast, the replication checkpoint is controlled by different players, including the conserved kinase WEE1 and likely the transcriptional repressor RBR1. These checkpoint mechanisms are called upon during developmental processes, in retrograde signaling pathways, and in response to biotic and abiotic stresses, including metal toxicity, cold, salinity, and phosphate deficiency. Additionally, the recent expansion of research from Arabidopsis to other model plants has revealed species‐specific aspects of the DDR. Overall, it is becoming evidently clear that the DNA damage checkpoint mechanisms represent an important aspect of the adaptation of plants to a changing environment, hence gaining more knowledge about this topic might be helpful to increase the resilience of plants to climate change. Significance Statement: To cope with stresses that might impede the replication process or cause DNA breaks, plants utilize DNA damage‐responsive cell cycle checkpoint mechanisms that help them adapt to the stress while maintaining genome integrity. Understanding these mechanisms might be pivotal to increase the resilience of plants to climate change. [ABSTRACT FROM AUTHOR]

SUMMARY: Coastal regions contribute an estimated 20% of annual gross primary production in the oceans, despite occupying only 0.03% of their surface area. Diatoms frequently dominate coastal sediments, where they experience large variations in light regime resulting from the interplay of diurnal and tidal cycles. Here, we report on an extensive diurnal transcript profiling experiment of the motile benthic diatom Seminavisrobusta. Nearly 90% (23 328) of expressed protein‐coding genes and 66.9% (1124) of expressed long intergenic non‐coding RNAs showed significant expression oscillations and are predominantly phasing at night with a periodicity of 24 h. Phylostratigraphic analysis found that rhythmic genes are enriched in highly conserved genes, while diatom‐specific genes are predominantly associated with midnight expression. Integration of genetic and physiological cell cycle markers with silica depletion data revealed potential new silica cell wall‐associated gene families specific to diatoms. Additionally, we observed 1752 genes with a remarkable semidiurnal (12‐h) periodicity, while the expansion of putative circadian transcription factors may reflect adaptations to cope with highly unpredictable external conditions. Taken together, our results provide new insights into the adaptations of diatoms to the benthic environment and serve as a valuable resource for the study of diurnal regulation in photosynthetic eukaryotes. [ABSTRACT FROM AUTHOR]

Casein kinase 2 (CK2) is a ubiquitous pleiotropic enzyme that is highly conserved across eukaryotic kingdoms. CK2 is singular amongst kinases as it is highly rigid and constitutively active. Arabidopsis thaliana is widely used as a model system in molecular plant research; the biological functions of A. thaliana CK2 are well studied in vivo and many of its substrates have been identified. Here, crystal structures of the α subunit of A. thaliana CK2 in three crystal forms and of its complex with the nonhydrolyzable ATP analog AMppNHp are presented. While the C‐lobe of the enzyme is highly rigid, structural plasticity is observed for the N‐lobe. Small but significant displacements within the active cleft are necessary in order to avoid steric clashes with the AMppNHp molecule. Binding of AMppNHp is influenced by a rigid‐body motion of the N‐lobe that was not previously recognized in maize CK2. [ABSTRACT FROM AUTHOR]

Summary: Galls induced by plant‐parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident.We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode‐induced galls.Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1‐knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls.Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1‐knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root‐knot nematodes. Together with data on DNA‐damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls. [ABSTRACT FROM AUTHOR]

Summary: Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development.We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle.Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large‐leaf mutants.Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general. [ABSTRACT FROM AUTHOR]

Cell cycle control in galls provoked by root-knot nematodes involves the activity of inhibitor genes like the Arabidopsis ICK/KRP members. Ectopic KRP1, KRP2 and KRP4 expression resulted in decreased gall size by inhibiting mitotic activity, whereas KRP6 induces mitosis in galls. Herein, we investigate the role of KRP3, KRP5 and KRP7 during gall development and compared their role with previously studied members of this class of cell cycle inhibitors. Overexpression of KRP3 and KRP7 culminated in undersized giant cells, with KRP3OE galls presenting peculiar elongated giant cells. Nuclei in KRP3OE and KRP5OE lines presented a convoluted and apparently connected phenotype. This appearance may be associated with the punctuated protein nuclear localization driven by specific common motifs. As well, ectopic expression of KRP3OE and KRP5OE affected nematode development and offspring. Decreased mitotic activity in galls of KRP3OE and KRP7OE lines led to a reduced gall size which presented distinct shapes - from more elongated like in the KRP3OE line to small rounded like in the KRP7OE line. Results presented strongly support the idea that induced expression of cell cycle inhibitors such as KRP3 and KRP7 in galls can be envisaged as a conceivable strategy for nematode feeding site control in crop species attacked by phytopathogenic nematodes. [ABSTRACT FROM AUTHOR]

• The mitotic checkpoint (MC) guards faithful sister chromatid segregation by monitoring the attachment of spindle microtubules to the kinetochores. When chromosome attachment errors are detected, MC delays the metaphase-to-anaphase transition through the inhibition of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. In contrast to yeast and mammals, our knowledge on the proteins involved in MC in plants is scarce. • Transient synchronization of root tips as well as promoter-reporter gene fusions were performed to analyze temporal and spatial expression of COPPER MODIFIED RESISTANCE1/PATRONUS1 (CMR1/PANS1) in developing Arabidopsis thaliana seedlings. Functional analysis of the gene was carried out, including CYCB1;2 stability in CMR1/PANS1 knockout and overexpressor background as well as metaphase-anaphase chromosome status. • CMR1/PANS1 is transcriptionally active during M phase. Its deficiency provokes premature cell cycle exit and in consequence a rapid consumption of the number of meristematic cells in particular under stress conditions that are known to affect spindle microtubules. Root growth impairment is correlated with a failure to delay the onset of anaphase, resulting in anaphase bridges and chromosome missegregation. CMR1/PANS1 overexpression stabilizes the mitotic CYCB1;2 protein. • Likely, CMR1/PANS1 coordinates mitotic cell cycle progression by acting as an APC/C inhibitor and plays a key role in growth adaptation to stress. [ABSTRACT FROM AUTHOR]

The establishment of galls and syncytia as feeding sites induced by root-knot and cyst nematodes, respectively, involves a progressive increase in nuclear and cellular size. Here we describe the functional characterization of endocycle activators CCS52A, CCS52B and a repressor of the endocycle, DEL1, during two types of nematode feeding site development in Arabidopsis thaliana. In situ hybridization analysis showed that expression of CCS52A1 and CCS52B was strongly induced in galls and syncytia and DEL1 was stably but weakly expressed throughout feeding site development. Down-regulation and over-expression of CCS52 and DEL1 in Arabidopsis drastically affected giant cell and syncytium growth, resulting in restrained nematode development, illustrating the need for mitotic activity and endo-reduplication for feeding site maturation. Exploiting the mechanism of endo-reduplication may be envisaged as a strategy to control plant-parasitic nematodes. [ABSTRACT FROM AUTHOR]

Post-embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB-RBR1 (retinoblastoma-related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FA?RB) lacking the RBR1-binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FA?RB and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways. [ABSTRACT FROM AUTHOR]

Summary To elucidate the epigenetic maintenance mechanism for functional plant centromeres, we studied transcriptional regulation of the centromere-specific histone H3 variant CENH3 in Arabidopsis thaliana. We focused on the structure and activity of the CENH3 promoter ( CENH3pro) and its regulation by E2F transcription factors. Use of CENH3pro::GUS reporter gene constructs showed that CENH3pro is active in dividing tissues, and that full expression in root meristems depends on intragenic regulatory elements within the second intron. Chromatin immunoprecipitation identified CENH3 as an E2F target gene. Transient co-expression of a CENH3pro::GUS reporter gene construct with various E2F transcription factors in A. thaliana protoplasts showed that E2Fa and E2Fb (preferentially with dimerization protein DPb) activate CENH3pro. Stable over-expression of E2Fa and E2Fb increased the CENH3 transcript level in planta, whereas over-expression of E2Fc decreased the CENH3 transcript level. Surprisingly, mutation of the two E2F binding sites of CENH3pro, in particular the more upstream one (E2F2), caused an increase in CENH3pro activity, indicating E2F-dependent transcriptional repression. CENH3pro repression may be triggered by the interplay of typical and atypical E2Fs in a cell cycle-dependent manner, and/or by interaction of typical E2Fs with retinoblastoma-related (RBR) protein. We speculate that E2Fs are involved in differential transcriptional regulation of CENH3 versus H3, as H3 promoters lack E2F binding motifs. E2F binding motifs are also present in human and Drosophila CENH3pro regions, thus cell cycle-dependent transcriptional regulation of CENH3 may be highly conserved. [ABSTRACT FROM AUTHOR]

In multicellular organisms, morphogenesis relies on a strict coordination in time and space of cell proliferation and differentiation. In contrast to animals, plant development displays continuous organ formation and adaptive growth responses during their lifespan relying on a tight coordination of cell proliferation. How developmental signals interact with the plant cell-cycle machinery is largely unknown. Here, we characterize plant A2-type cyclins, a small gene family of mitotic cyclins, and show how they contribute to the fine-tuning of local proliferation during plant development. Moreover, the timely repression of CYCA2;3 expression in newly formed guard cells is shown to require the stomatal transcription factors FOUR LIPS/MYB124 and MYB88, providing a direct link between developmental programming and cell-cycle exit in plants. Thus, transcriptional downregulation of CYCA2s represents a critical mechanism to coordinate proliferation during plant development. [ABSTRACT FROM AUTHOR]

Because of their sessile life style, plants have evolved the ability to adjust to environmentally harsh conditions. An important aspect of stress adaptation involves the reprogramming of the cell cycle to ensure optimal growth. The atypical E2F transcription factor DP-E2F-like 1 (E2Fe/DEL1) had been found previously to be an important regulator of the endocycle onset. Here, a novel role for E2Fe/DEL1 was identified as a transcriptional repressor of the type-II cyclobutane pyrimidine dimer-photolyase DNA repair gene PHR1. Upon ultraviolet-B (UV-B) treatment, plants knocked out for E2Fe/DEL1 had improved DNA repair abilities when compared with control plants, whereas those overexpressing it performed less well. Better DNA repair allowed E2Fe/DEL1 knockout plants to resume endoreduplication faster than control plants, contributing in this manner to UV-B radiation resistance by compensating the stress-induced reduction in cell number by ploidy-dependent cell growth. As E2Fe/DEL1 levels decreased upon UV-B treatment, we hypothesize that the coordinated transcriptional induction of PHR1 with the endoreduplication onset contributes to the adaptation of plants exposed to UV-B stress. [ABSTRACT FROM AUTHOR]

Synchronized cell cultures are an indispensable tool for the identification and understanding of key regulators of the cell cycle. Nevertheless, the use of cell cultures has its disadvantages, because it represents an artificial system that does not completely mimic the endogenous conditions that occur in organized meristems. Here, we present a new and easy method for Arabidopsis thaliana root tip synchronization by hydroxyurea treatment. A major advantage of the method is the possibility of investigating available Arabidopsis cell-cycle mutants without the need to generate cell cultures. As a proof of concept, the effects of over-expression of a dominant negative allele of the B-type cyclin-dependent kinase CDKB1;1 gene on cell-cycle progression were tested. The previously observed prolonged G phase was confirmed, but was found to be compensated for by a reduced G phase. Furthermore, altered S-phase kinetics indicated a functional role for CDKB1;1 during the replication process. [ABSTRACT FROM AUTHOR]

Summary As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration between cell division rates and fate specification is essential for normal organ formation and plant development. However, the inter-dependence of cell-cycle machinery and meristem-organizing genes is still poorly understood. To investigate this mechanism, we specifically inhibited the cell-cycle machinery in the shoot apex by expression of a dominant negative allele of the A-type cyclin-dependent kinase (CDK) CDKA;1 in meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven into the differentiation pathway, as indicated by premature cell expansion and onset of endo-reduplication. Although the meristem structure and expression patterns of the meristem identity genes were maintained in most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a threshold model in which the effect of the cell-cycle machinery on meristem organization is determined by the level of CDK activity. [ABSTRACT FROM AUTHOR]

The steady-state distribution of cell-cycle transcripts in Arabidopsis thaliana seedlings was studied in a broad in situ survey to provide a better understanding of the expression of cell-cycle genes during plant development. The 61 core cell-cycle genes analyzed were expressed at variable levels throughout the different plant tissues: 23 genes generally in dividing and young differentiating tissues, 34 genes mostly in both dividing and differentiated tissues and four gene transcripts primarily in differentiated tissues. Only 21 genes had a typical patchy expression pattern, indicating tight cell-cycle regulation. The increased expression of 27 cell-cycle genes in the root elongation zone hinted at their involvement in the switch from cell division to differentiation. The induction of 20 cell-cycle genes in differentiated cortical cells of etiolated hypocotyls pointed to their possible role in the process of endoreduplication. Of seven cyclin-dependent kinase inhibitor genes, five were upregulated in etiolated hypocotyls, suggesting a role in cell-cycle arrest. Nineteen genes were preferentially expressed in pericycle cells activated by auxin that give rise to lateral root primordia. Approximately 1800 images have been collected and can be queried via an online database. Our in situ analysis revealed that 70% of the cell-cycle genes, although expressed at different levels, show a large overlap in their localization. The lack of regulatory motifs in the upstream regions of the analyzed genes suggests the absence of a universal transcriptional control mechanism for all cell-cycle genes. [ABSTRACT FROM AUTHOR]

The Affymetrix ATH1 array provides a robust standard tool for transcriptome analysis, but unfortunately does not represent all of the transcribed genes in Arabidopsis thaliana. Recently, Affymetrix has introduced its Arabidopsis Tiling 1.0R array, which offers whole-genome coverage of the sequenced Col-0 reference strain. Here, we present an approach to exploit this platform for quantitative mRNA expression analysis, and compare the results with those obtained using ATH1 arrays. We also propose a method for selecting unique tiling probes for each annotated gene or transcript in the most current genome annotation, TAIR7, generating Chip Definition Files for the Tiling 1.0R array. As a test case, we compared the transcriptome of wild-type plants with that of transgenic plants overproducing the heterodimeric E2Fa-DPa transcription factor. We show that with the appropriate data pre-processing, the estimated changes per gene for those with significantly different expression levels is very similar for the two array types. With the tiling arrays we could identify 368 new E2F-regulated genes, with a large fraction including an E2F motif in the promoter. The latter groups increase the number of excellent candidates for new, direct E2F targets by almost twofold, from 181 to 334. [ABSTRACT FROM AUTHOR]

In contrast to animal growth, plant growth is largely post-embryonic. Therefore plants have developed new mechanisms to precisely regulate cell proliferation by means of internal and external stimuli whilst the general core cell cycle machinery is conserved between eukaryotes. In this work we demonstrate a role for the Arabidopsis thaliana DNA-binding-with-one-finger (DOF) transcription factor OBP1 in the control of cell division upon developmental signalling. Inducible overexpression of OBP1 resulted in a significant overrepresentation of cell cycle genes among the upregulated transcripts. Direct targets of OBP1, as verified by chromatin immunoprecipitation, include at least the core cell cycle gene CYCD3;3 and the replication-specific transcription factor gene AtDOF2;3. Consistent with our molecular data, short-term activation of OBP1 in cell cultures affected cell cycle re-entry, shortening the duration of the G1 phase and the overall length of the cell cycle, whilst constitutive overexpression of OBP1 in plants influenced cell size and cell number, leading to a dwarfish phenotype. Expression during embryogenesis, germination and lateral root initiation suggests an important role for OBP1 in cell cycle re-entry, operating as a transcriptional regulator of key cell cycle genes. Our findings provide significant input into our understanding of how cell cycle activity is incorporated into plant growth and development. [ABSTRACT FROM AUTHOR]

In multicellular organisms, organogenesis requires a tight control of the balance between cell division and cell differentiation. Distinct signalling pathways that connect both cellular processes with developmental cues might have evolved to suit different developmental plans. Here, we identified and characterized a novel protein that interacts with pre-replication complex (pre-RC) subunits, designated Armadillo BTB Arabidopsis protein 1 (ABAP1). Overexpression of ABAP1 in plants limited mitotic DNA replication and decreased cell proliferation in leaves, whereas ABAP1 downregulation increased cell division rates. Activity of ABAP1 in transcription was supported by its association with the transcription factor AtTCP24. The ABAP1–AtTCP24 complex bound specifically to the promoters of AtCDT1a and AtCDT1b in vitro and in vivo. Moreover, expression levels of AtCDT1a and AtCDT1b were reduced in ABAP1-overexpressing plants and they were increased in plants with reduced levels of ABAP1. We propose that ABAP1 participates in a negative feedback loop regulating mitotic DNA replication during leaf development, either by repressing transcription of pre-RC genes and possibly by regulating pre-RC utilization through direct association with pre-RC components. [ABSTRACT FROM AUTHOR]

Complete and accurate chromosomal DNA replication is essential for the maintenance of the genetic integrity of all organisms. Errors in replication are buffered by the activation of DNA stress checkpoints; however, in plants, the relative importance of a coordinated induction of DNA repair and cell cycle-arresting genes in the survival of replication mutants is unknown. In a systematic screen for Arabidopsis thaliana E2F target genes, the E2F TARGET GENE 1 (ETG1) was identified as a novel evolutionarily conserved replisome factor. ETG1 was associated with the minichromosome maintenance complex and was crucial for efficient DNA replication. Plants lacking the ETG1 gene had serrated leaves due to cell cycle inhibition triggered by the DNA replication checkpoints, as shown by the transcriptional induction of DNA stress checkpoint genes. The importance of checkpoint activation was highlighted by double mutant analysis: whereas etg1 mutant plants developed relatively normally, a synthetically lethal interaction was observed between etg1 and the checkpoint mutants wee1 and atr, demonstrating that activation of a G2 cell cycle checkpoint accounts for survival of ETG1-deficient plants. [ABSTRACT FROM AUTHOR]

In plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest. Inducible expression systems have been reported, but many of them show unwanted leaky expression. To circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the CRE- loxP site-specific recombination system of bacteriophage P1 and the subcellular targeting of proteins by a mammalian glucocorticoid receptor (GR). By fusing the receptor domain of the rat GR to the carboxyl terminus of the CRE recombinase, a double-lock conditional transcriptional induction system was created that is highly useful to overexpress genes whose expression may block transgenic regeneration. Furthermore, because the designed vector utilizes the GATEWAY™ recombination technology, cloning was restriction- and ligation-free, thus rendering the vector suitable for high-throughput research. The system was tested in Nicotiana tabacum bright yellow-2 (BY-2) cells and its efficiency was demonstrated for the controlled overexpression of the gus reporter gene and a mutant allele of the A-type cyclin-dependent kinase (CDKA), which is known to be a potent inhibitor of the cell cycle. [ABSTRACT FROM AUTHOR]

New plant cells arise at the meristems, where they divide a few times before they leave the cell-cycle program and start to differentiate. Here we show that the E2Fa-DPa transcription factor of Arabidopsis thaliana is a key regulator determining the proliferative status of plant cells. Ectopic expression of E2Fa induced sustained cell proliferation in normally differentiated cotyledon and hypocotyl cells. The phenotype was enhanced strongly by the co-expression of E2Fa with its dimerization partner, DPa. In endoreduplicating cells, E2Fa-DPa also caused extra DNA replication that was correlated with transcriptional induction of S phase genes. Because E2Fa-DPa transgenic plants arrested early in development, we argue that controlled exit of the cell cycle is a prerequisite for normal plant development. [ABSTRACT FROM AUTHOR]

Summary The SUC1/CKS1 proteins associate with cyclin-dependent kinases (CDKs) and play an essential role in the regulation of the cell cycle. Recently, an Arabidopsis thaliana SUC1/CKS1 homologous gene, designated CKS1At, has been cloned. Here, overexpression of CKS1At in Arabidopsis is shown to reduce leaf size and root growth rates. Reduced root growth resulted primarily from an increase of the cell-cycle duration and a shortening of the meristem. Endoreduplication was unaffected. The increased cell-cycle duration was associated with an equal extension of both the G1 and G2 phases. This inhibition was due to the binding of CDK subunits with CDKs. The reduced growth rates in response to altered cell-cycle gene expression demonstrates a direct dependence of plant growth rates on cell-cycle regulation. [ABSTRACT FROM AUTHOR]