Distinctive and complementary roles of E2F transcription factors during plant replication stress responses.

Survival of living organisms is fully dependent on their maintenance of genome integrity, being permanently threatened by replication stress in proliferating cells. Although the plant DNA damage response (DDR) regulator SOG1 has been demonstrated to cope with replication defects, accumulating evidence points to other pathways functioning independent of SOG1. Here, we report the roles of the Arabidopsis E2FA and EF2B transcription factors, two well-characterized regulators of DNA replication, in plant response to replication stress. Through a combination of reverse genetics and chromatin immunoprecipitation approaches, we show that E2FA and E2FB share many target genes with SOG1, providing evidence for their involvement in the DDR. Analysis of double- and triple-mutant combinations revealed that E2FB, rather than E2FA, plays the most prominent role in sustaining plant growth in the presence of replication defects, either operating antagonistically or synergistically with SOG1. Conversely, SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants. Collectively, our data reveal a complex transcriptional network controlling the replication stress response in which E2Fs and SOG1 act as key regulatory factors.

Constructing Cell-Free Expression Systems for Low-Cost Access.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Cell cycle checkpoint control in response to DNA damage by environmental stresses.

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.

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability.

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU.

During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase.

Maize ATR safeguards genome stability during kernel development to prevent early endosperm endocycle onset and cell death.

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

The plant DNA polymerase theta is essential for the repair of replication-associated DNA damage.

Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants' viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants' responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.

The Arabidopsis GRAS-type SCL28 transcription factor controls the mitotic cell cycle and division plane orientation.

Gene expression is reconfigured rapidly during the cell cycle to execute the cellular functions specific to each phase. Studies conducted with synchronized plant cell suspension cultures have identified hundreds of genes with periodic expression patterns across the phases of the cell cycle, but these results may differ from expression occurring in the context of intact organs. Here, we describe the use of fluorescence-activated cell sorting to analyze the gene expression profile of G2/M cells in the growing root. To this end, we isolated cells expressing the early mitosis cell cycle marker CYCLINB1;1-GFP from Arabidopsis root tips. Transcriptome analysis of these cells allowed identification of hundreds of genes whose expression is reduced or enriched in G2/M cells, including many not previously reported from cell suspension cultures. From this dataset, we identified SCL28, a transcription factor belonging to the GRAS family, whose messenger RNA accumulates to the highest levels in G2/M and is regulated by MYB3R transcription factors. Functional analysis indicates that SCL28 promotes progression through G2/M and modulates the selection of cell division planes.

Maize Thymidine Kinase Activity Is Present throughout Plant Development and Its Heterologous Expression Confers Tolerance to an Organellar DNA-Damaging Agent.

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

The Cyclin CYCA3;4 Is a Postprophase Target of the APC/CCCS52A2 E3-Ligase Controlling Formative Cell Divisions in Arabidopsis.

The anaphase promoting complex/cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CELL DIVISION PROTEIN20 (CDC20) and CELL CYCLE SWITCH52 (CCS52). Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis (Arabidopsis thaliana) CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhance the scored ccs52a2-1 phenotypes. Furthermore, whereas the CYCA3;4 protein is promptly broken down after prophase in wild-type plants, it remains present in later stages of mitosis in ccs52a2-1 mutant plants, marking it as a putative APC/CCCS52A2 substrate. Strikingly, increased CYCA3;4 levels result in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4 gain-of-function plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 contributes to disorganized formative divisions, possibly in part caused by the inactivation of RBR1.

Plant DNA Polymerases.

Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10-15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity.

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.

Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.

Chloroplasts around the plant cell cycle.

Plastids arose from an endosymbiosis between a host cell and free-living bacteria. One key step during this evolutionary process has been the establishment of coordinated cell and symbiont division to allow the maintenance of organelles during proliferation of the host. However, surprisingly little is known about the underlying mechanisms. In addition, due to their central role in the cell's energetic metabolism and to their sensitivity to various environmental cues such as light or temperature, plastids are ideally fitted to be the source of signals allowing plants to adapt their development according to external conditions. Consistently, there is accumulating evidence that plastid-derived signals can impinge on cell cycle regulation. In this review, we summarize current knowledge of the dialogue between chloroplasts and the nucleus in the context of the cell cycle.

Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis.

Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types.

Arabidopsis thaliana thymidine kinase 1a is ubiquitously expressed during development and contributes to confer tolerance to genotoxic stress.

Thymidine kinase catalyzes the first step in the nucleotide salvage pathway by transferring a phosphate group to a thymidine molecule. In mammals thymidine kinase supplies deoxyribonucleotides for DNA replication and DNA repair, and the expression of the gene is tightly regulated during the cell cycle. Although this gene is phylogenetically conserved in many taxa, its physiological function in plants remains unknown. The genome of the model plant Arabidopsis thaliana has two thymidine kinase genes (AtTK1a and AtTK1b) and microarray data suggest they might have redundant roles. In this study we analyzed the TK1a function by evaluating its expression pattern during development and in response to genotoxic stress. We also studied its role in DNA repair by the characterization of a mutant that contained the T-DNA insertion in the promoter region of the TK1a gene. We found that TK1a is expressed in most tissues during plant development and it was differentially induced by ultraviolet-C radiation because TK1b expression was unaffected. In the mutant, the T-DNA insertion caused a 40 % rise in transcript levels and enzyme activity in Arabidopsis seedlings compared to wild-type plants. This elevation was enough to confer tolerance to ultraviolet-C irradiation in dark conditions, as determined by root growth, and meristem length and structure. TK1a overexpression also provided tolerance to genotoxins that induce double-strand break. Our results suggest that thymidine kinase contributes to several DNA repair pathways by providing deoxythymidine triphosphate that serve as precursors for DNA repair and to balance deoxyribonucleotides pools.