The stem cell niche transcription factor ETHYLENE RESPONSE FACTOR 115 participates in aluminum-induced terminal differentiation in Arabidopsis roots.

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.

Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum.

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively, these data support the feasibility, safety, and immunogenicity of this treatment in resected NSCLC.

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.

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.

Suppressor of Gamma Response 1 Modulates the DNA Damage Response and Oxidative Stress Response in Leaves of Cadmium-Exposed Arabidopsis thaliana.

Cadmium (Cd) exposure causes an oxidative challenge and inhibits cell cycle progression, ultimately impacting plant growth. Stress-induced effects on the cell cycle are often a consequence of activation of the DNA damage response (DDR). The main aim of this study was to investigate the role of the transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) and three downstream cyclin-dependent kinase inhibitors of the SIAMESE-RELATED (SMR) family in the Cd-induced DDR and oxidative challenge in leaves of Arabidopsis thaliana. Effects of Cd on plant growth, cell cycle regulation and the expression of DDR genes were highly similar between the wildtype and smr4/5/7 mutant. In contrast, sog1-7 mutant leaves displayed a much lower Cd sensitivity within the experimental time-frame and significantly less pronounced upregulations of DDR-related genes, indicating the involvement of SOG1 in the Cd-induced DDR. Cadmium-induced responses related to the oxidative challenge were disturbed in the sog1-7 mutant, as indicated by delayed Cd-induced increases of hydrogen peroxide and glutathione concentrations and lower upregulations of oxidative stress-related genes. In conclusion, our results attribute a novel role to SOG1 in regulating the oxidative stress response and connect oxidative stress to the DDR in Cd-exposed plants.

The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls.

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.

A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Modification of DNA Checkpoints to Confer Aluminum Tolerance.

Although aluminum (Al) toxicity represents a global agricultural problem, the biochemical targets for Al remain elusive. Recently identified Arabidopsis mutants with increased Al tolerance provide evidence of DNA as one of the main targets of Al. This insight could lead the way for novel strategies to generate Al-tolerant crop plants.