RESUMO
Single-cell and single-nucleus RNA-sequencing technologies capture the expression of plant genes at an unprecedented resolution. Therefore, these technologies are gaining traction in plant molecular and developmental biology for elucidating the transcriptional changes across cell types in a specific tissue or organ, upon treatments, in response to biotic and abiotic stresses, or between genotypes. Despite the rapidly accelerating use of these technologies, collective and standardized experimental and analytical procedures to support the acquisition of high-quality data sets are still missing. In this commentary, we discuss common challenges associated with the use of single-cell transcriptomics in plants and propose general guidelines to improve reproducibility, quality, comparability, and interpretation and to make the data readily available to the community in this fast-developing field of research.
Assuntos
Perfilação da Expressão Gênica , Plantas , Reprodutibilidade dos Testes , Plantas/genética , Estresse Fisiológico/genética , Armazenamento e Recuperação da InformaçãoRESUMO
Plant roots possess remarkable regenerative potential owing to their ability to replenish damaged or lost stem cells. ETHYLENE RESPONSE FACTOR 115 (ERF115), one of the key molecular elements linked to this potential, plays a predominant role in the activation of regenerative cell divisions. However, the downstream operating molecular machinery driving wound-activated cell division is largely unknown. Here, we biochemically and genetically identified the GRAS-domain transcription factor SCARECROW-LIKE 5 (SCL5) as an interaction partner of ERF115 in Arabidopsis thaliana. Although nonessential under control growth conditions, SCL5 acts redundantly with the related PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) and SCL21 transcription factors to activate the expression of the DNA-BINDING ONE FINGER 3.4 (DOF3.4) transcription factor gene. DOF3.4 expression is wound-inducible in an ERF115-dependent manner and, in turn, activates D3-type cyclin expression. Accordingly, ectopic DOF3.4 expression drives periclinal cell division, while its downstream D3-type cyclins are essential for the regeneration of a damaged root. Our data highlight the importance and redundant roles of the SCL5, SCL21, and PAT1 transcription factors in wound-activated regeneration processes and pinpoint DOF3.4 as a key downstream element driving regenerative cell division.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Fitocromo A/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Divisão Celular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ciclinas/metabolismo , Transdução de Sinais/genética , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Brassinosteroides , Divisão Celular Assimétrica , Quinase 3 da Glicogênio Sintase , Transdução de Sinais , Diferenciação Celular , Arabidopsis/genética , Proteínas Quinases/genética , Proteínas de Arabidopsis/genéticaRESUMO
The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge on the APC/C by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis (Arabidopsis thaliana) plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits the growth of ccs52a2-1 plants, indicating the need for control of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its systemic overexpression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and the phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionarily conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific protein with a role in cell division that is likely proteolytically controlled by the CCS52A2-activated APC/C.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Divisão Celular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Arabidopsis/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Plantas/metabolismo , MitoseRESUMO
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.
RESUMO
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.
Assuntos
Reparo do DNA/genética , Endosperma/genética , Proteínas de Plantas/genética , Zea mays/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sistemas CRISPR-Cas , Morte Celular/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA/genética , Endosperma/citologia , Instabilidade Genômica , Mutação , Células Vegetais , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/citologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Zea mays/citologia , Zea mays/crescimento & desenvolvimentoRESUMO
Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.
Assuntos
Alumínio/toxicidade , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Caseína Quinase II/metabolismo , Fosfatos/metabolismo , Alumínio/farmacocinética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caseína Quinase II/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fosfatos/farmacologia , Fosforilação , Células Vegetais/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Proteínas de Ciclo Celular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Divisão Celular , Metanossulfonato de Etila/farmacologia , Regulação da Expressão Gênica de Plantas , Meristema/citologia , Meristema/genética , Mutação , Fosforilação , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Caules de Planta/citologia , Plantas Geneticamente Modificadas , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Plants are known for their outstanding capacity to recover from various wounds and injuries. However, it remains largely unknown how plants sense diverse forms of injury and canalize existing developmental processes into the execution of a correct regenerative response. Auxin, a cardinal plant hormone with morphogen-like properties, has been previously implicated in the recovery from diverse types of wounding and organ loss. Here, through a combination of cellular imaging and in silico modeling, we demonstrate that vascular stem cell death obstructs the polar auxin flux, much alike rocks in a stream, and causes it to accumulate in the endodermis. This in turn grants the endodermal cells the capacity to undergo periclinal cell division to repopulate the vascular stem cell pool. Replenishment of the vasculature by the endodermis depends on the transcription factor ERF115, a wound-inducible regulator of stem cell division. Although not the primary inducer, auxin is required to maintain ERF115 expression. Conversely, ERF115 sensitizes cells to auxin by activating ARF5/MONOPTEROS, an auxin-responsive transcription factor involved in the global auxin response, tissue patterning, and organ formation. Together, the wound-induced auxin accumulation and ERF115 expression grant the endodermal cells stem cell activity. Our work provides a mechanistic model for wound-induced stem cell regeneration in which ERF115 acts as a wound-inducible stem cell organizer that interprets wound-induced auxin maxima.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Ácidos Indolacéticos/metabolismo , Regeneração , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Divisão Celular , Autorrenovação Celular , Regulação da Expressão Gênica de Plantas , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Replicação do DNA , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myb/genética , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Genoma de Planta , Instabilidade Genômica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Adaptação Fisiológica/genética , Arabidopsis/fisiologia , Raízes de Plantas/genética , Poliploidia , Arabidopsis/citologia , Arabidopsis/genética , Tamanho Celular , DNA de Plantas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Células Vegetais/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes , Análise Espaço-Temporal , Estresse Fisiológico/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , DNA Polimerase I/genética , Resistência a Medicamentos/genética , Hidroxiureia/farmacologia , Mutação , Proteínas Serina-Treonina Quinases/deficiência , Antidrepanocíticos/farmacologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Ciclo Celular , Dano ao DNA , FosforilaçãoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/parasitologia , Ciclo Celular , Tumores de Planta/parasitologia , Proteínas Serina-Treonina Quinases/metabolismo , Tylenchoidea/fisiologia , Animais , Arabidopsis/genética , Ciclo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Células Gigantes/citologia , Glucuronidase/metabolismo , Solanum lycopersicum/genética , Mitose , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis (Arabidopsis thaliana), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/CCCS52A1 and APC/CCCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/CCCS52A1 and APC/CCCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/CCCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ciclo Celular , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas , Especificidade de Órgãos , Fenótipo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Ploidias , Fatores de Transcrição/genéticaRESUMO
Whereas our knowledge about the diverse pathways aiding DNA repair upon genome damage is steadily increasing, little is known about the molecular players that adjust the plant cell cycle in response to DNA stress. By a meta-analysis of DNA stress microarray data sets, three family members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) class of cyclin-dependent kinase inhibitors were discovered that react strongly to genotoxicity. Transcriptional reporter constructs corroborated specific and strong activation of the three SIM/SMR genes in the meristems upon DNA stress, whereas overexpression analysis confirmed their cell cycle inhibitory potential. In agreement with being checkpoint regulators, SMR5 and SMR7 knockout plants displayed an impaired checkpoint in leaf cells upon treatment with the replication inhibitory drug hydroxyurea (HU). Surprisingly, HU-induced SMR5/SMR7 expression depends on ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE1, rather than on the anticipated replication stress-activated ATM AND RAD3-RELATED kinase. This apparent discrepancy was explained by demonstrating that, in addition to its effect on replication, HU triggers the formation of reactive oxygen species (ROS). ROS-dependent transcriptional activation of the SMR genes was confirmed by different ROS-inducing conditions, including high-light treatment. We conclude that the identified SMR genes are part of a signaling cascade that induces a cell cycle checkpoint in response to ROS-induced DNA damage.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Proteínas de Ciclo Celular/fisiologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/fisiologia , Dano ao DNA , Espécies Reativas de Oxigênio/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Hidroxiureia/farmacologia , Estresse Oxidativo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
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.
Assuntos
Antígenos de Neoplasias , Vacinas Anticâncer , Carcinoma Pulmonar de Células não Pequenas , Diferenciação Celular , Células Dendríticas , Neoplasias Pulmonares , Linfócitos T , Vacinação , Humanos , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Masculino , Feminino , Pessoa de Meia-Idade , Antígenos de Neoplasias/imunologia , Diferenciação Celular/imunologia , Idoso , Linfócitos T/imunologiaRESUMO
Droplet-based single-cell RNA-sequencing (scRNA-seq) empowers transcriptomic profiling with an unprecedented resolution, facilitating insights into the cellular heterogeneity of tissues, developmental progressions, stress-response dynamics, and more at single-cell level. In this chapter, we describe the experimental workflow of processing Arabidopsis root tissue into protoplasts and generating single-cell transcriptomes. We also describe the general computational workflow of visualizing and utilizing scRNA-seq data. This protocol can be used as a starting point for establishing a scRNA-seq workflow.
Assuntos
Arabidopsis , Humanos , Arabidopsis/genética , Progressão da Doença , Perfilação da Expressão Gênica , Protoplastos , RNARESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
The effects of brassinosteroid signaling on shoot and root development have been characterized in great detail but a simple consistent positive or negative impact on a basic cellular parameter was not identified. In this study, we combined digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize root meristems and mature root segments of brassinosteroid-blind mutants and wild type. The resultant datasets demonstrate that brassinosteroid signaling affects neither cell volume nor cell proliferation capacity. Instead, brassinosteroid signaling is essential for the precise orientation of cell division planes and the extent and timing of anisotropic cell expansion. Moreover, we found that the cell-aligning effects of brassinosteroid signaling can propagate to normalize the anatomy of both adjacent and distant brassinosteroid-blind cells through non-cell-autonomous functions, which are sufficient to restore growth vigor. Finally, single-cell transcriptome data discern directly brassinosteroid-responsive genes from genes that can react non-cell-autonomously and highlight arabinogalactans as sentinels of brassinosteroid-dependent anisotropic cell expansion.
Assuntos
Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Brassinosteroides/metabolismo , Diferenciação Celular/efeitos dos fármacos , Raízes de Plantas/citologia , Arabidopsis/metabolismo , Brassinosteroides/farmacologia , Regulação da Expressão Gênica de Plantas , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcriptoma/efeitos dos fármacosRESUMO
Single-cell approaches are quickly changing our view on biological systems by increasing the spatiotemporal resolution of our analyses to the level of the individual cell. The field of plant biology has fully embraced single-cell transcriptomics and is rapidly expanding the portfolio of available technologies and applications. In this review, we give an overview of the main advances in plant single-cell transcriptomics over the past few years and provide the reader with an accessible guideline covering all steps, from sample preparation to data analysis. We end by offering a glimpse of how these technologies will shape and accelerate plant-specific research in the near future.