Mechanistic insights into DNA damage recognition and checkpoint control in plants.

The plant DNA damage response (DDR) pathway safeguards genomic integrity by rapid recognition and repair of DNA lesions that, if unrepaired, may cause genome instability. Most frequently, DNA repair goes hand in hand with a transient cell cycle arrest, which allows cells to repair the DNA lesions before engaging in a mitotic event, but consequently also affects plant growth and yield. Through the identification of DDR proteins and cell cycle regulators that react to DNA double-strand breaks or replication defects, it has become clear that these proteins and regulators form highly interconnected networks. These networks operate at both the transcriptional and post-transcriptional levels and include liquid-liquid phase separation and epigenetic mechanisms. Strikingly, whereas the upstream DDR sensors and signalling components are well conserved across eukaryotes, some of the more downstream effectors are diverged in plants, probably to suit unique lifestyle features. Additionally, DDR components display functional diversity across ancient plant species, dicots and monocots. The observed resistance of DDR mutants towards aluminium toxicity, phosphate limitation and seed ageing indicates that gaining knowledge about the plant DDR may offer solutions to combat the effects of climate change and the associated risk for food security.

A conserved graft formation process in Norway spruce and Arabidopsis identifies the PAT gene family as central regulators of wound healing.

The widespread use of plant grafting enables eudicots and gymnosperms to join with closely related species and grow as one. Gymnosperms have dominated forests for over 200 million years, and despite their economic and ecological relevance, we know little about how they graft. Here we developed a micrografting method in conifers using young tissues that allowed efficient grafting with closely related species and between distantly related genera. Conifer graft junctions rapidly connected vasculature and differentially expressed thousands of genes including auxin and cell-wall-related genes. By comparing these genes to those induced during Arabidopsis thaliana graft formation, we found a common activation of cambium, cell division, phloem and xylem-related genes. A gene regulatory network analysis in Norway spruce (Picea abies) predicted that PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) acted as a core regulator of graft healing. This gene was strongly up-regulated during both spruce and Arabidopsis grafting, and Arabidopsis mutants lacking PAT genes failed to attach tissues or successfully graft. Complementing Arabidopsis PAT mutants with the spruce PAT1 homolog rescued tissue attachment and enhanced callus formation. Together, our data show an ability for young tissues to graft with distantly related species and identifies the PAT gene family as conserved regulators of graft healing and tissue regeneration.

The regeneration conferring transcription factor complex ERF115-PAT1 coordinates a wound-induced response in root-knot nematode induced galls.

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.

The long non-coding RNA LINDA restrains cellular collapse following DNA damage in Arabidopsis thaliana.

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.

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.

SIAMESE-RELATED1 imposes differentiation of stomatal lineage ground cells into pavement cells.

The leaf epidermis represents a multifunctional tissue consisting of trichomes, pavement cells and stomata, the specialized cellular pores of the leaf. Pavement cells and stomata both originate from regulated divisions of stomatal lineage ground cells (SLGCs), but whereas the ontogeny of the stomata is well characterized, the genetic pathways activating pavement cell differentiation remain relatively unexplored. Here, we reveal that the cell cycle inhibitor SIAMESE-RELATED1 (SMR1) is essential for timely differentiation of SLGCs into pavement cells by terminating SLGC self-renewal potency, which depends on CYCLIN A proteins and CYCLIN-DEPENDENT KINASE B1. By controlling SLGC-to-pavement cell differentiation, SMR1 determines the ratio of pavement cells to stomata and adjusts epidermal development to suit environmental conditions. We therefore propose SMR1 as an attractive target for engineering climate-resilient plants.

PAT1-type GRAS-domain proteins control regeneration by activating DOF3.4 to drive cell proliferation in Arabidopsis roots.

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.

Evolution of wound-activated regeneration pathways in the plant kingdom.

Regeneration serves as a self-protective mechanism that allows a tissue or organ to recover its entire form and function after suffering damage. However, the regenerative capacity varies greatly within the plant kingdom. Primitive plants frequently display an amazing regenerative ability as they have developed a complex system and strategy for long-term survival under extreme stress conditions. The regenerative ability of dicot species is highly variable, but that of monocots often exhibits extreme recalcitrance to tissue replenishment. Recent studies have revealed key factors and signals that affect cell fate during plant regeneration, some of which are conserved among the plant lineage. Among these, several members of the ETHYLENE RESPONSE FACTOR (ERF) transcription factors have been implicated in wound signaling, playing crucial roles in the regenerative mechanisms after different types of wounding. An understanding of plant regeneration may ultimately lead to an increased regenerative potential of recalcitrant species, producing more high-yielding, multi-resistant and environmentally friendly crops and ensuring the long-term development of global agriculture.

Plant lineage-specific PIKMIN1 drives APC/CCCS52A2 E3-ligase activity-dependent cell division.

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.

SCARECROW-LIKE28 modulates organ growth in Arabidopsis by controlling mitotic cell cycle exit, endoreplication, and cell expansion dynamics.

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.

Endoreplication mediates cell size control via mechanochemical signaling from cell wall.

Endoreplication is an evolutionarily conserved mechanism for increasing nuclear DNA content (ploidy). Ploidy frequently scales with final cell and organ size, suggesting a key role for endoreplication in these processes. However, exceptions exist, and, consequently, the endoreplication-size nexus remains enigmatic. Here, we show that prolonged tissue folding at the apical hook in Arabidopsis requires endoreplication asymmetry under the control of an auxin gradient. We identify a molecular pathway linking endoreplication levels to cell size through cell wall remodeling and stiffness modulation. We find that endoreplication is not only permissive for growth: Endoreplication reduction enhances wall stiffening, actively reducing cell size. The cell wall integrity kinase THESEUS plays a key role in this feedback loop. Our data thus explain the nonlinearity between ploidy levels and size while also providing a molecular mechanism linking mechanochemical signaling with endoreplication-mediated dynamic control of cell growth.

Transcriptional activation of auxin biosynthesis drives developmental reprogramming of differentiated cells.

Plant cells exhibit remarkable plasticity of their differentiation states, enabling regeneration of whole plants from differentiated somatic cells. How they revert cell fate and express pluripotency, however, remains unclear. In this study, we demonstrate that transcriptional activation of auxin biosynthesis is crucial for reprogramming differentiated Arabidopsis (Arabidopsis thaliana) leaf cells. Our data show that interfering with the activity of histone acetyltransferases dramatically reduces callus formation from leaf mesophyll protoplasts. Histone acetylation permits transcriptional activation of PLETHORAs, leading to the induction of their downstream YUCCA1 gene encoding an enzyme for auxin biosynthesis. Auxin biosynthesis is in turn required to accomplish initial cell division through the activation of G2/M phase genes mediated by MYB DOMAIN PROTEIN 3-RELATED (MYB3Rs). We further show that the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19 and INDOLE-3-ACETIC ACID INDUCIBLE 3 (IAA3)/IAA18-mediated auxin signaling pathway is responsible for cell cycle reactivation by transcriptionally upregulating MYB3R4. These findings provide a mechanistic model of how differentiated plant cells revert their fate and reinitiate the cell cycle to become pluripotent.

The wound-activated ERF15 transcription factor drives Marchantia polymorpha regeneration by activating an oxylipin biosynthesis feedback loop.

The regenerative potential in response to wounding varies widely among species. Within the plant lineage, the liverwort Marchantia polymorpha displays an extraordinary regeneration capacity. However, its molecular pathways controlling the initial regeneration response are unknown. Here, we demonstrate that the MpERF15 transcription factor gene is instantly activated after wounding and is essential for gemmaling regeneration following tissue incision. MpERF15 operates both upstream and downstream of the MpCOI1 oxylipin receptor by controlling the expression of oxylipin biosynthesis genes. The resulting rise in the oxylipin dinor-12-oxo-phytodienoic acid (dn-OPDA) levels results in an increase in gemma cell number and apical notch organogenesis, generating highly disorganized and compact thalli. Our data pinpoint MpERF15 as a key factor activating an oxylipin biosynthesis amplification loop after wounding, which eventually results in reactivation of cell division and regeneration. We suggest that the genetic networks controlling oxylipin biosynthesis in response to wounding might have been reshuffled over evolution.

The regeneration factors ERF114 and ERF115 regulate auxin-mediated lateral root development in response to mechanical cues.

Plants show an unparalleled regenerative capacity, allowing them to survive severe stress conditions, such as injury, herbivory attack, and harsh weather conditions. This potential not only replenishes tissues and restores damaged organs but can also give rise to whole plant bodies. Despite the intertwined nature of development and regeneration, common upstream cues and signaling mechanisms are largely unknown. Here, we demonstrate that in addition to being activators of regeneration, ETHYLENE RESPONSE FACTOR 114 (ERF114) and ERF115 govern developmental growth in the absence of wounding or injury. Increased ERF114 and ERF115 activity enhances auxin sensitivity, which is correlated with enhanced xylem maturation and lateral root formation, whereas their knockout results in a decrease in lateral roots. Moreover, we provide evidence that mechanical cues contribute to ERF114 and ERF115 expression in correlation with BZR1-mediated brassinosteroid signaling under both regenerative and developmental conditions. Antagonistically, cell wall integrity surveillance via mechanosensory FERONIA signaling suppresses their expression under both conditions. Taken together, our data suggest a molecular framework in which cell wall signals and mechanical strains regulate organ development and regenerative responses via ERF114- and ERF115-mediated auxin signaling.

The Plant Anaphase-Promoting Complex/Cyclosome.

The anaphase-promoting complex/cyclosome (APC/C) represents a large multisubunit E3-ubiquitin ligase complex that controls the unidirectional progression through the cell cycle by the ubiquitination of specific target proteins, marking them for proteasomal destruction. Although the APC/C's role is largely conserved among eukaryotes, its subunit composition and target spectrum appear to be species specific. In this review, we focus on the plant APC/C complex, whose activity correlates with different developmental processes, including polyploidization and gametogenesis. After an introduction into proteolytic control by ubiquitination, we discuss the composition of the plant APC/C and the essential nature of its core subunits for plant development. Subsequently, we describe the APC/C activator subunits and interactors, most being plant specific. Finally, we provide a comprehensive list of confirmed and suspected plant APC/C target proteins. Identification of growth-related targets might offer opportunities to increase crop yield and resilience of plants to climate change by manipulating APC/C activity.

Cell-wall damage activates DOF transcription factors to promote wound healing and tissue regeneration in Arabidopsis thaliana.

Wound healing is a fundamental property of plants and animals that requires recognition of cellular damage to initiate regeneration. In plants, wounding activates a defense response via the production of jasmonic acid and a regeneration response via the hormone auxin and several ethylene response factor (ERF) and NAC domain-containing protein (ANAC) transcription factors. To better understand how plants recognize damage and initiate healing, we searched for factors upregulated during the horticulturally relevant process of plant grafting and found four related DNA binding with one finger (DOF) transcription factors, HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, whose expression rapidly activated at the Arabidopsis graft junction. Grafting or wounding a quadruple hca2, tmo6, dof2.1, dof6 mutant inhibited vascular and cell-wall-related gene expression. Furthermore, the quadruple dof mutant reduced callus formation, tissue attachment, vascular regeneration, and pectin methylesterification in response to wounding. We also found that activation of DOF gene expression after wounding required auxin, but hormone treatment alone was insufficient for their induction. However, modifying cell walls by enzymatic digestion of cellulose or pectin greatly enhanced TMO6 and HCA2 expression, whereas genetic modifications to the pectin or cellulose matrix using the PECTIN METHYLESTERASE INHIBITOR5 overexpression line or korrigan1 mutant altered TMO6 and HCA2 expression. Changes to the cellulose or pectin matrix were also sufficient to activate the wound-associated ERF115 and ANAC096 transcription factors, suggesting that cell-wall damage represents a common mechanism for wound perception and the promotion of tissue regeneration.

A long and stressful day: Photoperiod shapes aluminium tolerance in plants.

Aluminium (Al), a limiting factor for crop productivity in acidic soils (pH ≤ 5.5), imposes drastic constraints for food safety in developing countries. The major mechanisms that allow plants to cope with Al involve manipulations of organic acids metabolism and DNA-checkpoints. When assumed individually both approaches have been insufficient to overcome Al toxicity. On analysing the centre of origin of most cultivated plants, we hypothesised that day-length seems to be a pivotal agent modulating Al tolerance across distinct plant species. We observed that with increasing distance from the Equator, Al tolerance decreases, suggesting a relationship with the photoperiod. We verified that long-day (LD) species are generally more Al-sensitive than short-day (SD) species, whereas genetic conversion of tomato for SD growth habit boosts Al tolerance. Reduced Al tolerance correlates with DNA-checkpoint activation under LD. Furthermore, DNA-checkpoint-related genes are under positive selection in Arabidopsis accessions from regions with shorter days, suggesting that photoperiod act as a selective barrier for Al tolerance. A diel regulation and genetic diversity affect Al tolerance, suggesting that day-length orchestrates Al tolerance. Altogether, photoperiodic control of Al tolerance might contribute to solving the historical obstacle that imposes barriers for developing countries to reach a sustainable agriculture.

Single-cell transcriptomics sheds light on the identity and metabolism of developing leaf cells.

As the main photosynthetic instruments of vascular plants, leaves are crucial and complex plant organs. A strict organization of leaf mesophyll and epidermal cell layers orchestrates photosynthesis and gas exchange. In addition, water and nutrients for leaf growth are transported through the vascular tissue. To establish the single-cell transcriptomic landscape of these different leaf tissues, we performed high-throughput transcriptome sequencing of individual cells isolated from young leaves of Arabidopsis (Arabidopsis thaliana) seedlings grown in two different environmental conditions. The detection of approximately 19,000 different transcripts in over 1,800 high-quality leaf cells revealed 14 cell populations composing the young, differentiating leaf. Besides the cell populations comprising the core leaf tissues, we identified subpopulations with a distinct identity or metabolic activity. In addition, we proposed cell-type-specific markers for each of these populations. Finally, an intuitive web tool allows for browsing the presented dataset. Our data present insights on how the different cell populations constituting a developing leaf are connected via developmental, metabolic, or stress-related trajectories.

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.