The transition of plants from water to land required major evolutionary innovations, including mechanisms to cope with environmental stress. DNA repair via the homologous recombination (HR) pathway is central in maintaining genome integrity, and its evolutionary conservation highlights its importance across plant lineages. Our analysis supports conservation of genes involved in the main activities for HR across the Viridiplantae. Among them, the recombinase radiation sensitive protein 51 (RAD51) has a central role in the HR pathway due to its DNA binding, homology recognition, and DNA displacement activities. Here we describe the functional characterization of the RAD51 ortholog in common liverwort (Marchantia polymorpha). In silico, the mutations within critical RAD51 domains and phosphorylation motifs identified offered insights into the possible regulatory adaptations of RAD51's function through plant evolution. The main functions of Marchantia polymorpha RAD51 (MpRAD51) described in this pathway were confirmed biochemically. Nevertheless, beyond DNA repair, the loss of MpRAD51 showcased its role in the regulation of DNA damage-induced plant development and differentiation, highlighting its importance in preventing growth arrest under genotoxic conditions. This study provides evidence of the evolutionary importance of HR for plant development and genome maintenance under both normal and genotoxic conditions.

BACKGROUND:Ambystoma mexicanum is renowned for its extraordinary regenerative capacity. Regardless of whether progenitor cells arise via dedifferentiation or stem cell activation, these cells must replicate DNA to proliferate before they ultimately differentiate. This rapid cell division increases the likelihood of DNA damage, requiring efficient repair mechanisms. RESULTS:We focused on the non-homologous end joining pathway, responsible for repairing double-strand DNA breaks, and investigated the effects of 2 key components-KU70/80 and LIGIV- using chemical inhibitors STL127705 and SCR130, respectively. We assessed the impact of these inhibitors on axolotl limb regeneration, finding that treatment with the LIGIV inhibitor (SCR130) led to a positive growth trend in the regenerated area. Histological analysis of re-amputated limbs revealed no major morphological differences between treated and control animals. However, we observed a significant increase in the number of senescent cells in the regenerated tissue after KU70/80 inhibition (STL127705) at 32 days post-amputation, suggesting that DNA repair inhibition may promote senescence. Furthermore, we detected a dose-dependent increase in DNA damage, indicated by the γH2AX(Ser139) signal, specifically in cells' nuclei within the regenerated tissues. CONCLUSIONS:These findings suggest that DNA repair inhibition impacts regeneration, potentially through mechanisms linked to cellular senescence and DNA damage accumulation.

In Arabidopsis thaliana, one of the most important gene families involved in plant development is the one encoding the GRAS transcription factors. In this family, SHORT-ROOT (SHR) and SCARECROW (SCR) play key roles in asymmetric cell division and the maintenance of the root stem cell niche, while SCR is additionally regulated by RETINOBLASTOMA-RELATED (RBR) through the LxCxE motif. GRAS proteins can be traced back to Zygnematophyceae algae, indicating an early origin of this protein family; therefore, we sought to determine whether protein-protein interactions could have originated in this clade, including the SCR-SHR interaction, as well as the potential interaction between RBR and a GRAS protein carrying an LxCxE motif, and to assess how conserved are these interactions across land plants. We addressed this question by identifying all GRAS proteins in Zygnematophyceae and analyzing their domains, revealing that not all GRAS proteins contain the full set of characteristic GRAS elements and that only a few sequences harbor pseudo-LxCxE motifs. Through in silico prediction of protein-protein interactions between SCR-SHR and RBR-SCR_LxCxE across the Viridiplantae lineage, and by comparing the three-dimensional interactions of heterodimers from different species, our results suggest that both SCR-SHR and RBR-SCR_LxCxE interactions likely originated in Zygnematophyceae and were subsequently refined in land plants through amino acid changes that enhance protein-protein binding, leading to increasingly similar interactions along land plants.

Heat stress limits tomato yield and quality. Deciphering the key genes and metabolites related to heat tolerance is essential for selecting tolerant varieties. In this study, we profiled the transcriptomes and metabolomes of roots and shoots in response to heat stress from a Maya-land ecotype of heirloom tomato (Calkiní), which grows in lowlands and elevated temperatures. We compared them with those of another heirloom ecotype that grows in highlands and lower temperatures (Acaxochitlán). In our omics approach, several transcripts encode enzymes that participate in diverse biosynthetic pathways and produce differentially accumulated metabolites. Although both ecotypes display programs to deal with heat stress, the roots of the Calkiní ecotype showed an increased accumulation of diverse metabolites and the up-regulation of key genes involved in molecular and physiological strategies to cope with heat stress. One of the key findings of this study is the elevated accumulation of transcripts and metabolites associated with phenylpropanoid and suberin biosynthesis in the heat-resistant ecotype. Such increases correlate with substantial suberin deposition in the exodermis and endodermis of Calkiní roots, with enhanced gibberellin accumulation in the meristematic zone under heat stress. Additionally, we found differential expression and accumulation of metabolites and genes involved in the PA-GAPC-NFY heat stress tolerance pathway among both ecotypes. Our work strengthens the importance of studying Mexican native tomato varieties and ecotypes to identify traits that allow plants to cope with diverse biotic and abiotic stresses, specifically providing insights into the genetic and metabolic pathways linked to heat tolerance among ecotypes and tissues.

AINTEGUMENTA-LIKE/PLETHORA/BABYBOOM (APB) genes are considered part of the ancestral developmental toolkit in land plants. In Arabidopsis thaliana, these transcription factors are induced by auxin and are primarily expressed in tissues with actively dividing cells, where they play essential roles in organ development. Marchantia polymorpha, a liverwort that diverged from A. thaliana early in embryophyte evolution, possesses a single APB ortholog, MpAINTEGUMENTA (MpANT), encoded in its genome. In this study, we aimed to characterize the function of MpANT. Analysis of a transcriptional fusion line indicates that MpANT is predominantly expressed in the meristematic region. We report that the MpANT promoter region contains several cis-acting Auxin Responsive Elements (AREs) and demonstrate that its expression, which occurs predominantly in meristematic regions, is significantly altered by the addition of exogenous auxin and inhibition of auxin transport. These findings indicate that MpANT acts downstream of Auxin Response Factors (ARFs) and auxin signaling. Analyses of loss- and gain-of-function MpANT alleles highlight the importance of this transcription factor in meristem maintenance and cell proliferation. Additionally, we found that MpANT acts upstream of the auxin transporter MpPIN1 by influencing auxin distribution. Taken together, our findings reveal a feedforward regulatory loop involving auxin, MpANT, and MpPIN1 that is important for Marchantia development.

The mechanisms underlying the assembly and regulation of enzymatic complexes responsible for plant organellar DNA replication remain poorly characterized. Unlike the monophyletic origin of the gene products involved in animal mitochondrial replication, derived from T-odd bacteriophages, plant organellar DNA replication relies on genes either unique to plants or with origins traceable to bacteria and bacteriophages. Here, we demonstrate that the bacteriophage-related primase-helicase from Arabidopsis thaliana (AtTwinkle) is essential for double-stranded DNA unwinding. AtTwinkle functionally interacts with bacterial-related organellar DNA polymerases (AtPolIs), which lack the ability to unwind large regions of dsDNA, coupling DNA unwinding to processive DNA synthesis at the leading strand of the replisome. Analysis of two T-DNA insertion mutants of AtTwinkle reveals distinct phenotypic outcomes; these mutant lines are hereafter referred to as ph. The ph1 (-/-) mutant, which carries a T-DNA insertion in the 5´ UTR region, is viable and exhibits no noticeable developmental differences compared to wild-type plants. In contrast, the ph2 mutant, with a T-DNA insertion in the 19th exon, displays embryo lethality. Despite these differences, both ph1 (-/-) and heterozygous ph2 (+/-) mutants show a reduction in organellar DNA copy numbers under non-stress conditions and exhibit heightened sensitivity to DNA-damaging agents. In summary, our findings demonstrate that AtTwinkle is essential for organellar DNA replication. The heightened sensitivity of insertion mutants to organelle-specific genotoxic agents indicates that loss of AtTwinkle function reduces the availability of template DNA necessary for double-strand break (DSB) repair. Collectively, our findings reveal that two proteins of distinct evolutionary origins-AtTwinkle and plant organellar DNA polymerases-coevolved to coordinate DNA replication in plant organelles.

Essential genes of multicellular organisms encode products that are indispensable for proper development, reproduction, and survival of the organism. Knocking out essential genes often causes organismal death, which challenges the study of the role of these genes in traits. Here we conduct an Artificial MicroRNAs for Gene silencing and Overcome (AmiGO) strategy, using artificial microRNAs to generate cell type-specific loss-of-function mutants, to unravel cellular functions of essential genes in Arabidopsis thaliana (At). This strategy allows genetic complementation using wild-type genomic fragments or coding sequences (CDS). As a proof of concept for the AmiGO strategy, we describe its application to the gametophytic-lethal gene of RETINOBLASTOMA-RELATED (RBR). Mature AmiGO-RBR, which targets the three prime untranslated regions (3'UTR) of RBR messenger RNA (mRNA), was cloned into the backbone of AtmiR319a precursor. To dissect RBR functions in different cell types, we used tissue-specific promoters to drive the expression of AmiGO-RBR precursor, thereby repressing RBR expression in the corresponding tissues. By developing an AmiGO-RBR-sensor line, we showed that the AmiGO-RBR target gene silencing is efficient and tissue-specific. Moreover, since we were able to complement the AmiGO-RBR-induced phenotypes by expressing a RBR genomic fragment that lacks its mRNA 3'UTR region, we demonstrated that AmiGO-RBR silences its target in a sequence-specific manner. Although tested in Arabidopsis, this strategy can be applied to any plant or animal models, for which an endogenous precursor miRNA has been cloned.

Keywords: plant stem cell, stem cells signaling, stress response, regulatory network, plant regeneration

Main ConclusionOur study presents evidence for a novel mechanism for RBR function in transcriptional gene silencing by interacting with key players of the RdDM pathway in Arabidopsis and several plant clades.Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double-stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs serve as guides to direct AGO4-siRNA complexes to chromatin-bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. The Arabidopsis Retinoblastoma protein homolog (RBR) is a master regulator of the cell cycle, stem cell maintenance, and development. We in silico predicted and explored experimentally the protein-protein interactions (PPIs) between RBR and members of the RdDM pathway. We found that the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2, and SUVR2 contain canonical and non-canonical RBR binding motifs and several of them are conserved since algae and bryophytes. We validated experimentally PPIs between Arabidopsis RBR and several of the RdDM pathway proteins. Moreover, seedlings from loss-of-function mutants in RdDM and RBR show similar phenotypes in the root apical meristem. We show that RdDM and SUVR2 targets are up-regulated in the 35S:AmiGO-RBR background.

SummaryTransposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24 -nt siRNAs) by DCL3. 24-nt siRNAs are recruited by AGO4 and serve as guides to direct AGO4 - siRNA complexes to chromatin bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation.The Arabidopsis Retinoblastoma protein homolog is a master regulator of cell cycle, stem cell maintenance and development. In silico exploration of RBR protein partners revealed that several members of the RdDM pathway contain a motif that confers high affinity binding to RBR, including the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2 and SUVR2. We demonstrate that RBR binds to DRM2, DRD1 and SUVR2. We also report that seedlings from loss -of-function mutants in RdDM and in RBR show similar phenotypes in the root apical meristem. Furthermore, we show that RdDM and SUVR2 targets are up-regulated in the 35S::AmiGO-RBR background.Our results suggest a novel mechanism for RBR function in transcriptional gene silencing based on the interaction with key players of the RdDM pathway and opens several new hypotheses, including the convergence of RBR-DRM2 on the transcriptional control of TEs and several cell/tissue and stage -specific target genes.

Compared to other animals, the spontaneous occurrence of tumors in wild amphibians is relatively rare, generally limited to specific populations or species. The number of reports of spontaneous tumors in amphibians known up to 1986 was 491 cases in anurans and about 253 cases in urodeles. Similarly, there have been many, unsuccessful attempts to chemically or biologically induce tumors in amphibians. With these considerations, it is inevitable to wonder: do urodeles and anurans have an inherent resistance to cancer? Here, we review the spontaneous and induced occurrence of tumors in amphibians in a timeline, as well as failed attempts to induce tumors in these amphibians. Indeed, recent studies seem to indicate that there is a relationship between regeneration and cancer because regenerating tissues seem to resist tumorigenesis, as opposed to nonregenerative tissues of the same amphibian models. Although the mechanisms that allow regenerating tissues to resist tumorigenesis have not been elucidated, it is worth to note that, in addition to the apparent relationship between regeneration and cancer, amphibians possess characteristics that could contribute to their ability to resist the development of neoplastic events. The implications of these features in cancer susceptibility are discussed.

Fermentation is a biochemical process used by humans since thousands of years ago. Currently, this process is crucial for the production of diverse foods and beverages worldwide. Bacteria, yeasts and other fungi are reported as pivotal for different fermentation processes and pulque fermentation is not the exception. Historically most of the papers have focused on reporting and describing the presence and action of bacteria in the pulque production process. In the present review, we revisit the historically reported Yeast and other fungi in the literature since 1870 and the recently found ones using next-generation sequencing approaches. We also discuss its probable role in pulque production and the potential biotechnological role of such microorganisms.

The Mexican axolotl (Ambystoma mexicanum) is a well-established tetrapod model for regeneration and developmental studies. Remarkably, neotenic axolotls may undergo metamorphosis, a process that triggers many dramatic changes in diverse organs, accompanied by gradually decline of their regeneration capacity and lifespan. However, the molecular regulation and cellular changes in neotenic and metamorphosed axolotls are still poorly investigated. Here, we develop a single-cell sequencing method based on combinatorial hybridization to generate a tissue-based transcriptomic landscape of the neotenic and metamorphosed axolotls. We perform gene expression profiling of over 1 million single cells across 19 tissues to construct the first adult axolotl cell landscape. Comparison of single-cell transcriptomes between the tissues of neotenic and metamorphosed axolotls reveal the heterogeneity of non-immune parenchymal cells in different tissues and established their regulatory network. Furthermore, we describe dynamic gene expression patterns during limb development in neotenic axolotls. This system-level single-cell analysis of molecular characteristics in neotenic and metamorphosed axolotls, serves as a resource to explore the molecular identity of the axolotl and facilitates better understanding of metamorphosis.

The colonization of land by a single streptophyte algae lineage some 450 million years ago has been linked to multiple key innovations such as three-dimensional growth, alternation of generations, the presence of stomata, as well as innovations inherent to the birth of major plant lineages, such as the origins of vascular tissues, roots, seeds and flowers. Multicellularity, which evolved multiple times in the Chloroplastida coupled with precise spatiotemporal control of proliferation and differentiation were instrumental for the evolution of these traits. RETINOBLASTOMA-RELATED (RBR), the plant homolog of the metazoan Retinoblastoma protein (pRB), is a highly conserved and multifunctional core cell cycle regulator that has been implicated in the evolution of multicellularity in the green lineage as well as in plant multicellularity-related processes such as proliferation, differentiation, stem cell regulation and asymmetric cell division. RBR fulfills these roles through context-specific protein-protein interactions with proteins containing the Leu-x-Cys-x-Glu (LxCxE) short-linear motif (SLiM); however, how RBR-LxCxE interactions have changed throughout major innovations in the Viridiplantae kingdom is a question that remains unexplored. Here, we employ an in silico evo-devo approach to predict and analyze potential RBR-LxCxE interactions in different representative species of key Chloroplastida lineages, providing a valuable resource for deciphering RBR-LxCxE multiple functions. Furthermore, our analyses suggest that RBR-LxCxE interactions are an important component of RBR functions and that interactions with chromatin modifiers/remodelers, DNA replication and repair machinery are highly conserved throughout the Viridiplantae, while LxCxE interactions with transcriptional regulators likely diversified throughout the water-to-land transition.

Gene expression in roots has been assessed in different plant species in studies ranging from complete organs to specific cell layers, and more recently at the single cell level. While certain genes or functional categories are expressed in the root of all or most plant species, lineage-specific genes have also been discovered. An increasing amount of transcriptomic data is available for angiosperms, while a limited amount of data is available for ferns, and few studies have focused on fern roots. Here, we present a de novo transcriptome assembly from three different parts of the Ceratopteris richardii young sporophyte. Differential gene expression analysis of the root tip transcriptional program showed an enrichment of functional categories related to histogenesis and cell division, indicating an active apical meristem. Analysis of a diverse set of orthologous genes revealed conserved expression in the root meristem, suggesting a preserved role for different developmental roles in this tissue, including stem cell maintenance. The reconstruction of evolutionary trajectories for ground tissue specification genes suggests a high degree of conservation in vascular plants, but not for genes involved in root cap development, showing that certain genes are absent in Ceratopteris or have intricate evolutionary paths difficult to track. Overall, our results suggest different processes of conservation and divergence of genes involved in root development.

BACKGROUND:Limb regeneration in the axolotl is achieved by epimorphosis, thus depending on the blastema formation, a mass of progenitor cells capable of proliferating and differentiating to recover all lost structures functionally. During regeneration, the blastema cells accelerate the cell cycle and duplicate its genome, which is inherently difficult to replicate because of its length and composition, thus being prone to suffer double-strand breaks.RESULTS:We identified and characterized two remarkable components of the homologous recombination repair pathway (Amex.RAD51 and Amex.MRE11), which were heterologously expressed, biochemically characterized, and inhibited by specific chemicals. These same inhibitors were applied at different time points after amputation to study their effects during limb regeneration. We observed an increase in cellular senescent accompanied by a slight delay in regeneration at 28 days postamputation regenerated tissues; moreover, inhibitors caused a rise in the double-strand break signaling as a response to the inhibition of the repair mechanisms.CONCLUSIONS:We confirmed the participation and importance of homologous recombination during limb regeneration. The chemical inhibition induces double-strand breaks that lead to DNA damage associated senescence, or in an alternatively way, this damage could be possibly repaired by a different DNA repair pathway, permitting proper regeneration and avoiding senescence.

Lateral root (LR) formation is an example of a plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE (PKL) negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PKL interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) promoter activity. Since LBD16 function is required for the formative division of LR founder cells, repression mediated by the PKL–RBR1 complex negatively regulates formative division and LR formation. Inhibition of LR formation by PKL–RBR1 is counteracted by auxin, indicating that, in addition to auxin-mediated transcriptional responses, the fine-tuned process of LR formation is also controlled at the chromatin level in an auxin-signaling dependent manner.

PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3'-GTCG-5' DNA sequence, where the 3'-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.