RETINOBLASTOMA-RELATED interactions with key factors of the RNA-directed DNA methylation (RdDM) pathway and its influence on root development.

MAIN CONCLUSION: Our 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.

Predicted landscape of RETINOBLASTOMA-RELATED LxCxE-mediated interactions across the Chloroplastida.

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.

Transcriptional analysis of Ceratopteris richardii young sporophyte reveals conservation of stem cell factors in the root apical meristem.

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.

Cancer in Amphibia, a rare phenomenon?

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.

Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single-cell resolution.

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.

Evidence of requirement for homologous-mediated DNA repair during Ambystoma mexicanum limb regeneration.

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.

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast.

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.

Pickle Recruits Retinoblastoma Related 1 to Control Lateral Root Formation in Arabidopsis.

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.

DNA repair during regeneration in Ambystoma mexicanum.

The remarkable regenerative capabilities of the salamander Ambystoma mexicanum have turned it into one of the principal models to study limb regeneration. During this process, a mass of low differentiated and highly proliferative cells, called blastema, propagates to reestablish the lost tissue in an accelerated way. Such a process implies the replication of a huge genome, 10 times larger than humans, with about 65.6% of repetitive sequences. These features make the axolotl genome inherently difficult to replicate and prone to bear mutations. In this context, the role of DNA repair mechanisms acquires great relevance to maintain genomic stability, especially if we consider the necessity of ensuring the correct replication and integrity of such a large genome in the blastema cells, which are key for tissue regeneration. On the contrary, DNA damage accumulation in these cells may result in senescence, apoptosis and premature differentiation, all of them are mechanisms employed to avoid DNA damage perpetuation but with the potential to affect the limb regeneration process. Here we review and discuss the current knowledge on the implications of DNA damage responses during salamander regeneration.

Development and Cell Cycle Activity of the Root Apical Meristem in the Fern Ceratopteris richardii.

Ferns are a representative clade in plant evolution although underestimated in the genomic era. Ceratopteris richardii is an emergent model for developmental processes in ferns, yet a complete scheme of the different growth stages is necessary. Here, we present a developmental analysis, at the tissue and cellular levels, of the first shoot-borne root of Ceratopteris. We followed early stages and emergence of the root meristem in sporelings. While assessing root growth, the first shoot-borne root ceases its elongation between the emergence of the fifth and sixth roots, suggesting Ceratopteris roots follow a determinate developmental program. We report cell division frequencies in the stem cell niche after detecting labeled nuclei in the root apical cell (RAC) and derivatives after 8 h of exposure. These results demonstrate the RAC has a continuous mitotic activity during root development. Detection of cell cycle activity in the RAC at early times suggests this cell acts as a non-quiescent organizing center. Overall, our results provide a framework to study root function and development in ferns and to better understand the evolutionary history of this organ.

Functional Characterization of the Lin28/let-7 Circuit During Forelimb Regeneration in Ambystoma mexicanum and Its Influence on Metabolic Reprogramming.

The axolotl (Ambystoma mexicanum) is a caudate amphibian, which has an extraordinary ability to restore a wide variety of damaged structures by a process denominated epimorphosis. While the origin and potentiality of progenitor cells that take part during epimorphic regeneration are known to some extent, the metabolic changes experienced and their associated implications, remain unexplored. However, a circuit with a potential role as a modulator of cellular metabolism along regeneration is that formed by Lin28/let-7. In this study, we report two Lin28 paralogs and eight mature let-7 microRNAs encoded in the axolotl genome. Particularly, in the proliferative blastema stage amxLin28B is more abundant in the nuclei of blastemal cells, while the microRNAs amx-let-7c and amx-let-7a are most downregulated. Functional inhibition of Lin28 factors increase the levels of most mature let-7 microRNAs, consistent with an increment of intermediary metabolites of the Krebs cycle, and phenotypic alterations in the outgrowth of the blastema. In summary, we describe the primary components of the Lin28/let-7 circuit and their function during axolotl regeneration, acting upstream of metabolic reprogramming events.

Transcriptional and Morpho-Physiological Responses of Marchantia polymorpha upon Phosphate Starvation.

Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (e.g., PHR1-SPX1 and STOP1-ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration, and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes, and MpMYB14, which is an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting a preference for citrate exudation. An analysis of MpPHR1 binding sequences (P1BS) shows an enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.

Conservation analysis of core cell cycle regulators and their transcriptional behavior during limb regeneration in Ambystoma mexicanum.

Ambystoma mexicanum (axolotl) has been one of the major experimental models for the study of regeneration during the past 100 years. Axolotl limb regeneration takes place through a multi-stage and complex developmental process called epimorphosis that involves diverse events of cell reprogramming. Such events start with dedifferentiation of somatic cells and the proliferation of quiescent stem cells to generate a population of proliferative cells called blastema. Once the blastema reaches a mature stage, cells undergo progressive differentiation into the diverse cell lineages that will form the new limb. Such pivotal cell reprogramming phenomena depend on the fine-tuned regulation of the cell cycle in each regeneration stage, where cell populations display specific proliferative capacities and differentiation status. The axolotl genome has been fully sequenced and released recently, and diverse RNA-seq approaches have also been generated, enabling the identification and conservatory analysis of core cell cycle regulators in this species. We report here our results from such analyses and present the transcriptional behavior of key regulatory factors during axolotl limb regeneration. We also found conserved protein interactions between axolotl Cyclin Dependent Kinases 2, 4 and 6 and Cyclins type D and E. Canonical CYC-CDK interactions that play major roles in modulating cell cycle progression in eukaryotes.

Deep microbial community profiling along the fermentation process of pulque, a biocultural resource of Mexico.

The biggest non-tree perennial plant species endemic to Mexico were called metl in the Nahua culture; during colonial times, renamed with the Antillean word maguey. Carl von Linné finally renamed them as Agave, a Greek-Latin root word meaning admirable. Since pre-Columbian times, one of the major products obtained from some Agave species is the fermented beverage called pulque or octli. This beverage represents an ancient biotechnological development obtained by the natural fermentation of mead from such plants. Pulque played a central role in Mexican pre-Columbian cultures, while in recent times, there has been a renewed interest in it, due to its high content in nutrients and probiotics. In this study, we used massive sequencing of the 16S rRNA gene and the ribosomal internal transcribed spacer (ITS) to profile the pulque microbiome. We identified 2,855 bacteria operational taxonomic units (OTUs) and 1,494 fungi species in the pulque fermentation. Our results provide the most diverse catalog of microbes during pulque production reported so far. These findings allowed us to identify previously unidentified and core microbes resilient during pulque production, with the potential to be used as fermentation stage biomarkers. We confirmed previous reports of pulque microbes and discovered new ones like the bacteria Sphingomonas and Weisella. Among fungi we found that Saccharomyces cerevisiae was second to Candida zemplina in the studied pulque samples.

Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

Emergent Protective Organogenesis in Date Palms: A Morpho-Devo-Dynamic Adaptive Strategy during Early Development.

Desert plants have developed mechanisms for adapting to hostile desert conditions, yet these mechanisms remain poorly understood. Here, we describe two unique modes used by desert date palms (Phoenix dactylifera) to protect their meristematic tissues during early organogenesis. We used x-ray micro-computed tomography combined with high-resolution tissue imaging to reveal that, after germination, development of the embryo pauses while it remains inside a dividing and growing cotyledonary petiole. Transcriptomic and hormone analyses show that this developmental arrest is associated with the low expression of development-related genes and accumulation of hormones that promote dormancy and confer resistance to stress. Furthermore, organ-specific cell-type mapping demonstrates that organogenesis occurs inside the cotyledonary petiole, with identifiable root and shoot meristems and their respective stem cells. The plant body emerges from the surrounding tissues with developed leaves and a complex root system that maximizes efficient nutrient and water uptake. We further show that, similar to its role in Arabidopsis (Arabidopsis thaliana), the SHORT-ROOT homolog from date palms functions in maintaining stem cell activity and promoting formative divisions in the root ground tissue. Our findings provide insight into developmental programs that confer adaptive advantages in desert plants that thrive in hostile habitats.

CONSTITUTIVE TRIPLE RESPONSE1 and PIN2 act in a coordinate manner to support the indeterminate root growth and meristem cell proliferating activity in Arabidopsis seedlings.

The plant hormone ethylene induces auxin biosynthesis and transport and modulates root growth and branching. However, its function on root stem cells and the identity of interacting factors for the control of meristem activity remains unclear. Genetic analysis for primary root growth in wild-type (WT) Arabidopsis thaliana seedlings and ethylene-related mutants showed that the loss-of-function of CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) inhibits cell division and elongation. This phenotype is associated with an increase in the expression of the auxin transporter PIN2 and a drastic decrease in the expression of key factors for stem cell niche maintenance such as PLETHORA1, SHORT ROOT and SCARECROW. While the root stem cell niche is affected in ctr1 mutants, its maintenance is severely compromised in the ctr1-1eir1-1(pin2) double mutant, in which an evident loss of proliferative capacity of the meristematic cells leads to a fully differentiated root meristem shortly after germination. Root traits affected in ctr1-1 mutants could be restored in ctr1-1ein2-1 double mutants. These results reveal that ethylene perception via CTR1 and EIN2 in the root modulates the proliferative capacity of root stem cells via affecting the expression of genes involved in the two major pathways, AUX-PIN-PLT and SCR-SHR, which are key factors for proper root stem cell niche maintenance.

A Phylogenetic Study of the ANT Family Points to a preANT Gene as the Ancestor of Basal and euANT Transcription Factors in Land Plants.

Comparative genomics has revealed that members of early divergent lineages of land plants share a set of highly conserved transcription factors (TFs) with flowering plants. While gene copy numbers have expanded through time, it has been predicted that diversification, co-option, and reassembly of gene regulatory networks implicated in development are directly related to morphological innovations that led to more complex land plant bodies. Examples of key networks have been deeply studied in Arabidopsis thaliana, such as those involving the AINTEGUMENTA (ANT) gene family that encodes AP2-type TFs. These TFs play significant roles in plant development such as the maintenance of stem cell niches, the correct development of the embryo and the formation of lateral organs, as well as fatty acid metabolism. Previously, it has been hypothesized that the common ancestor of mosses and vascular plants encoded two ANT genes that later diversified in seed plants. However, algae and bryophyte sequences have been underrepresented from such phylogenetic analyses. To understand the evolution of ANT in a complete manner, we performed phylogenetic analyses of ANT protein sequences of representative species from across the Streptophyta clade, including algae, liverworts, and hornworts, previously unrepresented. Moreover, protein domain architecture, selection analyses, and regulatory cis elements prediction, allowed us to propose a scenario of how the evolution of ANT genes occurred. In this study we show that a duplication of a preANT-like gene in the ancestor of embryophytes may have given rise to the land plant-exclusive basalANT and euANT lineages. We hypothesize that the absence of euANT-type and basalANT-type sequences in algae, and its presence in extant land plant species, suggests that the divergence of pre-ANT into basal and eu-ANT clades in embryophytes may have influenced the conquest of land by plants, as ANT TFs play important roles in tolerance to desiccation and the establishment, maintenance, and development of complex multicellular structures which either became more complex or appeared in land plants.

Transcriptional profiling of the CAM plant Agave salmiana reveals conservation of a genetic program for regeneration.

In plants, the best characterized plant regeneration process is de novo organogenesis. This type of regeneration is characterized by the formation of a multicellular structure called callus. Calli are induced via phytohormone treatment of plant sections. The callus formation in plants like Agave species with Crassulacean Acid Metabolism (CAM) is poorly studied. In this study, we induced callus formation from Agave salmiana leaves and describe cell arrangement in this tissue. Moreover, we determined and analyzed the transcriptional program of calli, as well as those of differentiated root and leaf tissues, by using RNA-seq. We were able to reconstruct 170,844 transcripts of which 40,644 have a full Open Reading Frame (ORF). The global profile obtained by Next Generation Sequencing (NGS) reveals that several callus-enriched protein coding transcripts are orthologs of previously reported factors highly expressed in Arabidopsis calli. At least 62 genes were differentially expressed in Agave calli, 50 of which were up-regulated. Several of these are actively involved in the perception of, and response to, auxin and cytokinin. Not only are these the first results for the A. salmiana callus, but they provide novel data from roots and leaves of this Agave species, one of the largest non-tree plants in nature.