45S rDNA Diversity In Natura as One Step towards Ribosomal Heterogeneity in Arabidopsis thaliana.

The keystone of ribosome biogenesis is the transcription of 45S rDNA. The Arabidopsis thaliana genome contains hundreds of 45S rDNA units; however, they are not all transcribed. Notably, 45S rDNA units contain insertions/deletions revealing the existence of heterogeneous rRNA genes and, likely, heterogeneous ribosomes for rRNAs. In order to obtain an overall picture of 45S rDNA diversity sustaining the synthesis of rRNAs and, subsequently, of ribosomes in natura, we took advantage of 320 new occurrences of Arabidopsis thaliana as a metapopulation named At66, sampled from 0 to 1900 m of altitude in the eastern Pyrenees in France. We found that the 45S rDNA copy number is very dynamic in natura and identified new genotypes for both 5' and 3' External Transcribed Spacers (ETS). Interestingly, the highest 5'ETS genotype diversity is found in altitude while the highest 3'ETS genotype diversity is found at sea level. Structural analysis of 45S rDNA also shows conservation in natura of specific 5'ETS and 3'ETS sequences/features required to control rDNA expression and the processing of rRNAs. In conclusion, At66 is a worthwhile natural laboratory, and unraveled 45S rDNA diversity represents an interesting starting material to select subsets for rDNA transcription and alter the rRNA composition of ribosomes both intra- and inter-site.

Characterization of the complete plastome of Delphinium montanum, a polyploid, endemic and endangered Pyrenean Larkspur.

Delphinium montanum DC. 1815, is an endangered larkspur endemic to the Eastern Pyrenees. For biogeographic and conservation purpose, a hybrid assembly approach based on long- and short-read genomic data allowed us to successfully assemble whole plastid genome of Delphinium montanum. The complete plastome is 154,185 bp in length, consisting of a pair of inverted repeats (IRs) of 26,559 bp, a large single-copy (LSC) region and a small single-copy region (SSC) of 84,746 and 16,320 bp, respectively. It was found to contain 136 genes, including 84 protein-coding genes, 44 trRNA genes and 8 rRNA genes. The overall GC content of the plastid genome is 38.3%. Phylogenetic inference supports the polyphyly of the Delphinium genus.

Little hope for the polyploid endemic Pyrenean Larkspur (Delphinium montanum): Evidences from population genomics and Ecological Niche Modeling.

Species endemic to restricted geographical ranges represent a particular conservation issue, be it for their heritage interest. In a context of global change, this is particularly the case for plants which belong to high-mountain ecosystems and, because of their ecological requirements, are doomed to survive or disappear on their "sky islands". The Pyrenean Larkspur (Delphinium montanum, Ranunculaceae) is endemic to the Eastern part of the Pyrenees (France and Spain). It is now only observable at a dozen of localities and some populations show signs of decline, such as a recurrent lack of flowering. Implementing population genomics approach (e.g., RAD-seq like) is particularly useful to understand genomic patterns of diversity and differentiation in order to provide recommendations in term of conservation. However, it remains challenging for species such as D. montanum that are autotetraploid with a large genome size (1C-value >10 pg) as most methods currently available were developed for diploid species. A Bayesian framework able to call genotypes with uncertainty allowed us to assess genetic diversity and population structure in this system. Our results show evidence for inbreeding (mean G IS = 0.361) within all the populations and substantial population structure (mean G ST = 0.403) at the metapopulation level. In addition to a lack of connectivity between populations, spatial projections of Ecological Niche Modeling (ENM) analyses under different climatic scenarios predict a dramatic decrease of suitable habitat for D. montanum in the future. Based on these results, we discuss the relevance and feasibility of different conservation measures.

Phenotypic Trait Variation as a Response to Altitude-Related Constraints in Arabidopsis Populations.

Natural variations help in identifying genetic mechanisms of morphologically and developmentally complex traits. Mountainous habitats provide an altitudinal gradient where one species encounters different abiotic conditions. We report the study of 341 individuals of Arabidopsis thaliana derived from 30 natural populations not belonging to the 1001 genomes, collected at increasing altitudes, between 200 and 1800 m in the Pyrenees. Class III peroxidases and ribosomal RNA sequences were used as markers to determine the putative genetic relationships among these populations along their altitudinal gradient. Using Bayesian-based statistics and phylogenetic analyses, these Pyrenean populations appear with significant divergence from the other regional accessions from 1001 genome (i.e., from north Spain or south France). Individuals of these populations exhibited varying phenotypic changes, when grown at sub-optimal temperature (22 vs. 15°C). These phenotypic variations under controlled conditions reflected intraspecific morphological variations. This study could bring new information regarding the west European population structure of A. thaliana and its phenotypic variations at different temperatures. The integrative analysis combining genetic, phenotypic variation and environmental datasets is used to analyze the acclimation of population in response to temperature changes. Regarding their geographical proximity and environmental diversity, these populations represent a tool of choice for studying plant response to temperature variation. HIGHLIGHTS: -Studying the natural diversity of A. thaliana in the Pyrenees mountains helps to understand European population structure and to evaluate the phenotypic trait variation in response to climate change.

Nucleoredoxin guards against oxidative stress by protecting antioxidant enzymes.

Cellular accumulation of reactive oxygen species (ROS) is associated with a wide range of developmental and stress responses. Although cells have evolved to use ROS as signaling molecules, their chemically reactive nature also poses a threat. Antioxidant systems are required to detoxify ROS and prevent cellular damage, but little is known about how these systems manage to function in hostile, ROS-rich environments. Here we show that during oxidative stress in plant cells, the pathogen-inducible oxidoreductase Nucleoredoxin 1 (NRX1) targets enzymes of major hydrogen peroxide (H2O2)-scavenging pathways, including catalases. Mutant nrx1 plants displayed reduced catalase activity and were hypersensitive to oxidative stress. Remarkably, catalase was maintained in a reduced state by substrate-interaction with NRX1, a process necessary for its H2O2-scavenging activity. These data suggest that unexpectedly H2O2-scavenging enzymes experience oxidative distress in ROS-rich environments and require reductive protection from NRX1 for optimal activity.

Nuclear thiol redox systems in plants.

Thiol-disulfide redox regulation is essential for many cellular functions in plants. It has major roles in defense mechanisms, maintains the redox status of the cell and plays structural, with regulatory roles for many proteins. Although thiol-based redox regulation has been extensively studied in subcellular organelles such as chloroplasts, it has been much less studied in the nucleus. Thiol-disulfide redox regulation is dependent on the conserved redox proteins, glutathione/glutaredoxin (GRX) and thioredoxin (TRX) systems. We first focus on the functions of glutathione in the nucleus and discuss recent data concerning accumulation of glutathione in the nucleus. We also provide evidence that glutathione reduction is potentially active in the nucleus. Recent data suggests that the nucleus is enriched in specific GRX and TRX isoforms. We discuss the biochemical and molecular characteristics of these isoforms and focus on genetic evidences for their potential nuclear functions. Finally, we make an overview of the different thiol-based redox regulated proteins in the nucleus. These proteins are involved in various pathways including transcriptional regulation, metabolism and signaling.

NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells.

Thioredoxins (TRX) are key components of cellular redox balance, regulating many target proteins through thiol/disulfide exchange reactions. In higher plants, TRX constitute a complex multigenic family whose members have been found in almost all cellular compartments. Although chloroplastic and cytosolic TRX systems have been largely studied, the presence of a nuclear TRX system has been elusive for a long time. Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms. In contrast to mammals, which harbor a unique NRX, angiosperms generally possess multiple NRX organized in three subfamilies. Here, we show that Arabidopsis thaliana has two NRX genes (AtNRX1 and AtNRX2), respectively, belonging to subgroups I and III. While NRX1 harbors typical TRX active sites (WCG/PPC), NRX2 has atypical active sites (WCRPC and WCPPF). Nevertheless, both NRX1 and NRX2 have disulfide reduction capacities, although NRX1 alone can be reduced by the thioredoxin reductase NTRA. We also show that both NRX1 and NRX2 have a dual nuclear/cytosolic localization. Interestingly, we found that NTRA, previously identified as a cytosolic protein, is also partially localized in the nucleus, suggesting that a complete TRX system is functional in the nucleus. We show that NRX1 is mainly found as a dimer in vivo. nrx1 and nrx2 knockout mutant plants exhibit no phenotypic perturbations under standard growth conditions. However, the nrx1 mutant shows a reduced pollen fertility phenotype, suggesting a specific role of NRX1 at the haploid phase.

Thioredoxin and glutaredoxin systems in plants: molecular mechanisms, crosstalks, and functional significance.

Thioredoxins (Trx) and glutaredoxins (Grx) constitute families of thiol oxidoreductases. Our knowledge of Trx and Grx in plants has dramatically increased during the last decade. The release of the Arabidopsis genome sequence revealed an unexpectedly high number of Trx and Grx genes. The availability of several genomes of vascular and nonvascular plants allowed the establishment of a clear classification of the genes and the chronology of their appearance during plant evolution. Proteomic approaches have been developed that identified the putative Trx and Grx target proteins which are implicated in all aspects of plant growth, including basal metabolism, iron/sulfur cluster formation, development, adaptation to the environment, and stress responses. Analyses of the biochemical characteristics of specific Trx and Grx point to a strong specificity toward some target enzymes, particularly within plastidial Trx and Grx. In apparent contradiction with this specificity, genetic approaches show an absence of phenotype for most available Trx and Grx mutants, suggesting that redundancies also exist between Trx and Grx members. Despite this, the isolation of mutants inactivated in multiple genes and several genetic screens allowed the demonstration of the involvement of Trx and Grx in pathogen response, phytohormone pathways, and at several control points of plant development. Cytosolic Trxs are reduced by NADPH-thioredoxin reductase (NTR), while the reduction of Grx depends on reduced glutathione (GSH). Interestingly, recent development integrating biochemical analysis, proteomic data, and genetics have revealed an extensive crosstalk between the cytosolic NTR/Trx and GSH/Grx systems. This crosstalk, which occurs at multiple levels, reveals the high plasticity of the redox systems in plants.