Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass.

Long-term climate change and periodic environmental extremes threaten food and fuel security1 and global crop productivity2-4. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience5, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation6-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.

Plant telomere biology: The green solution to the end-replication problem.

Telomere maintenance is a fundamental cellular process conserved across all eukaryotic lineages. Although plants and animals diverged over 1.5 billion years ago, lessons learned from plants continue to push the boundaries of science, revealing detailed molecular mechanisms in telomere biology with broad implications for human health, aging biology, and stress responses. Recent studies of plant telomeres have unveiled unexpected divergence in telomere sequence and architecture, and the proteins that engage telomeric DNA and telomerase. The discovery of telomerase RNA components in the plant kingdom and some algae groups revealed new insight into the divergent evolution and the universal core of telomerase across major eukaryotic kingdoms. In addition, resources cataloging the abundant natural variation in Arabidopsis thaliana, maize (Zea mays), and other plants are providing unparalleled opportunities to understand the genetic networks that govern telomere length polymorphism and, as a result, are uncovering unanticipated crosstalk between telomeres, environmental factors, organismal fitness, and plant physiology. Here we recap current advances in plant telomere biology and put this field in perspective relative to telomere and telomerase research in other eukaryotic lineages.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Natural variation in plant telomere length is associated with flowering time.

Telomeres are highly repetitive DNA sequences found at the ends of chromosomes that protect the chromosomes from deterioration duringcell division. Here, using whole-genome re-sequencing and terminal restriction fragment assays, we found substantial natural intraspecific variation in telomere length in Arabidopsis thaliana, rice (Oryza sativa), and maize (Zea mays). Genome-wide association study (GWAS) mapping in A. thaliana identified 13 regions with GWAS-significant associations underlying telomere length variation, including a region that harbors the telomerase reverse transcriptase (TERT) gene. Population genomic analysis provided evidence for a selective sweep at the TERT region associated with longer telomeres. We found that telomere length is negatively correlated with flowering time variation not only in A. thaliana, but also in maize and rice, indicating a link between life-history traits and chromosome integrity. Our results point to several possible reasons for this correlation, including the possibility that longer telomeres may be more adaptive in plants that have faster developmental rates (and therefore flower earlier). Our work suggests that chromosomal structure itself might be an adaptive trait associated with plant life-history strategies.

Telomere biology and ribosome biogenesis: structural and functional interconnections.

Telomeres are nucleoprotein structures that play a pivotal role in the protection and maintenance of eukaryotic chromosomes. Telomeres and the enzyme telomerase, which replenishes telomeric DNA lost during replication, are important factors necessary to ensure continued cell proliferation. Cell proliferation is also dependent on proper and efficient protein synthesis, which is carried out by ribosomes. Mutations in genes involved in either ribosome biogenesis or telomere biology result in cellular abnormalities and can cause human genetic diseases, defined as ribosomopathies and telomeropathies, respectively. Interestingly, recent discoveries indicate that many of the ribosome assembly and rRNA maturation factors have additional noncanonical functions in telomere biology. Similarly, several key proteins and enzymes involved in telomere biology, including telomerase, have unexpected roles in rRNA transcription and maturation. These observations point to an intriguing cross-talk mechanism potentially explaining the multiple pleiotropic symptoms of mutations in many causal genes identified in various telomeropathy and ribosomopathy diseases. In this review, we provide a brief summary of eukaryotic telomere and rDNA loci structures, highlight several universal features of rRNA and telomerase biogenesis, evaluate intriguing interconnections between telomere biology and ribosome assembly, and conclude with an assessment of overlapping features of human diseases of telomeropathies and ribosomopathies.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Plasticity, pleiotropy and fitness trade-offs in Arabidopsis genotypes with different telomere lengths.

Telomere length has been implicated in the organismal response to stress, but the underlying mechanisms are unknown. Here we examine the impact of telomere length changes on the responses to three contrasting abiotic environments in Arabidopsis, and measure 32 fitness, developmental, physiological and leaf-level anatomical traits. We report that telomere length in wild-type and short-telomere mutants is resistant to abiotic stress, while the elongated telomeres in ku70 mutants are more plastic. We detected significant pleiotropic effects of telomere length on flowering time and key leaf physiological and anatomical traits. Furthermore, our data reveal a significant genotype by environment (G × E) interaction for reproductive fitness, with the benefits and costs to performance depending on the growth conditions. These results imply that life-history trade-offs between flowering time and reproductive fitness are impacted by telomere length variation. We postulate that telomere length in plants is subject to natural selection imposed by different environments.

Drought responsive gene expression regulatory divergence between upland and lowland ecotypes of a perennial C4 grass.

Climatic adaptation is an example of a genotype-by-environment interaction (G×E) of fitness. Selection upon gene expression regulatory variation can contribute to adaptive phenotypic diversity; however, surprisingly few studies have examined how genome-wide patterns of gene expression G×E are manifested in response to environmental stress and other selective agents that cause climatic adaptation. Here, we characterize drought-responsive expression divergence between upland (drought-adapted) and lowland (mesic) ecotypes of the perennial C4 grass,Panicum hallii, in natural field conditions. Overall, we find that cis-regulatory elements contributed to gene expression divergence across 47% of genes, 7.2% of which exhibit drought-responsive G×E. While less well-represented, we observe 1294 genes (7.8%) with transeffects.Trans-by-environment interactions are weaker and much less common than cis G×E, occurring in only 0.7% oft rans-regulated genes. Finally, gene expression heterosis is highly enriched in expression phenotypes with significant G×E. As such, modes of inheritance that drive heterosis, such as dominance or overdominance, may be common among G×E genes. Interestingly, motifs specific to drought-responsive transcription factors are highly enriched in the promoters of genes exhibiting G×E and transregulation, indicating that expression G×E and heterosis may result from the evolution of transcription factors or their binding sites.P. hallii serves as the genomic model for its close relative and emerging biofuel crop, switchgrass (Panicum virgatum). Accordingly, the results here not only aid in the discovery of the genetic mechanisms that underlie local adaptation but also provide a foundation to improve switchgrass yield under water-limited conditions.

Promises and Challenges of Eco-Physiological Genomics in the Field: Tests of Drought Responses in Switchgrass.

Identifying the physiological and genetic basis of stress tolerance in plants has proven to be critical to understanding adaptation in both agricultural and natural systems. However, many discoveries were initially made in the controlled conditions of greenhouses or laboratories, not in the field. To test the comparability of drought responses across field and greenhouse environments, we undertook three independent experiments using the switchgrass reference genotype Alamo AP13. We analyzed physiological and gene expression variation across four locations, two sampling times, and three years. Relatively similar physiological responses and expression coefficients of variation across experiments masked highly dissimilar gene expression responses to drought. Critically, a drought experiment utilizing small pots in the greenhouse elicited nearly identical physiological changes as an experiment conducted in the field, but an order of magnitude more differentially expressed genes. However, we were able to define a suite of several hundred genes that were differentially expressed across all experiments. This list was strongly enriched in photosynthesis, water status, and reactive oxygen species responsive genes. The strong across-experiment correlations between physiological plasticity-but not differential gene expression-highlight the complex and diverse genetic mechanisms that can produce phenotypically similar responses to various soil water deficits.

Evolution of the Telomere-Associated Protein POT1a in Arabidopsis thaliana Is Characterized by Positive Selection to Reinforce Protein-Protein Interaction.

Gene duplication is a major driving force in genome evolution. Here, we explore the nature and origin of the POT1 gene duplication in Arabidopsis thaliana. Protection of Telomeres (POT1) is a conserved multifunctional protein that modulates telomerase activity and its engagement with telomeres. Arabidopsis thaliana encodes two divergent POT1 paralogs termed AtPOT1a and AtPOT1b. AtPOT1a positively regulates telomerase activity, whereas AtPOT1b is proposed to negatively regulate telomerase and promote chromosome end protection. Phylogenetic analysis uncovered two independent POT1 duplication events in the plant kingdom, including one at the base of Brassicaceae. Tests for positive selection implemented in PAML revealed that the Brassicaceae POT1a lineage experienced positive selection postduplication and identified three amino acid residues with signatures of positive selection. A sensitive and quantitative genetic complementation assay was developed to assess POT1a function in A. thaliana. The assay showed that AtPOT1a is functionally distinct from single-copy POT1 genes in other plants. Moreover, for two of the sites with a strong signature of positive selection, substitutions that swap the amino acids in AtPOT1a for residues found in AtPOT1b dramatically compromised AtPOT1a function in vivo. In vitro-binding studies demonstrated that all three sites under positive selection specifically enhance the AtPOT1a interaction with CTC1, a core component of the highly conserved CST (CTC1/STN1/TEN1) telomere protein complex. Our results reveal a molecular mechanism for the role of these positively selected sites in AtPOT1a. The data also provide an important empirical example to refine theories of duplicate gene retention, as the outcome of positive selection here appears to be reinforcement of an ancestral function, rather than neofunctionalization. We propose that this outcome may not be unusual when the duplicated protein is a component of a multisubunit complex whose function is in part specified by other members.

Gene networks and chromatin and transcriptional regulation of the phaseolin promoter in Arabidopsis.

The complete lack of seed storage protein expression in vegetative tissues and robust expression during embryogenesis makes seed development an ideal system to study tissue-specific expression of genes. The promoter for the Phaseolin (phas) gene, which encodes the major seed storage protein in bean (Phaseolus vulgaris), is activated in two sequential steps: Phaseolus vulgaris ABI3-like factor (Pv-ALF)-dependent potentiation and abscisic acid-mediated activation. In this study, a heterologous in vivo Pv-ALF/phas-GUS (for ß-glucuronidase) expression system in transgenic Arabidopsis thaliana leaves was used in conjunction with the powerful RNA-Seq approach to capture transcriptional landscapes of phas promoter expression. Remarkably, expression of over 1300 genes from 11 functional categories coincided with changes in the transcriptional status of the phas promoter. Gene network analysis of induced genes and artificial microRNA-mediated loss-of-function genetic assays identified transcriptional regulators RINGLET 2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5) as being essential for phas transcription. Pv-ALF binding to the RLT2 and AIL5 promoter regions was confirmed by electrophoretic mobility shift assay. RLT2 and AIL5 knockdown lines displayed reduced expression of several endogenous seed genes, suggesting that these factors are involved in activation of endogenous Arabidopsis seed storage gene expression. Overall, the identification of these key factors involved in phas activation provides important insight into the two-step transcriptional regulation of seed-specific gene expression.

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1.

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus).

Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3x draft genome sequence of 'SunUp' papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica's distinguishing morpho-physiological, medicinal and nutritional properties.

Telomere Length Variation in Model Bryophytes.

The ends of linear chromosomes of most eukaryotes consist of protein-bound DNA arrays called telomeres, which play essential roles in protecting genome integrity. Despite general evolutionary conservation in function, telomeric DNA is known to drastically vary in length and sequence between different eukaryotic lineages. Bryophytes are a group of early diverging land plants that include mosses, liverworts, and hornworts. This group of ancient land plants recently emerged as a new model for important discoveries in genomics and evolutionary biology, as well as for understanding plant adaptations to a terrestrial lifestyle. We measured telomere length in different ecotypes of model bryophyte species, including Physcomitrium patens, Marchantia polymorpha, Ceratodon purpureus, and in Sphagnum isolates. Our data indicate that all analyzed moss and liverwort genotypes have relatively short telomeres. Furthermore, all analyzed ecotypes and isolates of model mosses and liverworts display evidence of substantial natural variation in telomere length. Interestingly, telomere length also differs between male and female strains of the dioecious liverwort M. polymorpha and dioecious moss C. purpureus. Given that bryophytes are extraordinarily well adapted to different ecological niches from polar to tropical environments, our data will contribute to understanding the impact of natural telomere length variation on evolutionary adaptations in this ancient land plant lineage.

Protection of Telomeres 1 is required for telomere integrity in the moss Physcomitrella patens.

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.

Identification and Analysis of Antimicrobial Activities from a Model Moss Ceratodon purpureus.

The emergence of bacterial drug resistance is often viewed as the next great health crisis of our time. While more antimicrobial agents are urgently needed, very few new antibiotics are currently in the production pipeline. Here, we aim to identify and characterize novel antimicrobial natural products from a model dioicous moss, Ceratodon purpureus. We collected secreted moss exudate fractions from two C. purpureus strains, male R40 and female GG1. Exudates from the female C. purpureus strain GG1 did not exhibit inhibitory activity against any tested bacteria. However, exudates from the male moss strain R40 exhibited strong inhibitory properties against several species of Gram-positive bacteria, including Staphylococcus aureus and Enterococcus faecium, though they did not inhibit the growth of Gram-negative bacteria. Antibacterial activity levels in C. purpureus R40 exudates significantly increased over four weeks of moss cultivation in liquid culture. Size fractionation experiments indicated that the secreted bioactive compounds have a relatively low molecular weight of less than 1 kDa. Additionally, the R40 exudate compounds are thermostable and not sensitive to proteinase K treatment. Overall, our results suggest that the bioactive compounds present in C. purpureus R40 exudates can potentially add new options for treating infections caused by antibiotic-resistant Gram-positive bacteria.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a - deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Antimicrobial Activities of Secondary Metabolites from Model Mosses.

Plants synthetize a large spectrum of secondary metabolites with substantial structural and functional diversity, making them a rich reservoir of new biologically active compounds. Among different plant lineages, the evolutionarily ancient branch of non-vascular plants (Bryophytes) is of particular interest as these organisms produce many unique biologically active compounds with highly promising antibacterial properties. Here, we characterized antibacterial activity of metabolites produced by different ecotypes (strains) of the model mosses Physcomitrium patens and Sphagnum fallax. Ethanol and hexane moss extracts harbor moderate but unstable antibacterial activity, representing polar and non-polar intracellular moss metabolites, respectively. In contrast, high antibacterial activity that was relatively stable was detected in soluble exudate fractions of P. patens moss. Antibacterial activity levels in P. patens exudates significantly increased over four weeks of moss cultivation in liquid culture. Interestingly, secreted moss metabolites are only active against a number of Gram-positive, but not Gram-negative, bacteria. Size fractionation, thermostability and sensitivity to proteinase K assays indicated that the secreted bioactive compounds are relatively small (less than <10 kDa). Further analysis and molecular identification of antibacterial exudate components, combined with bioinformatic analysis of model moss genomes, will be instrumental in the identification of specific genes involved in the bioactive metabolite biosynthesis.

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA.

Telomeric DNA terminates with a single-stranded 3' G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1-DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.