The meristem-associated endosymbiont Methylorubrum extorquens DSM13060 reprograms development and stress responses of pine seedlings.

Microbes living in plant tissues-endophytes-are mainly studied in crop plants where they typically colonize the root apoplast. Trees-a large carbon source with a high capacity for photosynthesis-provide a variety of niches for endophytic colonization. We have earlier identified a new type of plant-endophyte interaction in buds of adult Scots pine, where Methylorubrum species live inside the meristematic cells. The endosymbiont Methylorubrum extorquens DSM13060 significantly increases needle and root growth of pine seedlings without producing plant hormones, but by aggregating around host nuclei. Here, we studied gene expression and metabolites of the pine host induced by M. extorquens DSM13060 infection. Malic acid was produced by pine to potentially boost M. extorquens colonization and interaction. Based on gene expression, the endosymbiont activated the auxin- and ethylene (ET)-associated hormonal pathways through induction of CUL1 and HYL1, and suppressed salicylic and abscisic acid signaling of pine. Infection by the endosymbiont had an effect on pine meristem and leaf development through activation of GLP1-7 and ALE2, and suppressed flowering, root hair and lateral root formation by downregulation of AGL8, plantacyanin, GASA7, COW1 and RALFL34. Despite of systemic infection of pine seedlings by the endosymbiont, the pine genes CUL1, ETR2, ERF3, HYL, GLP1-7 and CYP71 were highly expressed in the shoot apical meristem, rarely in needles and not in stem or root tissues. Low expression of MERI5, CLH2, EULS3 and high quantities of ononitol suggest that endosymbiont promotes viability and protects pine seedlings against abiotic stress. Our results indicate that the endosymbiont positively affects host development and stress tolerance through mechanisms previously unknown for endophytic bacteria, manipulation of plant hormone signaling pathways, downregulation of senescence and cell death-associated genes and induction of ononitol biosynthesis.

Epigenetic Variability Among Saffron Crocus (Crocus sativus L.) Accessions Characterized by Different Phenotypes.

This work represents the first epigenomic study carried out on saffron crocus. Five accessions of saffron, showing differences in tepal pigmentation, yield of saffron and flowering time, were analyzed at the epigenetic level by applying a methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach. Five accession-specific hypomethylomes plus a reference hypomethylome, generated by combining the sequence data from the single accessions, were obtained. Assembled sequences were annotated against existing online databases. In the absence of the Crocus genome, the rice genome was mainly used as the reference as it is the best annotated genome among monocot plants. Comparison of the hypomethylomes revealed many differentially methylated regions, confirming the high epigenetic variability present among saffron accessions, including sequences encoding for proteins that could be good candidates to explain the accessions' alternative phenotypes. In particular, transcription factors involved in flowering process (MADS-box and TFL) and for the production of pigments (MYB) were detected. Finally, by comparing the generated sequences of the different accessions, a high number of SNPs, likely having arisen as a consequence of the prolonged vegetative propagation, were detected, demonstrating surprisingly high genetic variability. Gene ontology (GO) was performed to map and visualize sequence polymorphisms located within the GOs and to compare their distributions among different accessions. As well as suggesting the possible existence of alternative phenotypes with a genetic basis, a clear difference in polymorphic GO is present among accessions based on their geographic origin, supporting a possible signature of selection in the Indian accession with respect to the Spanish ones.

A first view on the unsuspected intragenus diversity of N-glycans in Chlorella microalgae.

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health-promoting food supplements. A mass spectrometry-based survey of N-glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N-glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N-acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan-type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4-O-methyl-N-acetylglucosamine), and substitution of the second N-acetylglucosamine. Analysis of barcode ITS1-5.8S-ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N-glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella-like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N-glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.

Elucidating Drought Stress Tolerance in European Oaks Through Cross-Species Transcriptomics.

The impact of climate change that comes with a dramatic increase of long periods of extreme summer drought associated with heat is a fundamental challenge for European forests. As a result, forests are expected to shift their distribution patterns toward north-east, which may lead to a dramatic loss in value of European forest land. Consequently, unraveling key processes that underlie drought stress tolerance is not only of great scientific but also of utmost economic importance for forests to withstand future heat and drought wave scenarios. To reveal drought stress-related molecular patterns we applied cross-species comparative transcriptomics of three major European oak species: the less tolerant deciduous pedunculate oak (Quercus robur), the deciduous but quite tolerant pubescent oak (Q. pubescens), and the very tolerant evergreen holm oak (Q. ilex). We found 415, 79, and 222 differentially expressed genes during drought stress in Q. robur, Q. pubescens, and Q. ilex, respectively, indicating species-specific response mechanisms. Further, by comparative orthologous gene family analysis, 517 orthologous genes could be characterized that may play an important role in drought stress adaptation on the genus level. New regulatory candidate pathways and genes in the context of drought stress response were identified, highlighting the importance of the antioxidant capacity, the mitochondrial respiration machinery, the lignification of the water transport system, and the suppression of drought-induced senescence - providing a valuable knowledge base that could be integrated in breeding programs in the face of climate change.

Drought Sensitivity of Norway Spruce at the Species' Warmest Fringe: Quantitative and Molecular Analysis Reveals High Genetic Variation Among and Within Provenances.

Norway spruce (Picea abies) is by far the most important timber species in Europe, but its outstanding role in future forests is jeopardized by its high sensitivity to drought. We analyzed drought response of Norway spruce at the warmest fringe of its natural range. Based on a 35-year old provenance experiment we tested for genetic variation among and within seed provenances across consecutively occurring strong drought events using dendroclimatic time series. Moreover, we tested for associations between ≈1,700 variable SNPs and traits related to drought response, wood characteristics and climate-growth relationships. We found significant adaptive genetic variation among provenances originating from the species' Alpine, Central and Southeastern European range. Genetic variation between individuals varied significantly among provenances explaining up to 44% of the phenotypic variation in drought response. Varying phenotypic correlations between drought response and wood traits confirmed differences in selection intensity among seed provenances. Significant associations were found between 29 SNPs and traits related to drought, climate-growth relationships and wood properties which explained between 11 and 43% of trait variation, though 12 of them were due to single individuals having extreme phenotypes of the respective trait. The majority of these SNPs are located within exons of genes and the most important ones are preferentially expressed in cambium and xylem expansion layers. Phenotype-genotype associations were stronger if only provenances with significant quantitative genetic variation in drought response were considered. The present study confirms the high adaptive variation of Norway spruce in Central and Southeastern Europe and demonstrates how quantitative genetic, dendroclimatic and genomic data can be linked to understand the genetic basis of adaptation to climate extremes in trees.

The promises of microalgae-still a long way to go.

Over the past decades, interest in microalgae has grown exponentially. Especially industrial applications, such as biodiesel production from microalgae were hyped in the early 2000s. But since the first attempts to commercialise microalgae-derived products for energy applications, only a few developments made it to the market. What is hindering the full exploitation of this valuable resource for food, feed, and energy applications? This opinion paper reflects on the state of the art of industrial microalgae culturing and the knowledge gaps which still need to be filled jointly by academia and industry.

Association genetics of phenolic needle compounds in Norway spruce with variable susceptibility to needle bladder rust.

KEY MESSAGE: Accumulation of phenolic needle metabolites in Norway spruce is regulated by many genes with small and additive effects and is correlated with the susceptibility against fungal attack. Norway spruce accumulates high foliar concentrations of secondary phenolic metabolites, with important functions for pathogen defence responses. However, the molecular genetic basis underlying the quantitative variation of phenolic compounds and their role in enhanced resistance of spruce to infection by needle bladder rust are unknown. To address these questions, a set of 1035 genome-wide single nucleotide polymorphisms (SNPs) was associated to the quantitative variation of four simple phenylpropanoids, eight stilbenes, nine flavonoids, six related arithmetic parameters and the susceptibility to infection by Chrysomyxa rhododendri in an unstructured natural population of Norway spruce. Thirty-one significant genetic associations for the flavonoids gallocatechin, kaempferol 3-glucoside and quercetin 3-glucoside and the stilbenes resveratrol, piceatannol, astringin and isorhapontin were discovered, explaining 22-59% of phenotypic variation, and indicating a regulation of phenolic accumulation by many genes with small and additive effects. The phenolics profile differed between trees with high and low susceptibility to the fungus, underlining the importance of phenolic compounds in the defence mechanisms of Norway spruce to C. rhododendri. Results highlight the utility of association studies in non-model tree species and may enable marker-assisted selection of Norway spruce adapted to severe pathogen attack.

How to Isolate a Plant's Hypomethylome in One Shot.

Genome assembly remains a challenge for large and/or complex plant genomes due to their abundant repetitive regions resulting in studies focusing on gene space instead of the whole genome. Thus, DNA enrichment strategies facilitate the assembly by increasing the coverage and simultaneously reducing the complexity of the whole genome. In this paper we provide an easy, fast, and cost-effective variant of MRE-seq to obtain a plant's hypomethylome by an optimized methyl filtration protocol followed by next generation sequencing. The method is demonstrated on three plant species with knowingly large and/or complex (polyploid) genomes: Oryza sativa, Picea abies, and Crocus sativus. The identified hypomethylomes show clear enrichment for genes and their flanking regions and clear reduction of transposable elements. Additionally, genomic sequences around genes are captured including regulatory elements in introns and up- and downstream flanks. High similarity of the results obtained by a de novo assembly approach with a reference based mapping in rice supports the applicability for studying and understanding the genomes of nonmodel organisms. Hence we show the high potential of MRE-seq in a wide range of scenarios for the direct analysis of methylation differences, for example, between ecotypes, individuals, within or across species harbouring large, and complex genomes.

Control of origin of sesame oil from various countries by stable isotope analysis and DNA based markers--a pilot study.

The indication of origin of sesame seeds and sesame oil is one of the important factors influencing its price, as it is produced in many regions worldwide and certain provenances are especially sought after. We joined stable carbon and hydrogen isotope analysis with DNA based molecular marker analysis to study their combined potential for the discrimination of different origins of sesame seeds. For the stable carbon and hydrogen isotope data a positive correlation between both isotope parameters was observed, indicating a dominant combined influence of climate and water availability. This enabled discrimination between sesame samples from tropical and subtropical/moderate climatic provenances. Carbon isotope values also showed differences between oil from black and white sesame seeds from identical locations, indicating higher water use efficiency of plants producing black seeds. DNA based markers gave independent evidence for geographic variation as well as provided information on the genetic relatedness of the investigated samples. Depending on the differences in ambient environmental conditions and in the genotypic fingerprint, a combination of both analytical methods is a very powerful tool to assess the declared geographic origin. To our knowledge this is the first paper on food authenticity combining the stable isotope analysis of bio-elements with DNA based markers and their combined statistical analysis.

The oak gene expression atlas: insights into Fagaceae genome evolution and the discovery of genes regulated during bud dormancy release.

BACKGROUND: Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. RESULTS: The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. CONCLUSION: In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes.

Epigenetic regulation of adaptive responses of forest tree species to the environment.

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular "memory". Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

Towards decoding the conifer giga-genome.

Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world's conifer forests.

Allele discovery of ten candidate drought-response genes in Austrian oak using a systematically informatics approach based on 454 amplicon sequencing.

BACKGROUND: Rise of temperatures and shortening of available water as result of predicted climate change will impose significant pressure on long-lived forest tree species. Discovering allelic variation present in drought related genes of two Austrian oak species can be the key to understand mechanisms of natural selection and provide forestry with key tools to cope with future challenges. RESULTS: In the present study we have used Roche 454 sequencing and developed a bioinformatic pipeline to process multiplexed tagged amplicons in order to identify single nucleotide polymorphisms and allelic sequences of ten candidate genes related to drought/osmotic stress from sessile oak (Quercus robur) and sessile oak (Q. petraea) individuals. Out of these, eight genes of 336 oak individuals growing in Austria have been detected with a total number of 158 polymorphic sites. Allele numbers ranged from ten to 52 with observed heterozygosity ranging from 0.115 to 0.640. All loci deviated from Hardy-Weinberg equilibrium and linkage disequilibrium was found among six combinations of loci. CONCLUSIONS: We have characterized 183 alleles of drought related genes from oak species and detected first evidences of natural selection. Beside the potential for marker development, we have created an expandable bioinformatic pipeline for the analysis of next generation sequencing data.

Sequence composition and gene content of the short arm of rye (Secale cereale) chromosome 1.

BACKGROUND: The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale) with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3%) being the most abundant. More than four thousand simple sequence repeat (SSR) sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice. CONCLUSIONS: The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye.

Characterization of variable EST SSR markers for Norway spruce (Picea abies L.).

BACKGROUND: Norway spruce is widely distributed across Europe and the predominant tree of the Alpine region. Fast growth and the fact that timber can be harvested cost-effectively in relatively young populations define its status as one of the economically most important tree species of Northern Europe. In this study, EST derived simple sequence repeat (SSR) markers were developed for the assessment of putative functional diversity in Austrian Norway spruce stands. RESULTS: SSR sequences were identified by analyzing 14,022 publicly available EST sequences. Tri-nucleotide repeat motifs were most abundant in the data set followed by penta- and hexa-nucleotide repeats. Specific primer pairs were designed for sixty loci. Among these, 27 displayed polymorphism in a testing population of 16 P. abies individuals sampled across Austria and in an additional screening population of 96 P. abies individuals from two geographically distinct Austrian populations. Allele numbers per locus ranged from two to 17 with observed heterozygosity ranging from 0.075 to 0.99. CONCLUSIONS: We have characterized variable EST SSR markers for Norway spruce detected in expressed genes. Due to their moderate to high degree of variability in the two tested screening populations, these newly developed SSR markers are well suited for the analysis of stress related functional variation present in Norway spruce populations.

A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation.

Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

Microsatellite markers in the tree peony, Paeonia suffruticosa (Paeoniaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed in the tree peony, Paeonia suffruticosa, to perform paternity tests as well as assignment to variety in special Austrian collections. • METHODS AND RESULTS: Using SSR-enriched libraries and EST-mining, 8 polymorphic primer sets were identified in Austrian collections of Paeonia sect. Moutan DC. The primers amplified di- and trinucleotide repeats with 2-6 alleles per locus. All primers also amplified in P. ostii, P. pontaninii var. trolloides, P. delavayi, and P. lutea, and in the herbaceous species P. peregrina and P. tenuifolia (Paeonia sect. Paeon). • CONCLUSIONS: These results show the usefulness of primers in P. suffruticosa for population genetic studies and their ability to cross amplify in related taxa across the genus.

Transcriptomic changes in wind-exposed poplar leaves are dependent on developmental stage.

Responses of plant tissue to environmental challenges can vary among different plant parts and among plants of different ages. Investment into defense has been proposed to be influenced by fitness value and/or allocation of available resources. Here we show at first time at transcriptome level that plant defense is non-linear. On very young, expanding, adult and old leaves of Populus nigra plants exposed to air perturbation, we studied the ontogenic trajectory of gene expression changes to such a low-dose factor similar to wind. Although plant responses to mechanical sensation (wind, touch) are described and summarized as thigmomorphogenesis, the knowledge on the molecular background of plant responses to wind is largely incomplete. Our data describe which genes are activated during a ubiquitous and continuous environmental factor such as wind, and based on existing knowledge complement the picture on ongoing processes.

Transcript profiling of poplar leaves upon infection with compatible and incompatible strains of the foliar rust Melampsora larici-populina.

To understand key processes governing defense mechanisms in poplar (Populus spp.) upon infection with the rust fungus Melampsora larici-populina, we used combined histological and molecular techniques to describe the infection of Populus trichocarpa x Populus deltoides 'Beaupré' leaves by compatible and incompatible fungal strains. Striking differences in host-tissue infection were observed after 48-h postinoculation (hpi) between compatible and incompatible interactions. No reactive oxygen species production could be detected at infection sites, while a strong accumulation of monolignols occurred in the incompatible interaction after 48 hpi, indicating a late plant response once the fungus already penetrated host cells to form haustorial infection structures. P. trichocarpa whole-genome expression oligoarrays and sequencing of cDNAs were used to determine changes in gene expression in both interactions at 48 hpi. Temporal expression profiling of infection-regulated transcripts was further compared by cDNA arrays and reverse transcription-quantitative polymerase chain reaction. Among 1,730 significantly differentially expressed transcripts in the incompatible interaction, 150 showed an increase in concentration > or =3-fold, whereas 62 were decreased by > or =3-fold. Regulated transcripts corresponded to known genes targeted by R genes in plant pathosystems, such as inositol-3-P synthase, glutathione S-transferases, and pathogenesis-related proteins. However, the transcript showing the highest rust-induced up-regulation encodes a putative secreted protein with no known function. In contrast, only a few transcripts showed an altered expression in the compatible interaction, suggesting a delay in defense response between incompatible and compatible interactions in poplar. This comprehensive analysis of early molecular responses of poplar to M. larici-populina infection identified key genes that likely contain the fungus proliferation in planta. Sequences from the SSH library described in this article can be retrieved in GenBank under accession numbers CT 027996 to CT 029994 and CT 033829.

A genomic approach to suberin biosynthesis and cork differentiation.

Cork (phellem) is a multilayered dead tissue protecting plant mature stems and roots and plant healing tissues from water loss and injuries. Cork cells are made impervious by the deposition of suberin onto cell walls. Although suberin deposition and cork formation are essential for survival of land plants, molecular studies have rarely been conducted on this tissue. Here, we address this question by combining suppression subtractive hybridization together with cDNA microarrays, using as a model the external bark of the cork tree (Quercus suber), from which bottle cork is obtained. A suppression subtractive hybridization library from cork tree bark was prepared containing 236 independent sequences; 69% showed significant homology to database sequences and they corresponded to 135 unique genes. Out of these genes, 43.5% were classified as the main pathways needed for cork biosynthesis. Furthermore, 19% could be related to regulatory functions. To identify genes more specifically required for suberin biosynthesis, cork expressed sequence tags were printed on a microarray and subsequently used to compare cork (phellem) to a non-suberin-producing tissue such as wood (xylem). Based on the results, a list of candidate genes relevant for cork was obtained. This list includes genes for the synthesis, transport, and polymerization of suberin monomers such as components of the fatty acid elongase complexes, ATP-binding cassette transporters, and acyltransferases, among others. Moreover, a number of regulatory genes induced in cork have been identified, including MYB, No-Apical-Meristem, and WRKY transcription factors with putative functions in meristem identity and cork differentiation.