Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map.

BACKGROUND: The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. RESULTS: The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. CONCLUSIONS: The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

Expression of the GAF Sensor, Carbohydrate-Active Enzymes, Elicitins, and RXLRs Differs Markedly Between Two Phytophthora cactorum Isolates.

The phytopathogen Phytophthora cactorum infects economically important herbaceous and woody plant species. P. cactorum isolates differ in host specificity; for example, strawberry crown rot is often caused by a specialized pathotype. Here we compared the transcriptomes of two P. cactorum isolates that differ in their virulence to garden strawberry (Pc407: high virulence; Pc440: low virulence). De novo transcriptome assembly and clustering of contigs resulted in 19,372 gene clusters. Two days after inoculation of Fragaria vesca roots, 3,995 genes were differently expressed between the P. cactorum isolates. One of the genes that were highly expressed only in Pc407 encodes a GAF sensor protein potentially involved in membrane trafficking processes. Two days after inoculation, elicitins were highly expressed in Pc407 and lipid catabolism appeared to be more active than in Pc440. Of the carbohydrate-active enzymes, those that degrade pectin were often more highly expressed in Pc440, whereas members of glycosyl hydrolase family 1, potentially involved in the metabolism of glycosylated secondary metabolites, were more highly expressed in Pc407 at the time point studied. Differences were also observed among the RXLR effectors: Pc407 appears to rely on a smaller set of key RXLR effectors, whereas Pc440 expresses a greater number of RXLRs. This study is the first step toward improving understanding of the molecular basis of differences in the virulence of P. cactorum isolates. Identification of the key effectors is important, as it enables effector-assisted breeding strategies toward crown rot-resistant strawberry cultivars.

Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

De novo transcriptome assemblies of four accessions of the metal hyperaccumulator plant Noccaea caerulescens.

Noccaea caerulescens of the Brassicaceae family has become the key model plant among the metal hyperaccumulator plants. Populations/accessions of N. caerulescens from geographic locations with different soil metal concentrations differ in their ability to hyperaccumulate and hypertolerate metals. Comparison of transcriptomes in several accessions provides candidates for detailed exploration of the mechanisms of metal accumulation and tolerance and local adaptation. This can have implications in the development of plants for phytoremediation and improved mineral nutrition. Transcriptomes from root and shoot tissues of four N. caerulescens accessions with contrasting Zn, Cd and Ni hyperaccumulation and tolerance traits were sequenced with Illumina Hiseq2000. Transcriptomes were assembled using the Trinity de novo assembler and were annotated and the protein sequences predicted. The comparison against the BUSCO plant early release dataset indicated high-quality assemblies. The predicted protein sequences have been clustered into ortholog groups with closely related species. The data serve as important reference sequences in whole transcriptome studies, in analyses of genetic differences between the accessions and other species, and for primer design.

Gene expression differences between Noccaea caerulescens ecotypes help to identify candidate genes for metal phytoremediation.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

Characterization of the glyoxalase 1 gene TcGLX1 in the metal hyperaccumulator plant Thlaspi caerulescens.

Stress tolerance is currently one of the major research topics in plant biology because of the challenges posed by changing climate and increasing demand to grow crop plants in marginal soils. Increased Zn tolerance and accumulation has been reported in tobacco expressing the glyoxalase 1-encoding gene from Brassica juncea. Previous studies in our laboratory showed some Zn tolerance-correlated differences in the levels of glyoxalase 1-like protein among accessions of Zn hyperaccumulator Thlaspi caerulescens. We have now isolated the corresponding gene (named here TcGLX1), including ca. 570 bp of core and proximal promoter region. The predicted protein contains three glyoxalase 1 motifs and several putative sites for post-translational modification. In silico analysis predicted a number of cis-acting elements related to stress. The expression of TcGLX1 was not responsive to Zn. There was no correlation between the levels of TcGLX1 expression and the degrees of Zn tolerance or accumulation among T. caerulescens accessions nor was there co-segregation of TcGLX1 expression with Zn tolerance or Zn accumulation among F3 lines derived from crosses between plants from accessions with contrasting phenotypes for these properties. No phenotype was observed in an A. thaliana T-DNA insertion line for the closest A. thaliana homolog of TcGLX1, ATGLX1. These results suggest that glyoxalase 1 or at least the particular isoform studied here is not a major determinant of Zn tolerance in the Zn hyperaccumulator plant T. caerulescens. In addition, ATGLX1 is not essential for normal Zn tolerance in the non-tolerant, non-accumulator plant A. thaliana. Possible explanations for the apparent discrepancy between this and previous studies are discussed.

Genetic and environmental influence on maize kernel proteome.

Comparative targeted compositional analysis is currently an important element in the safety assessment of genetically modified plants. Profiling methods have been suggested as nontargeted tools to improve the detection of possible unintended effects. In this study, the capability of 2-dimensional electrophoresis to detect significant differences among seven conventional maize (Zea mays) cultivars grown in six different locations in Germany during two consecutive seasons was evaluated. Besides maize genotype, both geographic location and season had a significant effect on protein profiles. Differences as high as 55- and 53-fold in the quantity of specific proteins were recorded, the median observed difference being around 6- and 5-fold between the genotypes and growing locations, respectively. Understanding the variation in the quantity of individual proteins should help to put the variation of endogenous proteins and the novel proteins in the genetically modified plants in perspective. This together with the targeted analyses the profiling methods, including proteomics, could also help to get a deeper insight into the unintended alterations that might have occurred during the genetic modification process.

Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry (Fragaria x ananassa).

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria x ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus.

Comparative study of sugar fermentation and protein expression patterns of two Lactobacillus plantarum strains grown in three different media.

A comparative study of two strains of Lactobacillus plantarum (REB1 and MLBPL1) grown in commercial medium (MRS broth), cucumber juice, and liquid pig feed was performed to explore changes to the metabolic pathways of these bacteria, using a proteomics approach (two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry) combined with analyses of fermentable sugars and fermentation end products. The protein expression showed that even with an excess of glucose in all media, both strains could metabolize different carbohydrates simultaneously and that hexoses could also be used via a phosphoketolase pathway with preferential expression in liquid feed. Sugar analyses showed that the fermentation of sugars was homolactic for all media, with some heterolactic activity in liquid feed, as shown by the production of acetate. Cucumber juice (the medium with the highest glucose content) showed the lowest hexose consumption (10%), followed by liquid feed (33%) and MRS broth (50%). However, bacterial growth was significantly higher in cucumber juice and liquid feed than in MRS broth. This discrepancy was due to the growth benefit obtained from the utilization of the malate present in cucumber juice and liquid feed. Despite different growth conditions, the synthesis of essential cellular components and the stress response of the bacteria were unaffected. This study has improved our understanding of the mechanisms involved in the growth performance of an appropriate lactic acid bacterium strain to be used for food and feed fermentation, information that is of crucial importance to obtain a high-quality fermented product.

Isolation of genes up-regulated by copper in a copper-tolerant birch (Betula pendula) clone.

Suppression subtractive hybridization (SSH) was used to isolate genes differentially expressed following exposure to copper (Cu) in a naturally selected Cu-tolerant birch (Betula pendula Roth.) clone originating from a disused lead/zinc smelter. Of the 352 cDNA fragments initially isolated, 108 were up-regulated by Cu, of which 55 showed over twofold induction by macroarray analysis. Searches against protein databases (Blastx) and sequence analysis provided the tentative identity of 21 genes. Three fragments lacked homology to any sequences in the databases. Most of the identified genes are involved in cellular transport, regulation or cell rescue and defense. Several genes have not previously been reported to be up-regulated by Cu, e.g., plasma intrinsic protein 2, glutamine synthetase and multi-drug resistance-associated protein (MRP4). The expression of MRP4, a vacuolar sorting receptor-like protein and an unidentified gene was studied in more detail by quantitative real-time PCR. These genes showed stronger up-regulation by Cu in the roots and shoots of the Cu-tolerant birch clone compared with a less tolerant clone. Clear clonal differences in gene expression were observed, e.g., for the regulator of chromosome condensation family protein, DnaJ protein homolog, vacuolar sorting receptor-like protein and MRP4. These findings contribute to our understanding of Cu tolerance in birch, a pioneer plant in metal-contaminated soils.

Comparison of growth-phase-dependent cytosolic proteomes of two Lactobacillus plantarum strains used in food and feed fermentations.

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium highly adapted to a wide variety of environments and widely used in food and feed fermentations. Proteomes of two strains of L. plantarum, one isolated from spontaneously fermented cereal-based feed (strain REB1), and the other from white cabbage (strain MLBPL1), were studied to elucidate the strain-specific variation and the physiological changes occurring between the growth (lag, early-exponential, late-exponential and early-stationary) phases of this bacterium when cultivated in a standard rich medium. A total of 231 protein spots were identified by LC-MS/MS. These proteins showed that strain MLBPL1 had more proteins with growth phase-dependent expression than REB1, which possesses a more constant expression profile. The proteins with growth phase-dependent expression in REB1 and MLBPL1 were mainly associated with energy metabolism (glycolysis, phosphoketolase pathway and ribose metabolism), all having preferential expression in the early-exponential phase, confirming the use of different carbohydrates simultaneously. Indication of energy production was also seen in lag and early-stationary phases.

Effects of agricultural production systems and their components on protein profiles of potato tubers.

A range of studies have compared the level of nutritionally relevant compounds in crops from organic and nonorganic farming systems, but there is very limited information on the effect of farming systems and their key components on the protein composition of plants. We addressed this gap by quantifying the effects of different farming systems and key components of such systems on the protein profiles of potato tubers. Tuber samples were produced in the Nafferton factorial systems study, a group of long-term, replicated factorial field experiments designed to identify and quantify the effect of fertility management methods, crop protection practices and rotational designs used in organic, low input and conventional production systems. Protein profiles were determined by 2-DE and subsequent protein identification by HPLC-ESI-MS/MS. Principal component analysis of 2-DE data showed that only fertility management practices (organic matter vs. mineral fertiliser based) had a significant effect on protein composition. Quantitative differences were detected in 160 of the 1100 tuber proteins separated by 2-DE. Proteins identified by MS are involved in protein synthesis and turnover, carbon and energy metabolism and defence responses, suggesting that organic fertilisation leads to an increased stress response in potato tubers.

Production of transgenic strawberries by temporary immersion bioreactor system and verification by TAIL-PCR.

BACKGROUND: Strawberry (Fragaria x ananassa) is an economically important soft fruit crop with polyploid genome which complicates the breeding of new cultivars. For certain traits, genetic engineering offers a potential alternative to traditional breeding. However, many strawberry varieties are quite recalcitrant for Agrobacterium-mediated transformation, and a method allowing easy handling of large amounts of starting material is needed. Also the genotyping of putative transformants is challenging since the isolation of DNA for Southern analysis is difficult due to the high amount of phenolic compounds and polysaccharides that complicate efficient extraction of digestable DNA. There is thus a need to apply a screening method that is sensitive and unambiguous in identifying the different transformation events. RESULTS: Hygromycin-resistant strawberries were developed in temporary immersion bioreactors by Agrobacterium-mediated gene transfer. Putative transformants were screened by TAIL-PCR to verify T-DNA integration and to distinguish between the individual transformation events. Several different types of border sequence arrangements were detected. CONCLUSION: This study demonstrates that temporary immersion bioreactor system suits well for the regeneration of transgenic strawberry plants as a labour-efficient technique. Small amount of DNA required by TAIL-PCR is easily recovered even from a small transformant, which allows rapid verification of T-DNA integration and detection of separate gene transfer events. These techniques combined clearly facilitate the generation of transgenic strawberries but should be applicable to other plants as well.

Benzothiadiazole induces the accumulation of phenolics and improves resistance to powdery mildew in strawberries.

Benzothiadiazole (BTH) enhanced the accumulation of soluble and cell-wall-bound phenolics in strawberry leaves and also improved the resistance to powdery mildew infection under greenhouse conditions. The most pronounced change was seen in the levels of ellagitannins, which increased up to 2- to 6-fold 4 days after the BTH application, but persisted only in the inoculated plants. The induction of phenolic metabolism by BTH was also reflected in the fruits, several compounds being increased in inoculated, BTH-treated plants. Basal salicylic acid (SA) content was high in strawberry leaves, but increased in a similar fashion to other phenolics after the treatments. Several phenolic compounds were identified in strawberries for the first time. For example, ellagic acid deoxyhexose, three agrimoniin-like ellagitannins, sanguiin H-10- and lambertianin C-like ellagitannins in the leaves, ellagic acid, p-coumaric acid, gallic acid, and kaempferol hexose in the cell-wall-bound fraction of the leaves, and kaempferol malonylglucoside in the fruits. The findings show that BTH can enhance the accumulation of phenolics in strawberry plants which may then be involved in the BTH-induced resistance to powdery mildew.

Proteomic analysis of the potato tuber life cycle.

The tuber of potato (Solanum tuberosum) is commonly used as a model for underground storage organs. In this study, changes in the proteome were followed from tuberization, through tuber development and storage into the sprouting phase. Data interrogation using principal component analysis was able to clearly discriminate between the various stages of the tuber life cycle. Moreover, five well-defined protein expression patterns were found by hierarchical clustering. Altogether 150 proteins showing highly significant differences in abundance between specific stages in the life cycle were highlighted; 59 of these were identified. In addition, 50 proteins with smaller changes in abundance were identified, including several novel proteins. Most noticeably, the development process was characterized by the accumulation of the major storage protein patatin isoforms and enzymes involved in disease and defense reactions. Furthermore, enzymes involved in carbohydrate and energy metabolism and protein processing were associated with development but decreased during tuber maturation. These results represent the first comprehensive picture of many proteins involved in the tuber development and physiology.

Multivariate analysis of protein profiles of metal hyperaccumulator Thlaspi caerulescens accessions.

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyperaccumulation, we used proteomic profiling to identify differences in protein intensities among three T. caerulescens accessions with pronounced differences in tolerance, uptake and root to shoot translocation of Zn and Cd. Proteins were separated using two-dimensional electrophoresis and stained with SYPRO Orange. Intensity values and quality scores were obtained for each spot by using PDQuest software. Principal component analysis was used to test the separation of the protein profiles of the three plant accessions at various metal exposures, and to detect groups of proteins responsible for the differences. Spot sets representing individual proteins were analysed with the analysis of variance and non-parametric Kruskal-Wallis test. Clearest differences were seen among the Thlaspi accessions, while the effects of metal exposures were less pronounced. The 48 tentatively identified spots represent core metabolic functions (e.g. photosynthesis, nitrogen assimilation, carbohydrate metabolism) as well as putative signalling and regulatory functions. The possible roles of some of the proteins in heavy metal accumulation and tolerance are discussed.

Antioxidant capacity and phenolic content of sweet rowanberries.

Sweet rowanberry cultivars adapted to northern climates have been developed from rowanberries (Sorbus aucuparia L.) and hybrids of rowanberry with Malus, Pyrus, Aronia, or Mespilus. The rowanberries studied here (cvs. Burka, Dessertnaja, Eliit, Granatnaja, Kubovaja, Rosina, Rubinovaja, Titan, and Zholtaja) have high antioxidant and phenolic contents. The phenolic content varied between 550 and 1014 mg/100 g of fresh weight in sweet rowanberries, whereas 846 and 717 mg were found in the well-characterized bilberry and lingonberry, respectively. Anthocyanins (6-80 mg) were mainly found from berries of hybrid cultivars. Of the other phenolics, chlorogenic (29-160 mg) and neochlorogenic (34-104 mg) acids constituted the major fraction in all rowanberries, the concentrations almost equaling those present in coffee. Antioxidant capacities of rowanberries were high, as measured with FRAP (61-105 micromol of Fe2+/g) and DPPH (21.3-9.7 g/g DPPH) methods. Principal component analysis was able to separate the cultivars of different origin into clusters on the basis of their phenolic profiles.

Birch PR-10c interacts with several biologically important ligands.

PR-10c is a unique member of PR-10 proteins in birch, since it is the only one known to be post-translationally modified by glutathione and is not constitutively expressed in pollen. Both reduced and S-glutathiolated forms of PR-10c show low ribonuclease activity. However, the major function of the protein is apparently not yet resolved. Our protein-ligand interaction studies with saturation transfer difference (STD) NMR revealed that PR-10c interacts with several biologically important molecules, including cytokinin, flavonoid glycosides, sterols and emodin. Competition study with deoxycholate and kinetin revealed no statistically significant binding interference, indicating that these ligands have different binding sites in PR-10c. Ligand docking studies with a molecular model of PR-10c support the STD NMR results of ligand binding and binding epitopes, suggesting that there are three potential binding sites in PR-10c: two in the hydrophobic cavity and one in the glycine-rich loop. Our docking calculations suggested that only kinetin interacts with the glycine-rich loop, the binding occurring through its adenine moiety. Clear ligand specificity could be observed in the binding of nucleotide derivatives. S-glutathiolation of PR-10c did not affect kinetin binding. The present results suggest that birch PR-10c is a multifunctional protein, which has diverse roles in plant stress responses.