Volatile-Mediated Induced and Passively Acquired Resistance in Sagebrush (Artemisia tridentata).

Plants produce a diversity of secondary metabolites including volatile organic compounds. Some species show discrete variation in these volatile compounds such that individuals within a population can be grouped into distinct chemotypes. A few studies reported that volatile-mediated induced resistance is more effective between plants belonging to the same chemotype and that chemotypes are heritable. The authors concluded that the ability of plants to differentially respond to cues from related individuals that share the same chemotype is a form of kin recognition. These studies assumed plants were actively responding but did not test the mechanism of resistance. A similar result was possible through the passive adsorption and reemission of repellent or toxic VOCs by plants exposed to damage-induced plant volatiles (DIPVs). Here we conducted exposure experiments with five chemotypes of sagebrush in growth chambers; undamaged receiver plants were exposed to either filtered air or DIPVs from mechanically wounded branches. Receiver plants exposed to DIPVs experienced less herbivore damage, which was correlated with increased expression of genes involved in plant defense as well as increased emission of repellent VOCs. Plants belonging to two of the five chemotypes exhibited stronger resistance when exposed to DIPVs from plants of the same chemotypes compared to when DIPVs were from plants of a different chemotype. Moreover, some plants passively absorbed DIPVs and reemitted them, potentially conferring associational resistance. These findings support previous work demonstrating that sagebrush plants actively responded to alarm cues and that the strength of their response was dependent on the chemotypes of the plants involved. This study provides further support for kin recognition in plants but also identified volatile-mediated associational resistance as a passively acquired additional defense mechanism in sagebrush.

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.

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.

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.

Effect of birch (Betula spp.) and associated rhizoidal bacteria on the degradation of soil polyaromatic hydrocarbons, PAH-induced changes in birch proteome and bacterial community.

Two birch clones originating from metal-contaminated sites were exposed for 3 months to soils (sand-peat ratio 1:1 or 4:1) spiked with a mixture of polyaromatic hydrocarbons (PAHs; anthracene, fluoranthene, phenanthrene, pyrene). PAH degradation differed between the two birch clones and also by the soil type. The statistically most significant elimination (p < or = 0.01), i.e. 88% of total PAHs, was observed in the more sandy soil planted with birch, the clearest positive effect being found with Betula pubescens clone on phenanthrene. PAHs and soil composition had rather small effects on birch protein complement. Three proteins with clonal differences were identified: ferritin-like protein, auxin-induced protein and peroxidase. Differences in planted and non-planted soils were detected in bacterial communities by 16S rRNA T-RFLP, and the overall bacterial community structures were diverse. Even though both represent complex systems, trees and rhizoidal microbes in combination can provide interesting possibilities for bioremediation of PAH-polluted soils.

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.

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.

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.

Birch PR-10c is induced by factors causing oxidative stress but appears not to confer tolerance to these agents.

• Expression of all known and newly found pathogenesis-related PR-10 proteins (PR-10a, b, c, d, e) was analysed from Cu-sensitive and -tolerant birch clones to find out whether they follow the same expression pattern. The relationship of PR-10 proteins, particularly PR-10c, to oxidative stress caused by metals or ozone was studied in tolerant and sensitive birch clones to find out possible linkages to tolerance. • Antibody developed to PR-10c was used in Western blot analysis. Other PR-10 proteins were studied with two-dimensional electrophoresis and mass spectrometry. Metal-sensitive yeasts were transformed with PR-10c. • Two new members of PR-10 family, PR-10d and PR-10e, were found. Various PR-10 proteins showed different expression patterns. The amount of PR-10c increased with increasing soil metal concentrations but was, in general, more prominent in Cu-sensitive than in Cu-tolerant clones. PR-10c did not alter metal tolerance in metal-sensitive yeasts. • The PR-10c protein appears not to confer metal- or ozone-tolerance in birch. However, this does not exclude the possibility that it is involved in the tolerance or sensitivity mechanism in an indirect manner.