Elevation of night-time temperature increases terpenoid emissions from Betula pendula and Populus tremula.

Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 degrees C) while daytime temperature was kept at a constant 22 degrees C. VOC emissions were collected during the daytime and analysed by gas chromatography-mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and beta-ocimene increased from 6 degrees C to 14 degrees C, while several other monoterpenes and the SQTs (Z,E)-alpha-farnesene and (E,E)-alpha-farnesene increased up to 18 degrees C. Total monoterpene and sesquiterpene emission peaked at 18 degrees C, whereas isoprene emissions decreased at 22 degrees C. Leaf area increased across the temperature range of 6-22 degrees C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees.

Proteomics of Thlaspi caerulescens accessions and an inter-accession cross segregating for zinc accumulation.

Metal hyperaccumulator plants have previously been characterized by transcriptomics, but reports on other profiling techniques are scarce. Protein profiles of Thlaspi caerulescens accessions La Calamine (LC) and Lellingen (LE) and lines derived from an LCxLE cross were examined here to determine the co-segregation of protein expression with the level of zinc (Zn) hyperaccumulation. Although hydrophobic proteins such as membrane transporters are not disclosed, this approach has the potential to reveal other proteins important for the Zn hyperaccumulation trait. Plants were exposed to metals. Proteins were separated using two-dimensional electrophoresis and those showing differences among accessions, lines or metal exposures were subjected to mass-spectrometric analysis for identification. Crossing decreased the number of different proteins in the lines compared with the parents, more so in the shoots than in the roots, but the frequencies of Zn-responsive proteins were about the same in the accessions and the selection lines. This supports the finding that the Zn accumulation traits are mainly determined by the root and that Zn accumulation itself is not the reason for the co-segregation. This study demonstrates that crossing accessions with contrasting Zn accumulation traits is a potent tool to investigate the mechanisms behind metal hyperaccumulation. Four tentatively identified root proteins showed co-segregation with high or low Zn accumulation: manganese superoxide dismutase, glutathione S-transferase, S-formyl glutathione hydrolase, and translation elongation factor 5A-2. However, these proteins may not be the direct determinants of Zn accumulation. The role of these and other tentatively identified proteins in Zn accumulation and tolerance is discussed.

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.

Transcription profiling of the metal-hyperaccumulator Thlaspi caerulescens (J. & C. PRESL).

Thlaspi caerulescens is a well-studied metal-hyperaccumulator of zinc, cadmium and nickel, belonging to the Brassicaceae family. Moreover it is one of the few hyperaccumulators that occur on different metalliferous soil types, as well as on nonmetalliferous soils. We are interested in the development of systems to improve phytoremediation of metal contaminated soils through improved metal-accumulation. About 1900 cDNAs isolated from T. caerulescens roots were hybridized with reverse transcribed RNA from zinc-treated T. caerulescens plants of two accessions originating from two different soil types. This comparative transcript profiling of T. caerulescens plants resulted in the identification of genes that are affected by heavy metals. The developed microarray proved to be an appropriate tool for a large scale analysis of gene expression in this metal-accumulator species.

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.

Comparison of two ecotypes of the metal hyperaccumulator Thlaspi caerulescens (J. & C. PRESL) at the transcriptional level.

This paper investigates differences in gene expression among the two Thlaspi caerulescens ecotypes La Calamine (LC) and Lellingen (LE) that have been shown to differ in metal tolerance and metal uptake. LC originates from a metalliferous soil and tolerates higher metal concentrations than LE which originates from a non-metalliferous soil. The two ecotypes were treated with different levels of zinc in solution culture, and differences in gene expression were assessed through application of a cDNA microarray consisting of 1,700 root and 2,700 shoot cDNAs. Hybridisation of root and shoot cDNA from the two ecotypes revealed a total of 257 differentially expressed genes. The regulation of selected genes was verified by quantitative reverse transcriptase polymerase chain reaction. Comparison of the expression profiles of the two ecotypes suggests that LC has a higher capacity to cope with reactive oxygen species and to avoid the formation of peroxynitrite. Furthermore, increased transcripts for the genes encoding for water channel proteins could explain the higher Zn tolerance of LC compared to LE. The higher Zn tolerance of LC was reflected by a lower expression of the genes involved in disease and defence mechanisms. The results of this study provide a valuable set of data that may help to improve our understanding of the mechanisms employed by plants to tolerate toxic concentrations of metal in the soil.

Correlation of foliar MT2b expression with Cd and Zn concentrations in hybrid aspen (Populus tremulaxtremuloides) grown in contaminated soil.

Metal uptake and its effect on foliar metallothionein 2b (MT2b) mRNA levels were studied in hybrid aspen (Populus tremulaxtremuloides) in field conditions. The trees were planted on a site contaminated with several metals, including cadmium (mean 5.1 mgkg(-1)), chromium (80 mgkg(-1)), copper (180 mgkg(-1)), nickel (81 mgkg(-1)), vanadium (240 mgkg(-1)) and zinc (520 mgkg(-1)). Of the ten trace elements analyzed, only Cd and Zn accumulated in the leaves with maximal foliar concentrations of 35 and 2400 mgkg(-1) (dry weight), respectively. There was a strong correlation between Cd and Zn concentrations and bioaccumulation factors (concentration in plant/soil) in the leaves, branches and roots, suggesting similar transport mechanisms for these two metals. The levels of MT2b correlated with Cd and Zn concentrations in the leaves, demonstrating that increased MT2b expression is one of the responses of hybrid aspen to chronic metal exposure.

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.