Arja Tervahauta

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.

Plant metallothioneins – metal chelators with ROS scavenging activity?

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.

Leaf proteome responses of Arabidopsis thaliana exposed to mild cadmium stress

The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1muMCd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10muMCd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F3 families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F3 sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.

Correlation of foliar MT2b expression with Cd and Zn concentrations in hybrid aspen (Populus tremula × tremuloides) 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.

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant Thlaspi caerulescens

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.

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.

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 °C) while daytime temperature was kept at a constant 22 °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 β-ocimene increased from 6 °C to 14 °C, while several other monoterpenes and the SQTs (Z,E)-α-farnesene and (E,E)-α-farnesene increased up to 18 °C. Total monoterpene and sesquiterpene emission peaked at 18 °C, whereas isoprene emissions decreased at 22 °C. Leaf area increased across the temperature range of 6–22 °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 LC×LE 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.

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.