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.

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 F(3) 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, F(3) 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.

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.

Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation for SvMT2b expression. Allele-specific PCR showed that low-expression F(3) plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent. SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F(3) plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

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.

Detection of p53 protein and Ki-67 proliferation antigen in human papillomavirus (HPV)-positive and HPV-negative cervical lesions by immunohistochemical double-staining.

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. Of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67. Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. The positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G1 to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered.

Expression of p53 protein related to the presence of human papillomavirus (HPV) DNA in genital carcinomas and precancer lesions.

The E6 protein of the high-risk human papillomavirus (HPV) types 16 and 18 is capable of complexing with the wild-type p53 tumor suppressor gene product, leading to loss of the normal p53 function as an anti-oncogene, whereas the low-risk HPV types 6 and 11 lack this binding property. The malignant potential of HPV 16 and 18 has been ascribed to this complexing of E6 with p53, which regularly leads to undetectable expression of the latter in HPV-positive lesions. To assess the role of p53 in HPV-associated genital carcinogenesis, the expression of p53 protein was studied immunohistochemically in 22 genital carcinomas and precancer lesions; 8 vulvar carcinomas, 1 VIN (vulvar intraepithelial neoplasia), 5 cervical carcinomas and 8 CIN (cervical intraepithelial neoplasia) using monoclonal antibody PAb 1801. Presence of HPV was demonstrated by PCR using HPV consensus primers, and amplified HPV-DNA was digested with the restriction enzymes giving distinct patterns for various HPV-types in gel electrophoresis. HPV-typing was confirmed by in situ hybridization with biotinylated DNA probes. Altogether, 17 of the 22 specimens (77%) showed p53 expression: 67% of the precancer lesions and 83% of carcinomas. Expression was more frequent (89%) in the vulvar than (70%) in cervical lesions. Using PCR,HPV DNA was detected in 19/22(86%) of the samples. The following HPV types were identified: HPV 6 (2 samples), HPV 11 (3 cases), HPV 16 (5 cases), HPV 33 (3 cases), and 6 contained unidentified HPV types. All HPV DNA-negative specimens showed p53 expression. Of the 19 HPV DNA-positive lesions, 5 were p53-negative, three of these being HPV 16 positive CIN lesions. The remaining two HPV 16 lesions were invasive carcinomas with a weak p53 expression. HPV 6 and 11-positive lesions showed a weak p53 expression more frequently than HPV-negative cases and HPV 33 lesions. The results indicate that p53 expression is detectable, but it is less frequent and less intense in HPV DNA-positive genital precancer lesions and carcinomas (particularly those with HPV 16 DNA) as compared with HPV DNA-negative lesions.

Immunohistochemistry, in situ hybridization and polymerase chain reaction (PCR) in detecting c-myc expression in human malignancies.

The assessment of oncogene expression at cellular level is important in understanding the role of those genes in carcinogenesis. Using in situ hybridization and immunohistochemistry, the expression of oncogenes can be visualized in topographic relation to tissue morphology. In the present study, c-myc overexpression was studied in ten carcinomas of different origin (6 mammary adenocarcinomas, 2 vulvar and 2 bronchial squamous cell carcinomas) by in situ hybridization (ISH) with 35S-labeled RNA probes and by immunohistochemistry (IHC). DNA amplification and transcription of c-myc oncogene were also studied with polymerase chain reaction (PCR) using beta-globin as an intrinsic standard for DNA amplification. The effect of formalin fixation of c-myc expression was simultaneously studied. Half of the tumours (5/10) demonstrated c-myc mRNA overexpression by ISH performed on frozen sections and two of the samples were shown to over-express c-myc protein by IHC. Only two samples fixed in formalin showed positive signals for c-myc mRNA. None of the biopsies showed DNA amplifications either with ISH or PCR. The present results suggest that ISH with RNA probes is a useful method for detecting the transcription of activated oncogenes in malignant tissues, especially when applied on frozen sections. The results also indicate that in some cases, c-myc gene may be adequately transcribed to mRNA but the latter is not translated to the appropriate oncoprotein.