Different patterns of regulation for the copper and cadmium metallothioneins of the ectomycorrhizal fungus Hebeloma cylindrosporum.

Metallothioneins (MTs) are small cysteine-rich peptides involved in metal homeostasis and detoxification. We have characterized two MT genes, HcMT1 and HcMT2, from the ectomycorrhizal fungus Hebeloma cylindrosporum in this study. Expression of HcMT1 and HcMT2 in H. cylindrosporum under metal stress conditions was studied by competitive reverse transcription-PCR analysis. The full-length cDNAs were used to perform functional complementation in mutant strains of Saccharomyces cerevisiae. As revealed by heterologous complementation assays in yeast, HcMT1 and HcMT2 each encode a functional polypeptide capable of conferring increased tolerance against Cd and Cu, respectively. The expression levels of HcMT1 were observed to be at their maximum at 24 h, and they increased as a function of Cu concentration. HcMT2 was also induced by Cu, but the expression levels were lower than those for HcMT1. The mRNA accumulation of HcMT1 was not influenced by Cd, whereas Cd induced the transcription of HcMT2. Zn, Pb, and Ni did not affect the transcription of HcMT1 or of HcMT2. Southern blot analysis revealed that both of these genes are present as a single copy in H. cylindrosporum. While the promoters of both HcMT1 and HcMT2 contained the standard stress response elements implicated in the metal response, the numbers and varieties of potential regulatory elements were different in these promoters. These results show that ectomycorrhizal fungi encode different MTs and that each of them has a particular pattern of expression, suggesting that they play critical specific roles in improving the survival and growth of ectomycorrhizal trees in ecosystems contaminated by heavy metals.

Isolation and characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria bicolor.

Ectomycorrhizae formed by the symbiotic interaction between ectomycorrhizal fungi and plant roots play a key role in maintaining and improving the health of a wide range of plants. Mycorrhizal initiation, development, and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes. We identified a gene, Lbras, in the ectomycorrhizal fungus Laccaria bicolor that belongs to the ras family of genes, which has been shown in other systems to be associated with signaling pathways controlling cell growth and proliferation. The Lbras cDNA complemented ras2 function in Saccharomyces cerevisiae and had the ability to transform mammalian cells. Expression of Lbras, present as a single copy in the genome, was dependent upon interaction with host roots. Northern analysis showed that expression was detectable in L bicolor 48 h after interaction as well as in the established mycorrhizal tissue. Phylogenetic analysis with other Ras proteins showed that Lbras is related most closely to Aras of Aspergillus nidulans.

Effects of elevated CO2 and O3 on aspen clones varying in O3 sensitivity: can CO2 ameliorate the harmful effects of O3?

To determine whether elevated CO2 reduces or exacerbates the detrimental effects of O3 on aspen (Populus tremuloides Michx.). aspen clones 216 and 271 (O3 tolerant), and 259 (O3 sensitive) were exposed to ambient levels of CO2 and O3 or elevated levels of CO2, O3, or CO2 + O3 in the FACTS II (Aspen FACE) experiment, and physiological and molecular responses were measured and compared. Clone 259. the most O3-sensitive clone, showed the greatest amount of visible foliar symptoms as well as significant decreases in chlorophyll, carotenoid, starch, and ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) concentrations and transcription levels for the Rubisco small subunit. Generally, the constitutive (basic) transcript levels for phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) and the average antioxidant activities were lower for the ozone sensitive clone 259 as compared to the more tolerant 216 and 271 clones. A significant decrease in chlorophyll a, b and total (a + b) concentrations in CO2, O3, and CO2 + O3 plants was observed for all clones. Carotenoid concentrations were also significantly lower in all clones; however. CHS transcript levels were not significantly affected, suggesting a possible degradation of carotenoid pigments in O3-stressed plants. Antioxidant activities and PAL and 1-aminocyclopropane-l-carboxylic acid (ACC)-oxidase transcript levels showed a general increase in all O3 treated clones, while remaining low in CO2 and CO2 + O3 plants (although not all differences were significant). Our results suggest that the ascorbate-glutathione and phenylpropanoid pathways were activated under ozone stress and suppressed during exposure to elevated CO2. Although CO2 + O2 treatment resulted in a slight reduction of O3-induced leaf injury, it did not appear to ameliorate all of the harmful affects of O3 and, in fact. may have contributed to an increase in chloroplast damage in all three aspen clones.

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus.

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-L-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.

In vitro translation of mushroom tyrosinase.

Mushroom tyrosinase was purified and antibodies prepared against the holo enzyme and a protein of 26,000 daltons. Both antibodies recognized the large subunit of the enzyme but only one recognized the 26,000 dalton protein. Poly A+ mRNA was isolated from mushrooms, translated in vitro, and a 41,000 dalton protein immunoprecipitated from the translation mix with either antibody. This 41,000 dalton protein presumably corresponds to the large subunit of the holoenzyme. Antibodies against the holoenzyme also immunoprecipitated another translation product with a molecular weight of 15,000 daltons corresponding to the small subunit of the holoenzyme. These results suggest that each subunit may be coded for by different genes and undergo posttranslational processing.

In vitro translation of the major capsid polypeptide from Ustilago maydis virus stain P1.

Double-stranded RNA (dsRNA) from Ustilago maydis virus strain P1 was translated in vitro using a nuclease-treated rabbit reticulocyte lysate system. Following heat denaturation of the H2 double-stranded RNA segment in 90% dimethyl sulfoxide and incubation in the cell free extract, a primary translation product was observed which showed the same molecular weight and co-migrated with viral coat protein on 10% SDS-polyacrylamide gels. The in vitro product of the H2 dsRNA segment could also be immunoprecipitated with antibodies prepared against viral coat protein. Limited proteolysis of the in vitro product and authentic viral coat protein using Staphylococcus aureus V8 protease produced similar peptide patterns on SDS gels. In vitro translation products from other dsRNA segments that make up the P1 viral genome could not be precipitated by antibody to viral coat protein. These results complement the genetic data that indicated that information for coat formation and maintenance was contained within the H segments of dsRNA.