Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean.

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.

Polyadenylation and decay of 26S rRNA as part of Nicotiana tabacum response to cadmium.

In contrast to mRNAs, ribosomal RNAs are generally not considered to be polyadenylated. Only a few recent reports describe non-abundant polyadenylated rRNA-related transcripts that have been detected and characterized in yeast and in human cells. Here we depict the phenomenon of 26S rRNA polyadenylation and degradation that was observed in shoots of Nicotiana tabaccum plants grown in the presence of cadmium. Fragments corresponding to 26S rRNA were identified using suppression subtractive hybridization during screening for genes induced in tobacco plants upon a three-week exposure to 15 microM cadmium chloride. Extracts prepared from the above-ground tissues of cadmium-treated tobacco plants were supposed to contain exclusively polyadenylated mRNAs. Surprisingly, numerous polyadenylated fragments matching parts of 26S rRNA were identified and their presence was confirmed by Northern blot and cDNA amplification techniques. To our knowledge this is the first report on rRNA polyadenylation in plants.

Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30) involved in the production of O-acetylserine, the cysteine precursor, (ii) gamma-glutamylcysteine synthetase (EC 6.3.2.2) involved in the production of gamma-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 microM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.

Overexpression of genes involved in phytochelatin biosynthesis in Escherichia coli: effects on growth, cadmium accumulation and thiol level.

In Escherichia coli, heterologous production of Schizosaccharomyces pombe phytochelatin synthase (PCS) along with overproduction of E. coli serine acetyltransferase (SAT) and gamma-glutamylcysteine synthase (gammaECS) was achieved and resulted in the accumulation of phytochelatins in bacterial cells. Overproduction of either gammaECS alone or simultaneous production of all three proteins in bacterial cells were accompanied by reduced growth rate in liquid cultures. Interestingly, bacteria overproducing either gammaECS or both SAT and gammaECS (with elevated level of gamma-glutamylcysteine but not of phytochelatins) were able to accumulate more cadmium per dry weight than the control. However, the most efficient cadmium accumulation was observed in bacteria with elevated levels of all three proteins: SAT, gammaECS and PCS. Therefore, "pushing" the entire pathway might be the most promising approach in modification of bacteria for potential bioremediation purposes because the level of intermediates, cysteine and glutathione, can limit the rate of production of phytochelatins. However, in such bacteria other metabolic process might become limiting for efficient growth.