MORGANTE M., PFEIFFER A., COSTACURTA A. & OLIVIERJ A. M. 1996. Molecular tools for population and ecological genetics in coniferous trees. Phyton (Horn, Austria) 36 (3): (129) (138). In order for molecular markers to be used in the population genetics of forest trees they have to meet certain requirements such as the ease and speed of genotyping, the codominance of alleles, the reproducibility over time and space, the high information content and the possibility of easily exchanging marker information. None of the many molecular marker systems available fully meets all these requirements. However after taking into account advantages and disadvantages of each of the systems we decided to focus our attention on the use of simple sequence repeats (SSRs or microsatellites) because they are codominant, reproducible, highly informative and easy to exchange. We have been isolating AC/GT and AG/CT SSRs from the Norway spruce (Picea abies K.) nuclear genome. We isolated several hundreds positive clones from a small-insert genomic library and following sequence analysis we designed primers for 36 of them, 24 containing AG and 12 AC SSRs. After testing them on a panel of spruce individuals 25% of the primer pairs produced a single-locus hypervariable pattern, with the remaining ones giving either a single monomorphic product (18%) or very poor amplification (19%) or amplification of multiple bands (38%). Segregation in accordance with a simple Mendelian model of inheritance was demonstrated for all the loci amplified with the primer pairs giving a simple variable pattern. We screened a panel of 19 spruce trees at these loci. The average number of alleles per locus was 14 and expected heterozygosity 0.80, with up to 23 alleles per locus and heterozygosities exceeding 0.94. This shows that nuclear SSRs can be very useful markers in the population genetics of trees even though the overall efficiency of the marker identification process is quite low due to the high percentage of primer pairs producing complex or "dirty" patterns. We attribute this phenomenon to the high complexity of the spruce genome. Other methods, including the construction of libraries highly enriched for SSR sequences, that we developed in order to make SSR retrieval and typing easier and faster will be discussed. We recently extended the use of PCR amplified SSR markers to the chloroplast genome. We demonstrated that mononucleotide poly(A/T) stretches are frequent in the

The proteoglycan decorin inhibits TGF‐β; therefore, it could antagonize progression of fibrotic diseases associated with activation of TGF‐β1. The effect of decorin transfection in human mesangial cells (HMCs) on the expression of genes related to kidney fibrosis was investigated. HMCs, isolated from glomeruli of healthy portions of human kidneys removed due to carcinoma, were histochemically typed. Decorin cDNA cloned in a eukaryotic expression vector was transfected into HMCs. Gene expression of fibrogenetic cytokines and fibrotic proteins TGF‐β1, PDGF‐β, α1 collagen type IV, α1 collagen type I, fibronectin, and tenascin was analyzed, by reverse transcription polymerase chain reaction (RT‐PCR), 24 hr after transfection. Immunoblotting analysis of protein extracts using anti‐decorin IgG, revealed a positive signal of about 52 MDa, corresponding to the molecular weight of decorin, in cultures transfected with the decorin gene. Decorin mRNA increased about 12 times in cultures transfected with the construct pCR3.1‐Deco. Cells with increased decorin synthesis showed a 61% decrease of TGF‐β1 mRNA, a 71% reduction of α1 collagen type IV mRNA, and a 29% reduction of fibronectin mRNA. This study is the first to investigate decorin transfection into human mesangial cells, and supports the use of the decorin gene to control the progression of glomerular and interstitial fibrosis in kidney diseases. © 2002 Wiley‐Liss, Inc.

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.

Indole-3-acetic acid production by Xanthomonas strains pathogenic to citrus was analyzed by reversed-phase high-performance liquid chromatography, thin-layer chromatography, UV spectroscopy and gas chromatography-mass spectrometry, and quantified by high-performance liquid chromatography with fluorescence detection. Amounts of auxin produced by Xanthomonas strains were low when compared with indole-3-acetic acid biosynthesis by other bacteria. In three different experiments it was shown that the addition of plant leaf extracts to bacterial cultures increased indole-3-acetic acid biosynthesis by Xanthomonas axonopodis pv. citri 223.

The genus Azospirillum comprises free-living N2 fixing rhizosphere bacteria that have been isolated from different soil types and from the roots of numerous wild and cultivated plants all over the world. Field trials, carried out at different locations, have demonstrated that under certain environmental and soil conditions, inoculation with Azospirillum has beneficial effects on plant yields. Bacterial phytohormone biosynthesis has often been proposed as being responsible for the observed plant growth promotion upon Azospirillum inoculation.

In order for molecular markers to be used in the population genetics of forest trees they have to meet certain requirements such as the ease and speed of genotyping, the codominance of alleles, the reproducibility over time and space, the high information content and the possibility of easily exchanging marker information. None of the many molecular marker systems available fully meets all these requirements. However after taking into account advantages and disadvantages of each of the systems we decided to focus our attention on the use of simple sequence repeats (SSRs or microsatellites) because they are codominant, reproducible, highly informative and easy to exchange. We have been isolating AC/GT and AG/CT SSRs from the Norway spruce (Picea abies K.) nuclear genome. We isolated several hundreds positive clones from a small-insert genomic library and following sequence analysis we designed primers for 36 of them, 24 containing AG and 12 AC SSRs. After testing them on a panel of spruce individuals 25% of the primer pairs produced a single-locus hypervariable pattern, with the remaining ones giving either a single monomorphic product (18%) or very poor amplification (19%) or amplification of multiple bands (38%). Segregation in accordance with a simple Mendelian model of inheritance was demonstrated for all the loci amplified with the primer pairs giving a simple variable pattern. We screened a panel of 19 spruce trees at these loci. The average number of alleles per locus was 14 and expected heterozygosity 0.80, with up to 23 alleles per locus and heterozygosities exceeding 0.94. This shows that nuclear-SSRs can be very useful markers in the population genetics of trees even though the overall efficiency of the marker identification process is quite low due to the high percentage of primer pairs producing complex or ''dirty'' patterns. We attribute this phenomenon to the high complexity of the spruce genome. Other methods, including the construction of Libraries highly enriched for SSR sequences, that we developed in order to make SSR retrieval and typing easier and faster will be discussed. We-recently extended the use of PCR amplified SSR markers to the chloroplast genome. We demonstrated that mononucleotide poly(APT) stretches are frequent in the chloroplast genomes of plants and show high levels of between and within population variation, making them ideal tools for cytoplasmic population genetics overcoming the difficulties in finding within species variation that are frequently encountered when analysing the cpDNA molecule by RFLPs or PCR-RFLPs. We will discuss the possible applications of such markers for studying gene flow and for paternity analysis.

The plant hormones, auxins and cytokinins, are involved in several stages of plant growth and development such as cell elongation, cell division, tissue differentiation, and apical dominance. The biosynthesis and the underlying mechanism of auxins and cytokinins action are subjects of intense investigation. Not only plants but also microorganisms can synthesize auxins and cytokinins. The role of phytohormone biosynthesis by microorganisms is not fully elucidated: in several cases of pathogenic fungi and bacteria these compounds are involved in pathogenesis on plants; auxin and cytokinin production may also be involved in root growth stimulation by beneficial bacteria and associative symbiosis. The genetic mechanism of auxin biosynthesis and regulation by Pseudomonas, Agrobacterium, Rhizobium, Bradyrhizobium, and Azospirillum, are well studied; in these bacteria several physiological effects have been correlated to the bacterial phytohormones biosynthesis. The pathogenic bacteria Pseudomonas and Agrobacterium produce indole-3-acetic acid via the indole-3-acetamide pathway, for which the genes are plasmid borne. However, they do possess also the indole-3-pyruvic acid pathway, which is chromosomally encoded. In addition, they have genes that can conjugate free auxins or hydrolyze conjugated forms of auxins and cytokinins. In Agrobacterium there are also several genes, located near the auxin and cytokinin biosynthetic genes, that are involved in the regulation of auxins and cytokinins sensibility of the transformed plant tissue. Symbiotic bacteria Rhizobium and Bradyrhizobium synthesize indole-3-acetic acid via indole-3-pyruvic acid; also the genetic determinants for the indole-3-acetamide pathway have been detected, but their activity has not been demonstrated. In the plant growth-promoting bacterium Azospirillum, as in Agrobacterium and Pseudomonas, both the indole-3-pyruvic acid and the indole-3-acetamide pathways are present, although in Azospirillum the indole-3-pyruvic acid pathway is of major significance. In addition, biochemical evidence for a tryptophan-independent indole-3-acetic acid pathway in Azospirillum has been presented.

The plant hormones, auxins and cytokinins, are involved in several stages of plant growth and development such as cell elongation, cell division, tissue differentiation, and apical dominance. The biosynthesis and the underlying mechanism of auxins and cytokinins action are subjects of intense investigation. Not only plants but also microorganisms can synthesize auxins and cytokinins. The role of phytohormone biosynthesis by microorganisms is not fully elucidated: in several cases of pathogenic fungi and bacteria these compounds are involved in pathogenesis on plants; auxin and cytokinin production may also be involved in root growth stimulation by beneficial bacteria and associative symbiosis.The genetic mechanism of auxin biosynthesis acid regulation by Pseudomonas, Agrobacterium, Rhizobium, Bradyrhizobium, and Azospirillum, are well studied; in these bacteria several physiological effects have been correlated to the bacterial phytohormones biosynthesis. The pathogenic bacteria Pseudomonas and Agrobacterium produce indole-3-acetic acid via the indole-3-acetamide pathway, for which the genes are plasmid borne. However, they do possess also the indole-3-pyruvic acid pathway, which is chromosomally encoded. In addition, they have genes that can conjugate free auxins or hydrolyze conjugated forms of auxins and cytokinins. In Agrobacterium there are also several genes, located near the auxin and cytokinin biosynthetic genes, that are involved in the regulation of auxins and cytokinins sensibility of the transformed plant tissue. Symbiotic bacteria rhizobium and Bradyrhizobium synthesize indole-3-acetic acid via indole-3-pyruvic acid; also, the genetic determinants for the indole-3-acetamide pathway have been detected, but their activity has not been demonstrated. In the plant growth-promoting bacterium Azospirillum, as in Agrobacterium and Pseudomonas, both the indole-3-pyruvic acid and the indole-3-acetamide pathways are present, although in Azospirillum the indole-3-pyruvic acid pathway is of major significance. In addition, biochemical evidence for a tryptophan-independent indole-3-acetic acid pathway in Azospirillum has been presented.

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading frame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.

Bacteria of the nitrogen-fixing genus Azospirillum live in association with roots of many plants. Bacterial phytohormone synthesis is proposed to influence the host plant root proliferation. Analysis of tryptophan (Trp), indole-3-acetamide (IAM), and indole-3-acetic acid (IAA) synthesis of the mutant Azospirillum brasilense strain SpM7918 showed an indoleacetamide accumulation concomitant with reduced indoleacetic acid synthesis. The IAA deficiency, and IAM accumulation could be reversed with a specific cosmid from an A. brasilense Sp245 library. The identity of the indoleacetic acid and indoleacetamide produced was confirmed by HPLC with on-line mass spectrometry. Specific radioactivities of tryptophan, indoleacetamide, and indoleacetic acid formed during H-3-IAM and H-3-Trp feeding experiments revealed multiple IAA biosynthetic pathways in Azospirillum: the indoleacetamide pathway, a second tryptophan-dependent, and a tryptophan-independent pathway, the latter being predominant in case no tryptophan was supplied to the medium. This report is the first to demonstrate tryptophan-independent indoleacetic acid synthesis in bacteria.

A. brasilense SpM7918, a previously isolated A. brasilense Sp6 Tn5 mutant, is shown to produce high amounts of indoleacetamide, which is a precursor of indole-3-acetic acid in the indoleacetamide pathway, and to excrete very low amounts of indole-3-acetic acid. From genomic DNA of the mutant, the Tn5 containing restriction fragment was cloned in pUC19 and used as a hybridization probe to screen a genomic library of A. brasilense Sp245. Seven positive clones were isolated and conjugated into the original mutant restoring IAA production. One transconjugant was further analyzed for indoleacetamide and indole-3-acetic acid production by HPLC.

Azospirillum is probably the best studied example of beneficial plant rhizosphere bacteria. Studies in our laboratory focus on the identification of bacterial genes and gene products that are of importance in the physical and metabolic interaction of Azospirillum brasilense with plant roots. Here we report for Azospirillum brasilense, flagellation, motility, the physical interaction with plant roots, the synthesis of indole-3-acetic acid, the expression of nif genes in plant-root associated bacteria, and the induction of gene expression with plant root exudates.