The stachydrine catabolism region in Sinorhizobium meliloti encodes a multi-enzyme complex similar to the xenobiotic degrading systems in other bacteria.

Stachydrine (proline betaine) can be used by Sinorhizobium meliloti as a source of carbon and nitrogen. Catabolism depends on an initial N-demethylation, after which the resultant N-methyl proline enters general metabolism. Deletion and insertion mutagenesis demonstrated that the information necessary for catabolism is carried on the symbiotic plasmid (pSym) distal to nodD2 and the nod-nif cluster. Sequencing of an 8.5kb fragment spanning this region revealed four open reading frames with functional homology to known proteins, including a putative monooxygenase and a putative NADPH-FMN-reductase, which were shown by insertional and frame-shift mutagenesis to be necessary for stachydrine catabolism. Other open reading frames, encoding a putative flavoprotein and a repressor, were judged not to be required for stachydrine catabolism, since they were not included in a fragment capable of complementing a deletion of the entire stc region. Sequence and mutagenesis data suggest that stachydrine is demethylated by an iron-sulfur monooxygenase of the Rieske type with a requirement for a specific reductase. The stc catabolic cluster, therefore, resembles xenobiotic degradation in other bacteria and recalls rhizopine catabolism in S. meliloti. Stachydrine appears to have multiple roles in osmoprotection, nutrition and nodulation. Genes involved in stachydrine catabolism are also necessary for carnitine degradation; thus, they could be important in the catabolism of a variety of root exudates and mediate other relationships.

Natural genetic transformation by agrobacterium rhizogenes . Annual flowering in two biennials, belgian endive and carrot

Genetic transformation of Belgian endive (Cichorium intybus) and carrot (Daucus carota) by Agrobacterium rhizogenes resulted in a transformed phenotype, including annual flowering. Back-crossing of transformed (R1) endive plants produced a line that retained annual flowering in the absence of the other traits associated with A. rhizogenes transformation. Annualism was correlated with the segregation of a truncated transferred DNA (T-DNA) insertion. During vegetative growth, carbohydrate reserves accumulated normally in these annuals, and they were properly mobilized prior to anthesis. The effects of individual root-inducing left-hand T-DNA genes on flowering were tested in carrot, in which rolC (root locus) was the primary promoter of annualism and rolD caused extreme dwarfism. We discuss the possible adaptive significance of this attenuation of the phenotypic effects of root-inducing left-hand T-DNA.

Biological activities of the nortropane alkaloid, calystegine B2, and analogs: structure-function relationships.

Calystegines, polyhydroxy nortropane alkaloids, are a recently discovered group of plant secondary metabolites believed to influence rhizosphere ecology as nutritional sources for soil microorganisms and as glycosidase inhibitors. Evidence is presented that calystegines mediate nutritional relationships under natural conditions and that their biological activities are closely correlated with their chemical structures and stereochemistry. Assays using synthetic (+)- and (-)-enantiomers of calystegine B2 established that catabolism by Rhizobium meliloti, glycosidase inhibition, and allelopathic activities were uniquely associated with the natural, (+)-enantiomer. Furthermore, the N-methyl derivative of calystegine B2 was not catabolized by R. meliloti, and it inhibited alpha-galactosidase, but not beta-glucosidase, whereas the parent alkaloid inhibits both enzymes. This N-methyl analog therefore could serve to construct a cellular or animal model for Fabry's disease, which is caused by a lack of alpha-galactosidase activity.

Attenuation of the Phenotype Caused by the Root-Inducing, Left-Hand, Transferred DNA and Its rolA Gene (Correlations with Changes in Polyamine Metabolism and DNA Methylation).

We present four examples of attenuation of the transformed phenotype caused by the root-inducing, left-hand, transferred DNA from Agrobacterium rhizogenes in tobacco (Nicotiana tabacum). The first was associated with a genetic variable (homozygosity for the T-DNA), and the second was induced at the physiological level by putrescine and tyramine, suggesting that the transformed phenotype depends on defective polyamine metabolism. Physiological attenuation is further illustrated in the third example, in which the inhibition of flowering caused by P35S-rolA, a gene from the root-inducing, left-hand, transferred DNA driven by a strong viral promoter, was attenuated by grafting the transformed shoot onto non-transformed rootstock that had been induced to flower. Infertility in the resulting flowers was corrected by a mixture of putrescine and tyramine, indicating that P35S-rolA inhibited flowering through interference with polyamine conjugation and that tyramine was essential to fertility. A fourth example of attenuation of the transformed phenotype occurred in lateral branches of plants expressing rolA under the control of its native promoter. In these branches, reduction in the accumulation of rolA transcripts was correlated with the methylation of a site 3[prime] to the rolA coding sequence; thus, the transformed plant seems capable of recognizing and repressing a gene that interferes with flowering.

Chemical synthesis, expression and mutagenesis of a gene encoding beta-cryptogein, an elicitin produced by Phytophthora cryptogea.

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of alpha-elicitins and beta-elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic beta-elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic alpha-elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, beta-cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.

Changing root system architecture through inhibition of putrescine and feruloyl putrescine accumulation.

Plant roots provide anchorage and absorb the water and minerals necessary for photosynthesis in the aerial parts of the plant. Since plants are sessile organisms, their root systems must forage for resources in heterogeneous soils through differential branching and elongation [(1988) Funct. Ecol. 2, 345-351; (1991) Plant Roots: The Hidden Half, pp. 3-25, Marcel Dekker, NY]. Adaptation to drought, for instance, can be facilitated by increased root growth and penetration. Root systems thus develop as a function of environmental variables and the needs of the plant [(1988) Funct. Ecol. 2, 345-351; (1986) Bot. Gaz. 147, 137-147; (1991) Plant Roots: The Hidden Half, pp. 309-330, Marcel Dekker, NY]. We show, in a model system consisting of excised tobacco roots, that both alpha-DL-difluoromethylornithine (an inhibitor of putrescine biosynthesis) and the rolA gene (from the root-inducing transferred DNA of Agrobacterium rhizogenes) stimulate overall root growth and cause a conversion in the pattern of root system formation, producing a dominant or 'tap' root. These morphological changes are correlated with a depression in the accumulation of polyamines and their conjugates.

Calystegins, a novel class of alkaloid glycosidase inhibitors.

The alkaloid extract from roots of naturally growing Convolvulus arvensis, purified by ion-exchange chromatography, showed significant inhibitory activity toward beta-glucosidase and alpha-galactosidase. The demonstrated occurrence of polyhydroxy-nortropane alkaloids, the calystegins, in C. arvensis and their structural similarity to known polyhydroxy alkaloid glycosidase inhibitors, suggested that these might be responsible for the observed activity. Pure calystegins, isolated from transformed root cultures of the related plant species Calystegia sepium, were tested for glycosidase inhibitory activity. The purity of the alkaloids was established by gas chromatography and their identity confirmed by their mass spectrometric fragmentation patterns. The trihydroxy alkaloid, calystegin A3, was a moderately good inhibitor of beta-glucosidase (Ki = 4.3 x 10(-5) M) and a weak inhibitor of alpha-galactosidase (Ki = 1.9 x 10(-4) M). An increased level of hydroxylation, as in the tetrahydroxy calystegins B, consisting of 27% calystegin B1 and 73% calystegin B2, resulted in greatly enhanced inhibitory activity. The calystegins B were potent inhibitors of beta-glucosidase (Ki = 3 x 10(-6) M) and alpha-galactosidase (Ki = 7 x 10(-6) M). These levels of activity are comparable with those of the polyhydroxy indolizidine alkaloids castanospermine and swainsonine toward alpha-glucosidase and alpha-mannosidase, respectively, and of the polyhydroxy pyrrolizidine alkaloid australine toward alpha-glucosidase. The calystegins therefore compose a new structural class of polyhydroxy alkaloids, the nortropanes, possessing potent glycosidase inhibitory properties.

Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones.

The apple rootstock, M26, was genetically and phenotypically transformed using the Agrobacterium wild-type strain, A4. First, chimeric plants were obtained having transformed roots and normal aerial parts. Transformed plants were then produced through regeneration from transformed roots. Transformation was demonstrated by molecular hybridization and opine analysis. The effects of hormones on organogenesis was altered in transformants: cytokinins were required to form roots, whereas auxin was toxic at the concentration used to induce rooting in the control.

Betaine use by rhizosphere bacteria: genes essential for trigonelline, stachydrine, and carnitine catabolism in Rhizobium meliloti are located on pSym in the symbiotic region.

Rhizobium meliloti is known to use betaines synthesized by its host, Medicago sativa, as osmoprotectants and sources of energy. It is shown in the present report that the symbiotic megaplasmid (pSym) of R. meliloti RCR2011 encodes functions essential to the catabolism of three betaines, trigonelline (nicotinic acid N-methylbetaine), stachydrine (proline betaine or dimethylproline), and carnitine (gamma-trimethyl-beta-hydroxybutyrobetaine). Preliminary evidence is presented showing that functions on pSym also influence the catabolism of choline and its oxidative product, glycine betaine. Genes implicated in betaine catabolism are found in the symbiotic region of pSym. Trigonelline catabolism functions lie between two clusters of symbiotic genes, nifKDH and nok/fixVI'. Stachydrine and carnitine functions lie to the right of trigonelline catabolism functions, immediately to the right of fixVI'. Information necessary to choline and glycine betaine catabolism is probably encoded to the right of stachydrine catabolism functions.

alpha-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes.

alpha-dl-Difluoromethylarginine (DFMA) and alpha-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.

Inverse Relationship between Polyamine Levels and the Degree of Phenotypic Alteration Induced by the Root-Inducing, Left-Hand Transferred DNA from Agrobacterium rhizogenes.

Floral induction in plants is a paradigm for signal perception, transduction, and physiological response. The introduction of root-inducing, left-hand transferred DNA (Ri T-DNA) into the genomes of several plants results in modifications of flowering (D Tepfer [1984] Cell 47: 959-967), including a delay in flowering in tobacco (Nicotiana tabacum). Conjugated polyamines are markers for flowering in numerous species of plants. In tobacco their accumulation is correlated with the onset of flowering (F Cabanne et al. [1981] Physiol Plant 53: 399-404). Using tobacco, we have explored the possibility of a correlation between the expression of Ri TL-DNA and changes in polyamine metabolism. We made use of two levels of phenotypic change, designated T and T', that retard flowering by 5 to 10 and 15 to 20 days, respectively. We show that delay in flowering is correlated with a reduction in polyamine accumulation and with a delay in appearance of conjugated polyamines, and we propose that genes carried by the Ri TL-DNA intervene either directly in polyamine metabolism or that polyamine metabolism is closely linked to direct effects of Ri T-DNA expression.

Use of roots transformed by Agrobacterium rhizogenes in rhizosphere research: applications in studies of cadmium assimilation from sewage sludges.

The use of roots transformed by Agrobacterium rhizogenes in models for the rhizosphere is discussed. A list of species for which transformed root cultures have been obtained is provided and the example of studies of cadmium assimilation from sewage sludges is given to illustrate how transformed root cultures can be used in physiological tests under non-sterile conditions.

Analysis of TR-DNA/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4.

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the "core" overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome.

A plasmid of Rhizobium meliloti 41 encodes catabolism of two compounds from root exudate of Calystegium sepium.

Our objectives were to identify substances produced by plant roots that might act as nutritional mediators of specific plant-bacterium relationships and to delineate the bacterial genes responsible for catabolizing these substances. We discovered new compounds, which we call calystegins, that have the characteristics of nutritional mediators. They were detected in only 3 of 105 species of higher plants examined: Calystegia sepium, Convolvulus arvensis (both of the Convolvulaceae family), and Atropa belladonna. Calystegins are abundant in organs in contact with the rhizosphere and are not found, or are observed only in small quantities, in aerial plant parts. Just as the synthesis of calystegins is infrequent in the plant kingdom, their catabolism is rare among rhizosphere bacteria that associate with plants and influence their growth. Of 42 such bacteria tested, only one (Rhizobium meliloti 41) was able to catabolize calystegins and use them as a sole source of carbon and nitrogen. The calystegin catabolism gene(s) (cac) in this strain is located on a self-transmissible plasmid (pRme41a), which is not essential to nitrogen-fixing symbiosis with legumes. We suggest that under natural conditions calystegins provide an exclusive carbon and nitrogen source to rhizosphere bacteria which are able to catabolize these compounds. Calystegins (and the corresponding microbial catabolic genes) might be used to analyze and possibly modify rhizosphere ecology.

Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens.

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1-7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5-10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.

Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames.

We have determined the nucleotide sequence of the Ri TL-DNA region from an Agrobacterium rhizogenes agropine-type plasmid using subcloned regions from the essentially identical Ri TL-DNAs from strains A4 and HRI. This sequenced region of 21,126 base pairs (bp) contains the complete TL-DNA region of the Ri plasmid as determined by analysis of TL-DNA borders in the genome of infected, clonal, Convolvulus arvensis plants. The left and right borders of the TL-DNA are flanked by 25-bp sequences which match the 25-bp terminal sequences found near the borders of T-DNA regions of Agrobacterium tumefaciens Ti plasmids. Other DNA sequences similar to these 25-bp terminal sequences are found within the TL region, and some of these sequences appear to be associated with Ri TL-DNA structures found in transformed tobacco plants. The TL-DNA region contains 18 open reading frames, many of which have 5' and 3' regulatory elements similar to those found in eukaryotic genes. In many cases, CCAAT and TATA elements were found upstream from putative transcriptional initiation codons, and poly(A) addition (AATAAA) elements were observed in presumed 3'-noncoding regions. Comparison of Ri TL-DNA coding and noncoding sequence regions with T-DNA sequence regions from octopine type Ti plasmid pTi15955 reveals no extensive sequence homologies.

Isolation and identification of TL-DNA/plant junctions in Convolvulus arvensis transformed by Agrobacterium rhizogenes strain A4.

We have constructed a Charon 4A phage library containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant (clone 7) regenerated from a root organ culture incited by Agrobacterium rhizogenes, strain A4. Using a subcloned region of the Ri plasmid as P-labeled probe, two lambda clones containing most of the 'left' T-DNA (TL) region were isolated. One of these lambda clones contains the left TL-DNA/plant junction, which was located by comparing nucleotide sequences from the appropriate regions of the Ri plasmid and this lambda clone. A 25-bp sequence found near this left TL-DNA/plant junction matches the 25-bp terminal sequence found at or near T-DNA/plant junctions of both nopaline- and octopine-type A. tumefaciens Ti plasmids. A possible location for the right Ri TL-DNA/plant junction in C. arvensis clone 7 was found by obtaining the nucleotide sequence surrounding its mapped location. Hybridization of plant DNA found adjacent to the left TL-DNA/plant junction against total C. arvensis DNA shows that this T-DNA integration occurred in a plant DNA region that does not contain highly repetitive DNA sequences. Nucleotide sequence analysis of 1004 bp of this plant DNA revealed no complete or partial open reading frames, but this plant DNA does have the potential to form various secondary structures which might play a role in the T-DNA integration event.

Structure and expression of Ri T-DNA from Agrobacterium rhizogenes in Nicotiana tabacum. Organ and phenotypic specificity.

The incorporation of transferred DNA (T-DNA) from the Ri plasmid of Agrobacterium rhizogenes into the chromosomal DNA of higher plants is correlated with the appearance of a complex phenotype. The transformed genotype and phenotype undergo Mendelian inheritance. Through studies of Ri T-DNA content and transcription in Nicotiana tabacum, we have delineated a particular part of this foreign DNA as the likely source of the transformed phenotype. One inducible/repressible aspect of the transformed phenotype is termed T' and is correlated with the presence of a supplementary Ri T-DNA-encoded transcript. This transcript is found specifically in leaves, whereas most of the other T-DNA transcripts are more abundant in roots. The T' phenotype does not appear to be due to structural changes in the Ri T-DNA. It is inherited in a dominant Mendelian fashion. We propose that the T' phenotype is caused by heritable changes in the regulation of Ri T-DNA expression. We comment on the potential of this system as a model for studying eukaryotic gene expression.

Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype.

The T-DNA of the Ri plasmid from Agrobacterium rhizogenes is compatible with the regeneration of whole plants from genetically transformed roots and is transmitted through meiosis to the progeny of genetically transformed plants in carrot, tobacco, and morning glory (Convolvulus arvensis). The presence of Ri T-DNA is correlated with a phenotype that in some respects is invariable from species to species and in other respects varies as a function of species, organ clone within species, or individual. The transformed phenotype concerns a variety of morphological and physiological traits, is dominantly inherited in tobacco, but does not in general appear to be deleterious. The Ri T-DNA may provide a molecular starting point for studying a number of basic phenomena in plant morphology and physiology.