Molecular analysis of a homogentisate phytyltransferase gene from Lactuca sativa L.

Tocochromanols, usually known as vitamin E, play a crucial role in human and animal nutrition. The enzyme homogentisate phytyltransferase (HPT) performs the first committed step of the vitamin E biosynthetic pathway. The full-length cDNA encoding HPT was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPT, was 1,670 bp long containing an open reading frame (ORF) of 1,185 bp which encoded a protein of 395 amino acids. Sequence analysis indicated that the deduced protein, named as LsHPT, shared high identity with other dicotyledonous HPTs. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPT was preferentially expressed in mature leaves compared with other tissues. When lettuce plants were subjected to drought and high-light stress treatments, LsHPT expression was markedly increased. Expression of LsHPT in Arabidopsis showed that LsHPT could enhance the α-tocopherol biosynthesis in Arabidopsis. Transient expression of LsHPT via agroinfiltration resulted in 9-fold increase in LsHPT mRNA level and nearly 18-fold enhancement in α-tocopherol content compared with the negative controls.

Molecular characterization of microbial mutations induced by ion beam irradiation.

A positive selection system for gene disruption using a sucrose-sensitive transgenic rhizobium was established and used for the molecular characterization of mutations induced by ion beam irradiations. Single nucleotide substitutions, insertions, and deletions were found to occur in the sucrose sensitivity gene, sacB, when the reporter line was irradiated with highly accelerated carbon and iron ion beams. In all of the insertion lines, fragments of essentially the same sequence and of approximately 1188bp in size were identified in the sacB regions. In the deletion lines, iron ions showed a tendency to induce larger deletions than carbon ions, suggesting that higher LET beams cause larger deletions. We found also that ion beams, particularly "heavier" ion beams, can produce single gene disruptions and may present an effective alternative to transgenic approaches.

Irradiance regulates genotype-specific responses of Rhizobium-nodulated soybean to increasing iron and two manganese concentrations in solution culture.

The growth of soybean plants were examined when subjected to three contrasting irradiance levels and to various combinations of nutrient solution Fe and Mn concentrations. Two Rhizobium-nodulated soybean genotypes (PI 227557 and Biloxi), which had been previously found to differ in their growth response to various Fe and Mn solutions, were studied. Both genotypes displayed the poorest growth, nodulation and the lowest chlorophyll and nodule ureide concentration at high irradiance (HI), regardless of the solution Fe and Mn concentrations. However, the genotypes differed under HI in their accumulation of Fe. For solution concentrations greater than 13 microM, PI 227557 accumulated up to 1200 microg Feg(-1) leaf dry wt mainly in the form of ferritin crystals within chloroplasts. In contrast, leaf Fe concentrations in Biloxi only reached 300 microg Feg(-1) dry wt and there were no ferritin crystals. Also, in PI 227557 HI induced more severe distortions in leaf cells and nodule ultrastructure than in Biloxi. Based on its poor growth under HI, PI 227557 could be categorized as an Fe-inefficient genotype prone to undergo photoinhibition at HI, in spite of the ferritin crystals in the chloroplasts. Enhanced growth, nodulation, chlorophyll and ureide concentrations in nodules as well as leaf ureide catabolism occurred in both genotypes grown at moderate irradiance (MI) in Fe solutions from 13 to 60 microM supplied with 20 microM Mn. At low irradiance (LI), plant growth and nodulation were lower than at MI values, but higher than those of plants at HI. Irradiance and solution Fe concentration did not alter leaf Cu and Zn concentration in either genotype, with the higher concentrations of these two elements detected in Biloxi. Solutions with Fe concentrations greater than 100 microM were deleterious for both genotypes at all irradiances. Low Fe and high Mn concentrations in leaves was bound to result in the best growth at HI.

Efectividad de cepas rizobianas de frijolbajo diferentes regímenes de fósforo / Effectiveness of cowpea rhizobial strains under different phosphorus regimes

En Venezuela, el frijol representa una alternativa a la proteína animal, debido a su alto consumo y valor nutritivo, por ello se ha estimulado la implementación de programas para reactivar la economía de los pequeños y medianos productores, a fin de incrementar su producción y así tener mayor disponibilidad de proteína de alta calidad a bajo costo; de manera que, los estudios encaminados a mejorar su cultivo, son acertados. Se evaluó la efectividad de cepas rizobianas de crecimiento lento (cl) y rápido (cr) en frijol (Vigna unguiculata (L.) Walp.) cultivar TC9-6 en varios regímenes de fósforo (0, 20, 40 y 80 kgP2O5 ha-1), con un diseño experimental de bloques al azar con arreglo factorial. Las plantas se cultivaron en 4 kg de suelo de sabana 45 días y las cepas en caldo de levadura y manitol: 5 (cr: JV91) y 10 (cl: JV94) días. La inoculación (2 ml cada vez) fue aplicada a la siembra y 6 días más tarde. La utilización de fósforo (40-80 kgP2O5 ha-1) incrementó la nodulación (número, peso seco total e individual de nódulos) y favoreció la aparición de nódulos rojos; así mismo, acrecentó el peso de la materia seca, la altura, el número de hojas y la concentración de nitrógeno del vástago. Los valores fueron similares con ambos tipos de cepas (efectividad similar) y para las dos concentraciones (40-80 kgP2O5 ha-1), con las menores estimaciones para 0 y 20 kgP2O5 ha-1. De acuerdo con los resultados las concentraciones de 40 y 80 kgP2O5 ha-1 fueron las más favorables para el crecimiento y la nodulación de frijol.

In Venezuela, cowpea is an alternative to animal protein due to its high consumption and nutritious value, so it has stimulated the implementation of programs to reactivate the small and medium producers economy, in order to increase its production and to have major high quality protein availability at low cost; so that, the studies carry on to improve its cultivation, are well-aimed. The effectiveness of slow (sg) and fast (fg) growing rhizobial strains was evaluated in cowpea (Vigna unguiculata (L.) Walp) cultivar TC9-6 at various phosphorus regimes (0, 20, 40 and 80 kgP2O5 ha-1): randomized block design with factorial arrangement. Plants were cultivated in 4 kg savannah soil: 45 days, and the strains in yeast and mannitol broth: 5 (fg: JV91) and 10 (sg: JV94) days. The inoculation (2 ml each time) was applied at sowing time and 6 days later. Phosphorus utilization (40-80 kgP2O5 ha-1) increased nodulation (nodule number, total and individual dry weight) and favoured nodule red colour appearance; also, incremented shoot dry matter weight, height, leaves number and nitrogen concentration. Values were similar with both strain types (similar effectiveness) and to the two doses (40-80 kgP2O5 ha-1), with lower estimations to 0 and 20 kgP2O5 ha-1. Accordingly with the results, the doses of 40 and 80 kgP2O5 ha-1 were the most favourable to cowpea growth and nodulation.

[Serologic properties of mutants of Rhizobium trifolii and their interrelationship with effectiveness]. / Serologicheskie svoistva mutantov Rhizobium trifolii i vzaimosviaz' ikh s éffektivnost'iu.

UV and gamma irradiation of Rhizobium trifolii, strains 347a and 311a, produced their mutants with changed antigenic properties and effectiveness. The extent of changes of antigens did not depend on the dose of irradiation because mutants that completely lost antigens typical of the parent strains were obtained at different doses of UV and gamma rays. No correlation was established between the antigenic properties of the mutants of Rhizobium trifolii and their effectiveness.

Low-intensity microwave radiation and the virulence of Agrobacterium tumefaciens strain B6.

When virulent cells of Agrobacterium tumefaciens strain B6 were exposed to low-level microwave radiation at a frequency of 10,000 MHz and an intensity of 0.58 mW/cm2 for 30 to 120 min, a 30 to 60% decrease in their ability to produce tumors on potato and turnip disks was observed. This microwave exposure did not affect the viability of these bacteria or their ability to attach to a tumor-binding site nor did it induce thermal shock. This loss of virulence was reversible within 12 h.

Ultraviolet-induced changes in the infectivity of Agrobacterium tumefaciens.

The infectivity of Agrobacterium tumefaciens strain B6 irradiated with short-wavelength ultraviolet light was followed as a function of dose. Previously reported enhancements of B6 infectivity by ultraviolet irradiation, in samples inoculated after 1.75 hr of dark incubation at 27 C, or immediately following irradiation, were found to occur most frequently after losses in cell viability of 60% and of 90% or more, respectively. Changes in colony-forming ability and tumor-initiating ability with increasing dose showed no obvious correlation until the maximal infectivity promotion of samples inoculated immediately after irradiation was reached. Thereafter, both bacterial responses typically decreased in parallel. With low dose rates, infectivity promotions were obtained with less than 10% loss in cell viability. Data for tumor appearance and tumor growth resulting from inoculations with irradiated cultures showed no significant differences from controls, nor did the age of the bacterial culture or age of the host plant influence the response. The infectivity promotion appears to result from an increase in the proportion of viable cells that will subsequently initiate tumors. The characteristics of this ultraviolet infectivity promotion are shown to be most similar to those found in prophage and bacteriocin induction.

Bacterial attachment to a specific wound site as an essential stage in tumor initiation by Agrobacterium tumefaciens.

The number of tumors initiated by Agrobacterium tumefaciens strain B6 on primary pinto bean leaves was decreased when cells of an avirulent strain (IIBNV6) were included in the inoculum. With sufficient B6 cells to initiate ca. 50% of the maximal number of tumors per leaf, inhibition was detected at a 1:1 ratio of B6 to IIBNV6 cells and increased linearly with the logarithm of the number of IIBNV6. Varying the number of B6 in the presence of a constant number of IIBNV6 or varying the number of both, while maintaining a constant ratio of B6 to IIBNV6, showed that the inhibition was a function of the absolute concentration of each cell type. The data fit a one-particle dose response curve, which indicates that a single IIBNV6 cell can prevent tumor initiation by a single B6 cell. Inhibition was obtained with mixed inocula and when the addition of IIBNV6 preceded B6, but not when B6 preceded IIBNV6. Heat-inactivated IIBNV6 inhibited, as did ultraviolet or heat-inactivated B6. Several unrelated bacteria and certain strains of Agrobacterium failed to inhibit, whereas other related strains gave inhibition. Attachment of IIBNV6 to a specific would site, thus excluding B6 from the site, is proposed to account for these data. A specific complementary binding of a virulent bacterium to a host wound site exposed by the inoculation procedure is suggested as an essential early event in the crown-gall tumor initiation process.

[The presence of Agrobacterium tumefaciens in lucerne root nodules]. / O nakhozhdenii Agrobacterium tumefaciens v kluben'kakh na korniakh liutserny

Agrobacterium tumefaciens does not penetrate into nodules on the roots of lucerne with the active strain of nodule bacterium as was established with the aid of genetic markers and plant selection. A nodule, whose shape was not typical and which did not fix nitrogen, was formed on the root of lucerne inoculated with the culture of Agrobacterium tumefaciens treated with UV. A bacterial strain isolated from the nodule was identical to A. tumefaciens according to its resistance to streptomycin and several cultural properties. Upon repeated inoculation with the isolated strain, nodules were formed in 50% cases.

[Effect of ultraviolet rays on the activity of clover nodule bacteria]. / Vliianie ul'trafioletovykh luchei na aktivnost' kluben'kovykh bakterii klevera

The effect of UV on the survival, morphological variability, and effectiveness was studied with nodule bacteria of clover, strain 374a. The survival of the strain directly depended on the dose of UV rays, being 43.3% at 130 erg/mm2 and 0.000022% at 22,900 erg/mm2. UV irradiation yielded two morphological types of colonies; they differed by the production of slime which was less at higher doses of UV (15,970 to 22,900 erg/mm2). Variants with either positive or negative effectiveness were obtained by the action of UV. The positive variants increased the crop of clover by 21-60%, as compared with the parent strain 347a, while the negative variants decreased it by 18.3-35%.

[Lysogeny of Rhizobium japonicum and the sensitivity of these cultures to phages isolated from soil]. / O lizogenii u Rhizobium japonicum i chuvstvitel'nosti étikh kul'tur k fagam, vydelennym iz pochvy.

Twenty cultures of Rhizobium japonicum of various origin were tested for lysogeny using a cross technique with a preliminary UV induction and without it as well as by electron microscopy. None of the cultures was found in the lysogenic state. Phages active against Rh. japonicum were detected in four soil samples on which soybean plants were grown; 27 phages were isolated by the enrichment method and 3 phages without enrichment. The phages were capable of lysis of only Rh. japonicum cultures and differed in the spectrum of lytic action. Some of them were very specific: 9 phages caused lysis of only one among the 28 tested cultures, and 10 phages lysed 2 cultures. Phages with a broad spectrum of lytic action induced lysis of 7-9 cultures. Some of the phages isolated from soils of different type were identical and some were similar in the spectrum of lytic action. Among 28 cultures of Rh. japonicum, 8 cultures were sensitive to all of the 30 phages isolated from soil and 2 cultures were sensitive to most of the phages; the remaining cultures were sensitive to 1-8 phages. Phages and cultures have been selected which can be used for experimental preparation of lysogenic systems and further studies of lysogeny in Rh. japonicum.

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens.

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens V-1 is temperature-sensitive. At 25 C and 30 C, phage was released in a ratio of 1 plaque-forming unit per 100 bacteria; at 35 C, although bacterial growth was not inhibited, phage release was suppressed. Phage synthesis was induced by heat shock, 42 C for 30 min, ultraviolet irradiation, and mitomycin C. Induction by ultraviolet light was unusual-an immediate rise in phage titer followed irradiation. A large increase occurred after a 90-min latent period. The lysogenic strain was cured of the phage by incubation at 37 C, and the cured strain produced plant tumors.

Screening for mutants of Rhizobium japonicum with defects in nitrogen fixing ability.

Mutants of Rhizobium japonicum with reduced ex planta nitrogenase activity could be isolated with high frequency by direct screening of ultra-violet mutagenized bacteria growing as spots on the surface of an appropriate agar medium permitting derepression of nitrogenase synthesis. Small glass chambers fitted with a serum cap were pushed into the agar around each spot of growth, forming a small enclosed gas space which was made to 10% acetylene, permitting assessment of nitrogenase activity by the acetylene reduction test. Four mutants were isolated from a total of 305 screened spots. Two mutants had almost no ex planta activity, one of which had no symbiotic activity despite normal nodulation (ineffective), the other had only somewhat reduced activity symbiotically. Two other mutants with less than half wild-type activity ex planta were normal in symbiosis.

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene.

A recombinant lambda phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec- phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.

Isolation of a recombination deficient Agrobacterium tumefaciens mutant.

The isolation of a recombination deficient (Rec-) strain of Agrobacterium tumefaciens is described. Strain LBA 4011 was mutagenized with nitrosoguanidine and after segregation 18,000 colonies were replica plated and UV irradiated. Twentytwo UV sensitive strains were isolated and tested for methylmethanesulphonate (MMS) sensitivity. Six of these strains were more MMS-sensitive than LBA 4011. A Ti plasmid that was genetically marked with Tn 1 (CbR) was introduced in these strains and the rescue of the CbR marker during superinfection with an incompatible cointegrate plasmid Ti::R 702 was determined. One strain exhibited a large reduction in rescue frequency. It is concluded that the latter strain was recombination deficient. This property did not influence the induction of plant tumours.

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae.

DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA- Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 bp coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA- mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.

Isolation and characterization of the recA gene of Rhizobium meliloti.

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.

Formation of tumor-like structures on legume roots by Rhizobium.

Tumor-like structures appeared on the roots of Medicago sativa, Alysicarpus vaginalis, and Trifolium pratense inoculated with a non-nodulating strain of Rhizobium trifolii or with irradiated cultures of either of two nodulating Rhizobium strains. The structures were composed of disorganized plant tissues which, on the basis of microscopic examination, were devoid of bacterial cells. Rhizobia which could nodulate legumes of one cross-inoculation group and which were able to induce formation of such tumor-like structures on plants of a second cross-inoculation group were isolated from extracts of these root growths. The apparent tumorogenic activity of some of the rhizobia, but not their nodulating capacity, was lost when the bacteria were transferred in laboratory media.