Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Internode length and anatomical changes in Pisum genotypes crys and cryc in response to extended daylength and applied gibberellin A1

Dwarf pea (Pisum sativum L.) plants with genotypes cry^c and cry^s responded differently when an 8 h photoperiod (8 h daylight, 16 h dark) was extended to 24 h (8 h daylight, 16 h incandescent light). Genotype cry^c showed up to a 4-fold increase in internode length, sustained by increases in both cell length (particularly of epidermal cells) and cell number (particularly of cortical cells) while cry^s plants showed up to a 2-fold increase in internode length sustained mostly by an increase in cell number. Under an 8 h (daylight) photoperiod the two genotypes did not differ in their sensitivity to applied gibberellin A₁ (GA₁) and they showed a similar pattern of response. GA₁ significantly increased internode length, cell length and cell number in both genotypes. Incandescent light did not increase the size of the response to GA₁ except for cry^s plants at high dose rates of GA₁ (29–58 nmol). At saturating doses of GA₁ the two genotypes attained a similar peak internode length; incandescent light increased the peak by about 40%. GA₁ increased the rate of leaf appearance by up to 33% while incandescent light reduced the rate by 4–7%. The elongation response of the more mature internodes of cry^c plants to GA₁ or incandescent light was due primarily to an increase in cell length whereas increased cell number made a significant contribution in the case of internodes which were relatively immature at the time the stimulus was applied. The progressive increase in internode length of both genotypes during ontogeny was due primarily to an increase in cell number. In conclusion, alleles cry^c and cry^s (background le La) do not confer a difference in sensitivity to GA₁ and the increase in internode length in response to incandescent light is probably not the result of a real or perceived increase in GA₁ level. Allele cry^s may partially block a phytochrome mediated response to light and the key difference between genotypes cry^s and cry^c may lie in the greater elongation (extensibility?) of cry^c epidermal cells in incandescent light.     

Soluble carbohydrates in roots of leek (Allium porrum) plants in relation to phosphorus supply and VA mycorrhizas

Leek plants (Allium porrum L.) inoculated with Glomus mosseae were raised on sterilized soil/sand medium amended with Ca(H₂PO₄)₂.H₂O to test the hypothesis that high concentration of soil P inhibits formation of vesicular-arbuscular (VA) mycorrhizas by reducing concentration of soluble carbohydrate in the root. When P supply was increased, from either P addition or VA mycorrhizal infection, there was initially also an increase in concentration of soluble carbohydrate in the root. At the concentration of soil P at which infection was reduced, concentration of soluble carbohydrate was at its maximum. Therefore the above hypothesis is discounted. An increased delay in infection establishment and a greater number of abortive entry points would suggest that high concentration of soil P reduces VA mycorrhizal infection by changing the anatomy of the root to make it resistant to fungal penetration.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

Quantitative analysis of resistance in cotton to three new isolates of the bacterial blight pathogen

Summary Genetic variability for virulence of the bacterial blight pathogen [Xanthomonas campestris pv malvacearum (Smith) Dye] on cotton (Gossypium hirsutum L.) has been shown by the identification of 19 races of the pathogen based on disease reactions of a set of ten host differentials. This study was conducted to determine the inheritance of host resistance to three recently identified isolates of X. campestris pv malvacearum, which are virulent on the entire set of differentials. True leaves of Tamcot CAMD-E, LEBOCAS-3-80, Stoneville 825, and their f₁, F₂, and backcross progenies were wound-inoculated in the field with separate bacterial suspensions of the virulent HV3, HV7, and Sudan isolates of the pathogen. LEBOCAS-3-80 was replaced with S295, a new immune cultivar, for a greenhouse study in which both cotyledons and true leaves were inoculated. Disease reactions were rated on a scale of 1–10, and genetic models were proposed utilizing generation means analysis. Dominance, when significant, was in the direction of resistance in all but one cross-isolate combination. Digenic interaction components indicated a duplicate type. Narrow-sense heritability for resistance ranged from 0.59 to 0.68; therefore, primarily additive-genetic variability among the selected cutlivars was detected, indicating that breeding for improved resistance to these isolates is a practical goal.Contribution of the Department of Soil and Crop Sciences and the Texas Agricultural Experiment Station     

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F₁ primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F₁ hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.     

High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Summary Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned -subunit of F₁-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.     

Salinity tolerance of Kosteletzkya virginica. I. Shoot growth, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicotyledonous halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m^-3 NaCl, and the effects of external salinity on shoot growth and ion content of individual leaves were studied in successive harvests. Growth was stimulated by 85 mol m^-3 NaCl and was progressively reduced at the two higher salinities. Growth suppression at high salinity resulted principally from decreased leaf production and area, not from accelerated leaf death. As is characteristic of halophytic dicots. K. virginica accumulated inorganic ions in its leaves, particularly Na⁺ and K⁺. However, the Na⁺ concentration of individual leaves did not increase with time, but remained constant or even declined, seeming to be well-coordinated with changes in water content. A striking feature of the ion composition of salinized plants was the development of a dramatic gradient in sodium content, with Na⁺ partitioned away from the most actively growing leaves. Salt-treated plants exhibited a strong potassium affinity, with foliar K⁺ levels higher in salinized plants than unsalinized plants after an initial decrease. These results suggest that selective uptake and transport, foliar compartmentation of Na⁺ and K⁺ in opposite directions along the shoot axis, and the regulation of leaf salt loads over time to prevent build-up of toxic concentrations are whole-plant features which enable K. virginica to establish favourable K⁺-Na⁺ relations under saline conditions.