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Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti.

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.

Ammonia

The utilization of chopped and pelleted lucerne (Medicago sativa) by growing lambs.

1. The efficiency of utilization of the dietary energy and nitrogen contained in a dried lucerne (Medicago sativa cv. Chartainvilliers) given either chopped (CL) or ground (1.96 mm screen) and pelleted (PL), was measured in a comparative slaughter experiment. Growing lambs were given equal amounts of digestible energy in the chopped or pelleted form at each of the three planes of nutrition for a period of 100 d. 2. The initial energy, fat and protein content of both the carcass and the total body of the test lambs was estimated from regression equations between fasted (18 h) live weight and these components, derived from a group of twenty-three comparable lambs. The final energy, fat and protein content of the test lambs was determined directly by chemical analyses. 3. The metabolizable energy (ME) content of the diets was derived at each plane of nutrition from measured faecal and urinary losses and estimated methane losses. The depression in ME content with grinding and pelleting the dried lucerne was small (CL 8.69 MJ/kg dry matter (DM), PL 8.42 MG/kg DM). 4. The efficiency of utilization of the ME of the dried lucerne for growth and fattening was higher (P less than 0.01) when given in the ground pelleted form (0.533), than in the chopped form (0.284). The net energy value of the PL (3.5 MJ/kg DM) was higher than that of CL (2.2 MJ/kg DM). 5. Thus lambs fed on PL grew faster and had a higher caracass weight gain, carcass protein and fat retention than lambs fed on CL. The composition of the carcass was not altered by the physical processing treatment. 6. Digestion studies with these same CL and PL diets had shown that grinding and pelleting depressed digestion in the forestomachs and increased digestion in the small intestine compared with the chopped form. The increased efficiency of utilization of the gross energy and ME and the higher net energy value of PL was attributed primarily to a change in the site of digestion within the alimentary tract. Associated with this change was a higher value for absorbed amino acids : absorbed energy and an increased apparent absorption of methionine for lambs fed on PL. The difference in the energy costs of eating and ruminating the CL and PL was small.

Animal Nutritional Physiological Phenomena

Effects of interactions between different culture fractions of 'phosphobacteria' and Rhizobium on mycorrhizal infection, growth, and nodulation of Medicago sativa.

Interactions between cell-free culture supernatants, cells, and the whole cultures of Rhizobium and phosphobacteria with endomycorrhizal fungi and their effects on growth and nutrition of Medicago sativa grown in a low-phosphate soil were studied. A satisfactory nodulation was greatly dependent on the mycorrhizal symbiosis. Cell-free supernatants of Rhizobium and phosphobacteria improved plant growth, nodulation and mycorrhiza formation. The activity of phosphobacterial culture seemed to be due mainly to the supernatant and the possibility of plant hormones contained in this culture fraction being involved in such interactions is discussed. An increase of the overall pool of soluble P in soil by the inoculated phosphobacteria cells was not found in the conditions of this experiment. It was noteworthy that the best positive effect was achieved by the treatment which consisted of the whole cultures of Rhizobium, phosphobacteria, and the mycorrhizal fungi applied all together.

Fungi

[Enzymologic characterization of adenosine nucleosidase of medicinal plants (Medicago sativa)].

Adenosine nucleosidase (adenosine ribohydrolase, E C 3.2.2.7) was purified from alfalfa leaf juice. The final preparation shows a single band on polyacrylamide gel electrophoresis; the enzyme activity is stable for 12 hrs between pH 5.5 and pH 8.5, but is completely lost on heating at 55 degrees C for 10 min. Optimal pH for the hydrolysis of adenosine is between pH 5 and pH 6. Among nine purine nucleosides tested, only adenosine, 2'-deoxyadenosine and purine riboside were hydrolyzed by the enzyme preparation. A Km value of 7 x 10(-6) M was found with adenosine as substrate at pH 7.4. Of the two reaction products, adenine exerted a weak inhibitory effect, while D-ribose was without effect on the initial rate of adenosine hydrolysis. The data reported are compared with those obtained on the enzymes from other plant sources.

Adenosine

Quality and rate of extracellular polysaccharides produced by Rhizobium meliloti and their inducing effect on polygalacturonase production in legume roots as derived from the presence of extrachromosomal DNA.

The ability of extracellular polysaccharides of different strains of Rhizobium meliloti to induce the production of polygalacturonase by roots of Medicago sativa seedlings has been studied. Some strains showed a high inducing activity while those derived from them, after treatment with acridine orange and in which extrachromosomal DNA was absent, did not show this character. A comparative study of polysaccharide production and preliminary studies on the chemical composition of the active fractions obtained after Sephadex G-25 filtration indicated that the monomers which form the active fractions are qualitatively and quantitatively different according to their origin.

Acridine Orange

Methane production in the rumen and lower gut of sheep given lucerne chaff: effect of level of intake.

1. Methane production rates were estimated simultaneously in the rumen and caecum of sheep given 200, 400, 600, 800 and 1000 g lucerne (Medicago sativa) chaff/d using isotope dilution techniques. Estimates were also made of volatile fatty acid (VFA) production in the rumen at each level of feeding. In all studies three to four animals were used at each level of intake. 2. Production of VFA and of methane were both related to digestible energy (DE) intake. Regression lines for both VFA production and methane production v. DE intake had significant intercepts indicating an input of endogenous, fermentable organic matter into the rumen in excess of 50 g/d. 3. The values obtained for rates of methane production were compared with those calculated from stoicheiometric equations relating rates of methane and VFA production. Comparisons of methane production with that predicted from DE intake were also made. 4. Balances for digestion of food determined for the rumen indicated that the energies in the end-products were more than 100% of the DE intakes of lucerne chaff. Correction for fermentation of apparent endogenous materials resulted in more realistic values. Endogenous materials appeared to make a significant contribution to VFA and methane production, particularly at low levels of intake.

Animals

The dirigent protein MsDIR6 functions in drought tolerance and modulates reactive oxygen species scavenging and secondary metabolite biosynthesis in alfalfa.

Alfalfa (Medicago sativa L.) is a globally significant forage crop essential for ensuring global food security. However, soil water deficit leads to a substantial decline in its yield, posing a severe threat to sustainable forage production. Dirigent (DIR) proteins play important roles in lignan biosynthesis and plant stress responses. Here, we identified 52 MsDIR genes in alfalfa through a genome-wide analysis, and screened MsDIR6 as a key candidate gene associated with drought tolerance. The results of qRT-PCR showed that MsDIR6 transcription was significantly induced by drought stress in alfalfa. MsDIR6 was preferentially expressed in roots and leaves, and its protein was localized in the nucleus and plasma membrane. Heterologous expression of MsDIR6 in yeast improved tolerance to mannitol-triggered osmotic stress. Heterologous overexpression of MsDIR6 in Arabidopsis significantly increased seed germination rate, seedling survival rate, and antioxidant capacity under drought stress, while improving leaf water-holding capacity by regulating stomatal movement. In transgenic alfalfa hairy roots, MsDIR6 alleviated drought-induced growth inhibition and enhanced reactive oxygen species (ROS) scavenging mediated by the antioxidant defense system under drought stress. Transcriptomic analysis revealed that MsDIR6 activated key genes in the phenylpropanoid and flavonoid biosynthesis pathways, which are crucial for ROS scavenging during drought adaptation. Additionally, we observed elevated flavonoid and lignin contents in MsDIR6-overexpressing alfalfa. Collectively, our findings offer novel insights into alfalfa's drought tolerance mechanisms and identify MsDIR6 as a promising genetic resource for molecular breeding strategies to improve this vital forage crop.

Alfalfa

Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro.

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

Enzyme Activation

Quantitative aspects of the transformations of sulphur in sheep.

1. [35S]sulphate was used to obtain quantitative estimates of the transfer of sulphur between the blood, rumen and postruminal tract of four sheep given brome grass (Bromus inermis) pellets or lucerne (Medicago sativa) pellets at the rate of 33 or 66 g/h. Sodium sulphate (0-4 g S/d) was infused into the rumen or abomasum of sheep given brome grass during four periods of 19 d and was not infused into the sheep during a subsequent period in which lucerne was given. The flow of sulphide, sulphate, microbial S and non-microbial organic S from the abomasum was estimated using 103Ru and 51Cr. 2. The concentration of inorganic sulphate in serum was increased to maximum values of 35-46 mg S/l by infusion of sulphate into the rumen or abomasum. The rate of irreversible loss of serum sulphate and rumen sulphide was positively related to the amount of sulphate infused. 3. Reabsorption of sulphate by the kidney reached a maximum of 0.69-1.1 mmol sulphate/l glomerular filtrate. 4. The transfer of sulphate from blood to the rumen was related to the concentration of inorganic sulphate in serum, attaining maximum values of 133 (+/- 13) mg S/d for sheep given brome grass plus sulphate, and 127-159 mg S/d for sheep given lucerne. 5. Bacteria derived 0.52-0.67 of organic S from rumen sulphide in sheep given brome grass, and approximately 0.45 of bacterial organic S was derived from sulphide for sheep given lucerne. Protozoa derived approximately 0.90 of organic S from bacteria. 6. It was estimated that endogenous organic S contributed 300-340 mg S/d to the rumen, and that 0.24-0.45 of S digested in the rumen was derived from endogenous sources.

Animals

Transfer of urea from the blood to the rumen of sheep.

1. The rate of transfer of plasma urea-nitrogen to rumen ammonia was measured by infusion of 15NH4Cl and [15N]urea into sheep given brome grass (Bromus inermis) or lucerne (Medicago sativa) pellets. Urea was infused into the rumen or abomasum of two sheep given brome grass in order to increase the concentration of rumen ammonia. 2. From 6.2 to 9.8 g/d of plasma urea-N were transferred to the rumen of sheep given brome grass pellets and a measurement of 1.3 g nitrogen/d was obtained for a sheep given lucerne pellets. When urea was infused into the rumen of sheep given brome grass pellets the transfer was only 2.8--3.7 g N/d. 3. There was a significant negative correlation between the rate of transfer of plasma urea-N to the rumen and the concentration of rumen ammonia.

Ammonia

Adsorption of bile salts from aqueous solution by plant fibre and cholestyramine.

1. Adsorption of bile salts by dietary fibre is believed to promote their excretion and hence to reduce the serum cholesterol level in man and experimental animals. 2. We have tested a number of plant fibre fractions and other related materials for their ability to adsorb bile salts from aqueous solution. The "insoluble" plant fractions were from "dry grain" (a residue from brewing), apple, wheat bran, lucerne (Medicago sativa), soya beans, mung beans (Phaseolus mungo), chick peas (Cicer arietinum), rolled oats, spinach (Spinacia oleracea), sunflower seeds, sawdust and sheep faeces. The other materials were cholestyramine, pectin and lignins prepared from wheat bran and from sawdust. 3. Only cholestyramine and the fibre from lucerne, soya beans, mung beans, chick peas, spinach, and sunflower seeds adsorbed enough of either sodium cholate or sodium deoxycholate for adsorption to be detectable. 4. This result conflicts with a report that the lignin component of dietary fibre is responsible for adsorption of bile salts. 5. Adsorption of bile salts, when it occurs, may depend on the presence of saponins bound to the fibre.

Adsorption

Factors affecting the voluntary intake of food by sheep. 1. The role of distension, flow-rate of digesta and propulsive motility in the intestines.

1. Twelve sheep fitted with abomasal cannulas were given ad lib. access to a diet of chopped lucerne (Medicago sativa) hay to a ascertain if the amount of digesta being transported by the intestines was limiting intake. If this was the situation, pumping a solution of the bulk-laxative methylcellulose (100 g/kg; MC) into the abomasum should have reduced intake in proportion to the mass of digesta attributable to the MC and associated water. In preliminary experiments faecal water and wet matter outputs increased by 6.2 and 7.4 g/g MC powder respectively. 2. The infusion of 2.95 kg MC/d did not affect food intake even though wet faecal output increased from a control value of 2.436 to 4.616 kg/d. The transit time of 51 Cr-EDTA through the intestines decreased only slightly during the infusion indicating that MC produced a marked increase in the mass of intestinal contents. Increasing the rate of infusion to 5.336 kg/d increased wet faecal output to 5.437 kg/d, did not change transit time but significantly decreased food intake presumably to protect the intestines from overdistension. After stopping the infusion, food intakes and faecal outputs returned to control values but transit time remained unchanged. The intake of chopped lucerne hay by sheep was not limited by the capacity of the small and large intestines to transport bulk as intake was maintained even through wet faecal output was doubled and the intestines apparently became markedly distended.

Animals

Secondary fermentation in the runen of a sheep given a diet based on molasses.

1. The extent of conversion of acetate-carbon to carbon dioxide in the rumen of a 40 kg wether consuming 1 kg molasses/d was estimated using isotope-tracer-dilution techniques. 2. There was a high rate of conversion of acetate to CO2 (6.0 g C/d) in the rumen. There were high concentrations in the rumen of Methanosarcina approximately 6 x 10(9)/ml which represents a significant proportion of the rumen bacterial biomass. These organisms are usually found in mud and sludge and are capable of oxidizing acetate. 3. The most likely explanation of these results was that there was an extensive secondary or sludge-type fermentation occurring in the rumen which results in volatile fatty acids being converted to CO2 and methane. In similar studies with sheep given lucerne (Medicago sativa) diets, conversion of acetate-C to CO2 within the rumen was not evident. 4. It is suggested that a major effect of the presence of secondary fermentation processes in the rumen may be to reduce availability of energy nutrients to the animal, and to alter the ratio protein:energy in the absorbed nutrients.

Acetates