Preparations of plant plasma membrane vesicles were obtained by partition in dextran-polyethyIene glycol two-phase systems. By this procedure particles are separated according to their surface properties, and an iso-osmotic environment is maintained throughout. The vesicles thus produced are right-side-out and sealed, as measured by enzyme [(K+ + Mg2+)-ATPase and glucan synthetase II] latency on addition of Triton X-100.
Plasmalemma from 8‐day old oat (Avena sativa L. cv. Brighton) and spring wheat (Triticum aestivum L. cv. Drabant), grown in the dark at 18°C, was prepared from the 10000 g (10 min) – 30 000 g (60 min) root homogenate by two‐phase separation in three steps with 6.5% (w/w) Dextran T 500 and 6.5% (w/w) polyethylene glycol 4 000. Biochemically and with respect to activation by Mg2+ as well as by (Mg2++ K+), the oat preparations clearly appeared as ATPase(s) in the pH range 5–8. They showed high specificity for ATP, temperature optima between 38 and 40°C, and were inhibited by vanadate, DCCD (dicyclohexylcarbodiimide) and SH‐reagents, but not by oligomycin, ammonium molybdate or ouabain. In contrast, the preparations from wheat contained more than one type of MgATPase/ nucleotidase, as revealed by complex dependence on both pH and temperature as well as by comparatively low specificity towards nucleotides. However, no unspecific phosphatase was present, and the effect of K+ over and above that of Mg2+ was almost as specific as in oat by all criteria used. The data available from this and earlier investigations from our group would indicate that the complex reactions of preparations of wheat plasmalemma may not be due to contamination but, rather, expressions of the many biological functions that must be associated with the plasmalemma in vivo and which may be located in sub‐units that are more firmly attached to wheat than to oat plasmalemma.
The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots (Tritium aestivum L. cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g, 10,000 g, 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two‐phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter‐current distribution id a 6.3/6.3% two‐phase system. Protein and activities of Ca2+, Mg2+, and Mn2+ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured.
The partition of ATPase activities stimulated by Ca2+, Mg2+ or Mn2+ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3‐system was selected for further separation of the membranes in the 30 KP fraction by counter‐current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.
The abundance of bacteria in the rhizoplane of barley varieties was investigated at different soil nitrogen levels. Increased amendments of nitrogen resulted in higher bacterial numbers in the rhizoplane of barley seedlings of different varieties. A negative correlation was found between nitrogen level in the soil and the growth rate of the seedling roots. The effect of nitrogen on the bacterial abundances could be indirect through changed root growth and thereby changed exudation. The exudation of soluble organic carbon compounds from barley seedling roots were measured in hydroponic culture. The effect of natural variation in root growth rate and of different concentrations of nitrogen in the nutrient solution was investigated. The amount of exudates constituted 2-66% of the dry weight increase in root biomass, depending on the root growth. Slower growing roots released considerably more organic carbon per unit root weight than faster growing roots. The variation in root exudation appeared to be mainly explained by differences in root growth, rather than of the nitrogen concentration in the nutrient solution. A significantly higher exudation rate was found during day time compared to night.
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