ABSTRACT
The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20 degrees C. If inhibitors of ethylene formation or action (Co(2+), aminoethoxyvinylglycine, or Ag(+)) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag(+) is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag(+) for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle.
ABSTRACT
Mutational analysis of Pisum sativum L. was used to search for constitutive proteins that might function in nodule formation. The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation. Comparison of two-dimensional polyacrylamide gels of in vitro-translated root RNA showed a consistent difference in the migrational pattern of one peptide. In the nodulating parental cultivar 'Sparkle', a 66 kDa peptide had a pI of 5.9. In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8. This 66 kDa peptide is found in lateral root, tap root, and shoot. Its expression was independent of rhizobial inoculation, root temperature, or light.
Subject(s)
Fabaceae/genetics , Mutation , Plant Proteins/genetics , Plants, Medicinal , Symbiosis/genetics , Electrophoresis, Gel, Two-Dimensional , Genes, Recessive , Molecular Weight , Phenotype , Plant Proteins/isolation & purification , Protein Biosynthesis , RNA/genetics , RNA/isolation & purification , Rhizobium/geneticsABSTRACT
Phorbol esters specifically reduce the binding of epidermal growth factor to surface receptors in intact cells, but not when added directly to isolated membranes. We show that after treatment of intact cells with phorbol myristate acetate, 125I-EGF binding is reduced in membranes prepared subsequently. High-affinity binding of 125I-EGF is modulated by an intracellular calcium-dependent regulatory process. Preventing calcium entry with EGTA or enhancing intracellular calcium with A23187 in intact cells modulates EGF receptor affinity in membranes isolated subsequently. Also, EGTA attenuates the usual inhibition of EGF binding caused by phorbol esters. Membrane preparations do not respond to phorbol ester treatment because the calcium- and phospholipid-dependent protein kinase C is removed or inactivated during membrane isolation. Reconstitution of unresponsive membranes with purified C kinase alters phosphorylation of the EGF receptor and restores the inhibitory effect of phorbol esters on 125I-EGF binding previously observed only in intact cells. Thus, activation of the Ca++-dependent enzyme, C kinase, modulates EGF receptor affinity, possibly via altered receptor phosphorylation.
