Profilin in Phaseolus vulgaris is encoded by two genes (only one expressed in root nodules) but multiple isoforms are generated in vivo by phosphorylation on tyrosine residues.

Actin-binding proteins such as profilins participate in the restructuration of the actin cytoskeleton in plant cells. Profilins are ubiquitous actin-, polyproline-, and inositol phospholipid-binding proteins, which in plants are encoded by multigene families. By 2D-PAGE and immunoblotting, we detected as much as five profilin isoforms in crude extracts from nodules of Phaseolus vulgaris. However, by immunoprecipitation and gel electrophoresis of in vitro translation products from nodule RNA, only the most basic isoform of those found in nodule extracts, was detected. Furthermore, a bean profilin cDNA probe hybridised to genomic DNA digested with different restriction enzymes, showed either a single or two bands. These data indicate that profilin in P. vulgaris is encoded by only two genes. In root nodules only one gene is expressed, and a single profilin transcript gives rise to multiple profilin isoforms by post-translational modifications of the protein. By in vivo 32P-labelling and immunoprecipitation with both, antiprofilin and antiphosphotyrosine-specific antibodies, we found that profilin is phosphorylated on tyrosine residues. Since chemical (TLC) and immunological analyses, as well as plant tyrosine phosphatase (AtPTP1) treatments of profilin indicated that tyrosine residues were phosphorylated, we concluded that tyrosine kinases must exist in plants. This finding will focus research on tyrosine kinases/tyrosine phosphatases that could participate in novel regulatory functions/pathways, involving not only this multifunctional cytoskeletal protein, but other plant proteins.

Purification, characterization, and cDNA cloning of profilin from Phaseolus vulgaris.

Profilin from common bean (Phaseolus vulgaris L.) was purified to homogeneity by poly-L-Pro affinity chromatography and gel filtration. The hypocotyl and symbiotic root nodule protein was detected as a single isoform with a 14.4-kD molecular mass and an isoelectric point of 5.3. Partial amino acid and DNA sequencing of a full-length cDNA clone confirmed its identity as profilin. An antibody generated against the purified protein binds to a protein with the same molecular mass in leaves and nodules. Immunolocalization of the protein showed a diffuse distribution in the cytoplasm of hypocotyls and nodules but enhanced staining at the vascular bundles. The strong identity of the sequence among the profilins of birch, maize, and bean suggests that it may play an important role in the signal transduction mechanism of plant cells and plant-bacterial symbioses.

Isolation and characterization of a gene involved in ethylene biosynthesis from Arabidopsis thaliana.

The ethylene forming enzyme (EFE) is a key factor in ethylene biosynthesis. To understand better the regulation of ethylene biosynthesis in vegetative tissues, we set out to isolate and characterize a complementary DNA (cDNA) encoding the EFE from Arabidopsis thaliana. An A. thaliana cDNA library was screened with pTOM 13, a tomato cDNA coding for the EFE. A cDNA clone (pEAT1) was isolated. The cDNA is 1200 nucleotides (nt) in length and predicts a protein of M(r) 36,663. The insert includes the complete open reading frame of 972 bp and shows strong homology with several reported sequences, both at the nt and amino acid level. In whole seedlings, expression of pEAT1 was enhanced by wounding, ethrel, Fe2+, and 1-amino-cyclopropane-carboxylic acid (ACC) treatments. In contrast, heat shock had no effect on the expression.

A study of erythropoiesis and iron metabolism in the rabbit in vivo. II. Dependence of the response on iron storage and transport.

Rabbits subjected to a daily bloodletting schedule of 10 ml blood per kg body weight increase four- or fivefold their erythropoietic production compared to normal non-bled animals. The maximum response they can reach under these conditions mobilizes more than 9 mg of iron per day per rabbit into hemoglobin. When fed ad libitum with their regular diet, they do not need any further iron supplement for full erythropoiesis. The experimental increment in iron body stores and/or serum iron levels does not enhance their erythropoietic response, demonstrating that iron is not rate limiting under the conditions studied. Furthermore, although serum iron levels are elevated onefold in the controls under chronic anemia with respect to non-bled animals, the concentration of serum transferrin is only slightly increased; hence, the iron saturation of this protein changes from a 50% to an 80% level. In the absence of an extra supplement of iron, rabbits subjected to chronic bloodletting show no signs of body iron depletion, as judged by their continuous macrocytic RBC production.

A study of erythropoiesis and iron metabolism in the rabbit in vivo. I. Characterization and limits of the physiological response.

1. An experimental model system was developed in the rabbit to study the transport of iron and the erythropoietic response to chronic bloodletting. 2. This model presents certain novel features as the capacity to measure total red blood cell production and total hemoglobin production in a daily basis. 3. Daily red blood cell production and output of hemoglobin are directly proportional to the volume of blood extracted. The limits of the erythropoietic response were determined. 4. When only minimal bloodletting was performed (3-6 ml blood/kg body weight) a normocytic response was obtained; with the removal of larger volumes of blood, a switch to macrocytic anemia was observed. Cats and guinea pigs responded in a similar fashion. 5. After induction of macrocytic anemia, the diameter of erythroid precursor cells in the bone marrow of the long bones increased and their numbers increased 11.5-fold.