M. R. de Felipe

Essentiality of boron for symbiotic dinitrogen fixation in pea (Pisum sativum) Rhizobium nodules

The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum sativum) was examined. The absence of boron in the culture medium resulted in a decrease of the number of nodules and an alteration of nodule development leading to an inhibition of nitrogenase activity. Examination of boron-deficient nodules showed dramatic changes in cell walls and in both peribacteroid and infection thread membranes, suggesting a role for boron in the stability of these structures. These results indicate that boron is a requirement for normal nodule development and functionality.

Effects of Inoculation with PGPR Bacillus and Pisolithus tinctorius on Pinus pinea L. Growth, Bacterial Rhizosphere Colonization, and Mycorrhizal Infection

The effect of co-inoculation with Pisolithus tinctorius and a PGPR belonging to the genus Bacillus (Bacillus licheniformis CECT 5106 and Bacillus pumilus CECT 5105) in enhancing growth of Pinus pinea plants and the changes that occurred in rhizosphere microbial communities and the degree of mycorrhization were evaluated. Both bacterial strains of Bacillus promote the growth of Pinus pinea seedlings, but this biological effect does not imply a synergic effect with mycorrhizal infection. However, the positive response to mycorrhiza in a longer-term experiment it could be expected. The introduction of both inocula causes an lateration in the microbial rhizosphere composition, despite the low levels of inocula that were found at the end of the assay.

Immunolocalization of ferritin in determinate and indeterminate legume root nodules

SummaryIn eukaryotic organisms ferritin is a protein involved in the storage of iron. The occurrence of ferritin and its relationship to the effectiveness of the nitrogen-fixing activity have been previously studied during the early stages of the nodule development by biochemical methods. We have used immunocytochemistry techniques to determine the precise location of ferritin and the behavior of this protein along the nodule development. The major localization was found in plastids and amyloplasts of infected and uninfected cells of the three legume nodules studied. A decrease of the immunolabelling was observed in infected cells of lupin and soybean senescing nodules and in the senescent zone of indeterminate alfalfa nodules. In the cortex of soybean and lupin nodules, ferritin increased during nodule ageing and the immunogold particles were mainly located in crystalline structures. The putative role of ferritin and plastids during nodule development is discussed.

Cytochemical Study of Catalase and Peroxidase in the Mesophyll of Lolium rigidum Plants Treated with Isoproturon

Summary A study was made of the number of peroxisomes appearing in the mesophyll of Lolium rigidum in control and isoproturon-treated plants and also, using the diamino benzidine (DAB) technique, the cytochemical localization of catalase and peroxidase enzymes in the same tissues. The number of peroxisomes increased when growing in the presence of a 3.4x10-5 M isoproturon concentration. Isoproturon also somewhat changed the normal position of the peroxisomes. With regard to the cytochemical results, DAB precipitate (osmium black reaction product) was located mainly in the cell wall, vacuole membrane, and in the chloroplasts of the isoproturon-treated mesophyll. However, there was a decrease in DAB in the microbodies of these treated plants. In this study, microbody-like structures associated with a kind of network were observed, and we believe these to be microbodies in development, the Golgi vesicles contributing to their formation. They also presented DAB precipitate in their small internal compartments, probably corresponding to catalase activity. The results are discussed in relation to the senescence of the tissues studied.

Effects of Cyanazine and Linuron on Chloroplast Development, Nodule Activity and Protein Metabolism in Lupinus albus L.

Summary The effects of cyanazine and linuron on development, mesophyll and nodule ultrastructure, nitrogen fixation and protein metabolism of Lupinus albus L. cv. Multolupa inoculated with Bradyrbizobium sp. ( Lupinus ) OR-14, grown under controlled conditions, were investigated. Cyanazine and linuron decreased the weight of both roots and shoots. Cyanazine was more toxic than linuron with regard to the photosynthetic apparatus, nodule ultrastructure, and nitrogen fixation. Concentrations above 4μM cyanazine and 12μM linuron completely inhibited nodulation. The adverse effects on nitrogen fixation due to herbicide application were indirectly caused through action on the photosynthetic apparatus, since these herbicides did not affect the microsymbiont in vitro culture. Leaf protein metabolism was also altered, as determined by SDS-PAGE. When the concentration applied was 20μM cyanazine or 80μM linuron a polypeptide of 17 kDa, which was not found in control plants, appeared. Furthermore, a band of 30 kDa could be observed in the cyanazine treatments. On the other hand, staining of the large subunit of Ribulose-1,5-bisphosphate carboxylase became fainter when the cyanazine and linuron concentrations were increased.

SUBCELLULAR LOCALIZATION OF GLYCOPROTEIN EPITOPES DURING THE DEVELOPMENT OF LUPIN ROOT NODULES

SummaryThe monoclonal antibodies MAC236 and MAC265, raised against a soluble component of pea nodules, were used to elucidate the presence and subcellular localization of glycoprotein epitopes during the development of lupin (Lupinus albus L. cv. Multolupa) nodules, by means of immunocytochemistry and Western blot analysis. These antibodies recognize a single band of 95 kDa in pea, soybean and bean nodules, whilst two different bands of 240 and 135 kDa cross-react with MAC236 and MAC265 respectively in lupin nodules. This fact may indicate that the recognized epitopes can be present in different subcellular compartments and/or play different roles through the development of functional nodules. The results show that MAC265 is mainly associated with Bradyrhizobium infection and with the development of nodule primordium, in the first stages of nodulation. MAC265 is also detected when glycoprotein transport takes place across the cytoplasm and the cell wall, and also in the intercellular spaces of the middle cortex, attached to cell walls. The amount of MAC265 remains constant through nodule development. In contrast the amount of MAC236 increases with nodule age, parallel to the establishment of nitrogenase activity. This antibody is localized in cytoplasmic globules attached to the inner side of cell walls in the middle cortex, and mainly in the matrix filling the intercellular spaces of the middle and inner cortex. This main site of localization of MAC236 may indicate a role in the functioning of the oxygen diffusion barrier.

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds.

SummaryImmunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K+ in alkaloids transport is discussed.

Effect of Drought Stress on Carbohydrate Metabolism in Nodules of Lupinus angustifolius

In this work we have investigated the effect of drought stress on nitrogenase activity (C2H2 reduction activity), carbohydrate metabolism, and nodule structure of Lupinus angustifolius plants. Nitrogenase activity was inhibited around 50%. Water deficit increased the concentration of total soluble sugars, reducing sugars and sucrose more than twice and also increased the total amylase and the acid invertase activity. The possible physiological significance of these results is discussed.

Intercropping of Coinoculated Soybean and Sugar Cane Productivity

Trabajo presentado en el 12th International Congress on Nitrogen Fixation, celebrado en Foz do Iguacu, Parana (Brasil), del 12 al 17 de septiembre de 1999