Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

Cole latent virus (CoLV), genus Carlavirus, was studied by electron microscopy and biochemical approaches with respect both to the ultrastructure of the Chenopodium quinoa infected cells and to its association with chloroplasts. The CoLV was observed to be present as scattered particles interspersed with membranous vesicles and ribosomes or as dense masses of virus particles. These virus particles reacted by immunolabelling with a polyclonal antibody to CoLV. Morphologically, chloroplasts, mitochondria and nuclei appeared to be unaltered by virus infection and virus particles were not detected in these organelles. However, virus particle aggregates were frequently associated with the outer membrane of chloroplasts and occasionally with peroxisomes. Chloroplasts were purified by Percoll gradient, and the coat protein and virus-associated RNAs were extracted and analyzed by Western and Northern blots respectively. Coat protein and CoLV-associated RNAs were not detected within this organelle. The results presented in this work indicate that the association CoLV/chloroplasts, observed in the ultrastructural studies, might be a casual event in the host cell, and that the virus does not replicate inside the organelle.

Estudos químico-agrícolas sôbre o enxofre

1. Analyses of soluble sulphates in 2 N ammonium chloride extracts of 24 samples of soils of the state of São Paulo, Brazil, S. A., showed a sulphur content varying from 0,0013 g per 100 g (found in the b layer of a genuine "terra roxa") to 0,007 g per 100 g of soil (b layer of a soil of depression without definite characteristics). (The results are expressed as elemental sulphur). Determinations of total sulphur in 56 samples of soils of the same state using the method of fusion with sodium carbonate and sodium nitrate revealed 0.007 g of elemental S per 100 g of soil as the lowest value (found in several soil types) and 0.096 g as the highest one (found in the b layer of an ar-quean soil). Apparently soluble sulphates accumulate in the upper layers and total sulphur does the opposite. It was found a strong correlation between total S and carbon content. 2. Under laboratory conditions, in a compost of fresh soil, powdered sulphur and apatite, it was verified after a three months period of incubation that the pH value lowered from 6.30 to 3.23; the citric acid solubility of apatite increased to 271.1 per cent of the original one. Lupinus sp. grown in soil manured with sulphur and apatite has showed fresh and dry weights higher than the plants in control pots; the results are significant at 5% level of probability; phosphorus content is also higher in the manured plants. It was observed a net influence of the apatite plus sulphur treatment on the weight of root nodosities that was four times greater than in the control plants. 3. Nearly five hundred determinations of S, N and P were carried out in 35 species of plants cultivated in the state of São Paulo. A great variation in the amounts of these elements was observed. As a general rule, the leaves contain more sulphur than the stems and roots show the lowest percentages. The conjunct roots and stem of guar (Cyamopsis psoraloides) revealed only 0.019 per cent sulphur; the leaves of kale showed the highest sulphur content, i. e., 2.114%. Apparently there is no correlation between the amounts of S, N and P. The ratio S/N increases from 0.006 (guar) to 0.485 (kale). The ratio S/P, always higher than the corresponding S/N, increases from 0.082 (guar) to 6.381 (older leaves of tomato plants). It is interesting to mention that several among the most important crops in the state of São Paulo namely, cotton, rice, coffee and sugar cane contain more sulphur than phosphorus. 4. Tomato plants cultivated in nutrient solution lacking sulphur showed the following visual symptons of deficiency : chlorosis first in the younger leaves and afterwards in all the leaves; anthocyanin pigments in the petioles and stems; absence of fruits; primary roots stunted and secondary ones longer than in the control plants; stems slender, hard, woody. The histological study of petioles suffering from sulphur deficiency revealed anthocyanin in the parenchyme layer instead of clo-rophyll pigments observed in normal petioles; in the chlorotic leaves the large chloroplasts present only the stroma but the small ones have a little amount of green pigments. Chemical analysis revealed in the abnormal plants : less sulphur and an increased proportion of phosphorus; older leaves contain more sulphur and less phosphorus than the younger ones probably due to physiological difficulties in translocation of sulphur bearing material; increased amount of total N attributed to accumulation of nitrates; marked decrease in ash, sugars and starch; increased proportion of crude fiber and dry material. In the plants suffering from sulphur deficiency photosyntetic rate decreased 34 per cent. 5. Tomato plants were succesfully cultivated in nutrient solution in absence of mineral sulphur but in presence of cysteine. The plants absorbed sulphur, under that form and were able to grow up quite well; the fruiting was normal. In this way rested cleary demonstrated the possibility of absorption of organic sulphur without previous mineralization and its utilization in the building up of protein molecules.

Alterações morfológicas e citológicas do cafeeiro (Coffea arabica L, var. Bourbon (B. Rodr.) Choussy) cultivado em solução nutritiva decorrentes das deficiências e excessos dos macronutrientes

The present work was carried out in order to study: (1) the symptoms of deficiency and excess of macronutrients (N, P, K, Ca, S, Mg) in the coffee plant (Coffea arabica L. var. Mundo novo); (2) the modifications induced by those treatments in the hystological make up of the leaves; (3) the effects of deficiency and excess on the growth and in the chemical composition of the plants. Young coffee plants were grown in nutrient solution, three treatments being used, namely: complete solution (HOAGLAND & ARNON, 1950), deficient solution, in which a giVen element was omitted, and solution with 3 times the concentration of the element under study. The main conclusions can be summaryzed as follows. 1. SYMPTOMS. Clear cut symptoms of malnutrition were observed in the treatments: -N, -P, +P, -Ca, -Mg, -S and +S; the signals - and + stared respectively for deficient and excess level. 2. HISTOLOGICAL EFFECTS. The most definite alterations took place in the treatments +P, -Mg and +S. Usually the characteristics of the chloroplasts were affected: loss of the green color and coalescence into irregular bodies.

Sintomas externos (morfológicos) e internos (anatômicos), observados em folhas de goiabeira (Psidium guajava L.), de plantas cultivadas em solução nutritiva com carência dos macronutrientes

The present work was carried out in order to study: (1) The symptoms of deficiency of macronutrients (N, P, K, Ca, Mg, S) in guava (Psidium guajava L.). (2) The modifications induced by those treatments in the histological make up of the leaves. Young guava plants were grown in nutrient solution two treatments being used, namely: complete solution (HOAGLAND and ARNON, 1950), deficient solution, in which a given element was ommitted. The main conclusions can be summaryzed as follows. a) Symptoms Clear cut symptoms of malnutrition were observed in the treatments: -N, -P, -Ca, -K, -Mg, and -S. The signal - stared respectively for deficient level. b) Histological effects Usually the characteristics of the chloroplasts were affected: loss of the green and coalescence into irregular bodies.

Changes in anatomy and root cell ultrastructure of soybean genotypes under manganese stress

The deleterious effects of both Mn deficiency and excess on the development of plants have been evaluated with regard to aspects of shoot anatomy, ultrastructure and biochemistry, focusing mainly on the manifestation of visual symptoms. However, there is little information in the literature on changes in the root system in response to Mn supply. The objective of this study was to evaluate the effects of Mn doses (0.5, 2.0 and 200.0 μmol L-1) in a nutrient solution on the anatomy of leaves and roots of the Glycine max (L.) cultivars Santa Rosa, IAC-15 and IAC-Foscarin 31. Visual deficiency symptoms were first observed in Santa Rosa and IAC-15, which were also the only cultivars where Mn-toxicity symptoms were observed. Only in IAC-15, a high Mn supply led to root diameter thickening, but without alteration in cells of the bark, epidermis, exodermis and endodermis. The degree of disorganization of the xylem vessels, in particular the metaxylem, differed in the cultivars. Quantity and shape of the palisade parenchyma cells were influenced by both Mn deficiency and toxicity. A reduction in the number of chloroplasts was observed in the three Mn-deficient genotypes. The anatomical alterations in IAC-15 due to nutritional stress were greater, as expressed in extensive root cell cytoplasm disorganization and increased vacuolation at high Mn doses. The degree of changes in the anatomical and ultrastructural organization of roots and leaves of the soybean genotypes studied differed, suggesting the existence of tolerance mechanisms to different intensities of Mn deficiency or excess.

Deficiency symptoms and uptake of micronutrients by castor bean grown in nutrient solution

Castor bean is a nutrient-demanding species, but there is still little information on its micronutrient requirements. The objectives of this study were to evaluate the effects of levels of B (2.5, 12.5 and 25.0 µmol L-1), Cu (0.05, 0.25 and 0.50 µmol L-1), Mn (0.2, 1.0 and 2.0 µmol L-1) and Zn (0.2, 1.0 and 2.0 µmol L-1) in a nutrient solution on plant B, Cu, Mn and Zn concentrations and uptake, vegetative growth and fruit yield of castor bean "Iris", grown in greenhouse. The experiment was arranged in a completely randomized block design with three replicates. The first deficiency symptoms were observed for B, followed by Zn, Cu and Mn. The main changes in the cell ultrastructure due to lack of B were thickening of the cell walls and middle lamellae, distorted chloroplasts and tightly stacked thylakoids, besides the absence of starch grains. The Mn, Zn and Cu deficiencies led to disruption of chloroplasts, disintegration of thylakoids and absence of amyloplasts. The concentration and uptake of B, Cu, Mn, and Zn in castor bean plants increased with micronutrient supply in the solution. Fruit yield was drastically reduced by B and Mn deficiencies. On the other hand, the dry matter yield of the shoot and root of castor bean plants was not. In the treatment with full nutrient solution, the leaves accumulated 56 and 48 % of the total B and Mn taken up by the plants, respectively, and the seeds and roots 85 and 61 % of the total Cu and Zn taken up, respectively. This shows the high demand of castor bean Iris for B and Mn for fruit yield.

Cytopathological characterization of Mal de Río Cuarto virus in corn, wheat and barley

The Mal de Río Cuarto disease is caused by Mal de Río Cuarto virus (MRCV) transmitted by Delphacodes kuscheli. Comparative studies were carried out on the cytopathological alterations produced by MRCV in corn (Zea mays), wheat (Triticum aestivum) and barley (Hordeum vulgare), as seen with a transmission electron microscope. Corn plants were infected with viruliferous D. kuscheli collected from the endemic disease area (i.e. Río Cuarto County, Córdoba, Argentina). For the viral transmission to small grain cereal plants, laboratory rared insects were used. In this case, the inoculum source was wheat and barley plants infected with MRCV isolate grown in a greenhouse. Leaf samples with conspicuous symptoms were collected: enations and size reduction in corn; crenatures, swelling veins and dark green color in small grain cereals. Viral infection was corroborated by DAS-ELISA. Viroplasms containing complete and incomplete virus particles and fibrillar material were found in the cytoplasm of infected cells in all species. Mature virions were between 60 and 70 nm diameter. In wheat and barley, viroplasms and dispersed particles were observed only in phloem, while in corn virions were also found in cells of the bundle sheath. Crystalline arrays of particles were detected in corn enation constitutive cells. Tubular inclusions were found only in wheat samples. The three species showed abnormalities in the chloroplasts of affected cells. The results showed that MRCV cytopathology has similarities with other viruses from the genus Fijivirus, family family Reoviridae, but slight differences depending upon the host plant.

Anatomical changes on coffee leaves infected by Pseudomonas syringae pv. garcae

ABSTRACTAlthough poorly studied, the bacterial halo blight is an important disease in the major coffee-producing states of Brazil. External damage and anatomical changes on leaves were measured in seedlings of Coffea arabica cv. Mundo Novo, susceptible to Pseudomonas syringae pv. garcae, by using histological sections obtained at 10 and 20 days after inoculation (DAI). The changes on the epidermis were smaller than the lesions measured in the mesophyll, irrespective of the evaluated colonization period, showing that the internal damage caused by the bacterium represent twice the damage observed externally. From the inoculation site, lysis occurred on the epidermal cells and on the palisade and spongy parenchyma cells, with strong staining of their cellular contents, as well as abnormal intercellular spaces in the palisade parenchyma, hypertrophy and hyperplasia of mesophyll cells and partial destruction of chloroplasts. Additionally, this study revealed the presence of inclusion bodies in epidermal and mesophyll cells. Bacterial masses were found in the apoplast between and within mesophyll cells. Bacteria were also observed in the bundle sheath and vascular bundles and were more pronounced at 20 DAI, not only near the inoculation site but also in distant areas, suggesting displacement through the vascular system. These results can be useful to understand this plant-pathogen interaction.

Effects of Corynespora cassiicola on Lantana camara

The present study combines the examination of toxins produced by C. cassiicola and the effects of the fungus colonization on L. camara. C. cassiicola was cultivated on solid media and the crude extracts CAE and CE were produced. Both extracts were submitted to a seed germination and growth assay utilizing Physalis ixocarpa, Trifolium alexandrinum, Lolium multiflorum and Amaranthus hypochodriacus. The effect of the extracts on the ATP-synthesis in isolated spinach chloroplasts was also tested. Bioassay guided chromatographic fractionation identified the most active extract (CAE). From this extract ergosta-4,6,8(14),22-tetraen-3-one (C1) and fatty acids were isolated. The C1 compound reduce ATP synthesis in isolated spinach chloroplasts. The interference of fatty acids with ATP synthesis and also with weed growth provides one explanation of the phytogrowth-inhibitory properties of such fungal extracts. Histological observations involving fungus-plant interaction were made on L. camara plants inoculated with C. cassiicola conidia suspension. After inoculations, fragments of the leaf blades were prepared for observation by light and scanning electron microscopy. Fungal colonization of Lantana camara was typical of a necrotroph and penetration initiated a hypersensitive response. L. camara reacted to the pathogen penetration through thickening of the epidermis walls, cytoplasm granulation and a cicatrisation tissue.

Origin and structure of the Kranz tissue in bracts of Cyperus giganteus Vahl (Cyperaceae)

Cyperus giganteus shows Kranz anatomy of the clorocyperoid type or with two sheaths, one internal, adjacent to the vascular system and known as Kranz sheath, with thin-walled cells and a large number of organelles, mainly chloroplasts; and an external sheath, the mestome sheath or endodermis, the cells of which present thickened walls, are without chloroplasts and possess a suberin lamella, together with the casparian strip which are detected in early stages of differentiation. The development of the vascular bundles shows the Kranz sheath originating from the procambial as well as the mestome sheath. The chloroplasts of the Kranz cells are relatively larger, with convoluted thylakoids and a prominent peripheral reticulum, while the chloroplasts of the mesophyll cells are relatively smaller, with thylakoids forming grana and a sparse peripheral reticulum. These ultrastructural characteristics show similarities to those of other species of the genus Cyperus.

Leaf anatomy of two Anemia Sw. species (Schizaeaceae-Pteridophyte) from a rocky outcrop in Niterói, Rio de Janeiro, Brazil

The ferns Anemia tomentosa (Sav.) Sw. var. anthriscifolia (Schrad.) Mickel and Anemia villosa Humb. & Bonpl. ex Willd. are widely associated with vegetation islands on rocky outcrops in Rio de Janeiro. Both species are desiccation tolerant. The leaf anatomy of these species was examined aiming to identify morphological characteristics that would allow the establishment of these species in water-scarce environments. The plants were harvested on "Pedra de Itacoatiara" and prepared according to the usual procedures. The petiole has a uniseriate epidermis with lignified cell walls, conical stegmata, and uniseriate multicelular and glandular trichomes. In A. villosa, the stomata protrude in a respiratory line. Under the epidermis the cells have thick, lignified walls. The parenchyma has phenolic compounds and starch grains. The petiole vascular bundles are surrounded by endodermis with Casparian strips and the xylem is V-shaped (A. villosa) or arc-shaped (A. tomentosa var. anthriscifolia). The leaf blades have a uniseriate epidermis with sinuous anticlinal and convex periclinal walls, conical stegmata and chloroplasts on both surfaces. The leaf margins of A. villosa have lignified cells. The guard cells of the stomata on the abaxial surface are on the same level or are raised above ordinary epidermal cells. Multicelular uniseriate trichomes and glandular hairs were observed. The dorsiventral mesophyll has loosely packed chlorenchyma with arm-shaped and H-shaped cells. The vascular bundles are surrounded by endodermis with Casparian strips and with parenchymatic extensions towards the epidermis. Anatomical results were analyzed considering the interaction of these plants with abiotic factors.

Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.