Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: An electron microscopy and P-31 and C-13 nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using C-13- and P-31-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N-2 fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max. L.)

Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.

Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

Germination, senescence and pathogenic attack in soybean (Glycine max. L.): Identification of the cytosolic aconitase participating in the glyoxylate cycle

During our study of the glyoxylate cycle in soybean (Glycine max. L. var. Maple arrow), two mitochondrial and three cytosolic aconitase molecular species (EC 4.2.1.3) were detected, designated as M1, M2, C1, C2 and C3 isoforms, respectively, according to their intracellular locations and electrophoretic mobilities. Using the glyoxylate cycle marker enzymes isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), the activity of this pathway providing the essential link between P-oxidation and gluconeogenesis was confirmed during germination (cotyledons) and senescence (leaves). It was then established that, in both cases, the activity of the CI aconitase isoform developed concomitantly with the transcription and translation levels of the icl and ms genes. This strongly suggests that C1 aconitase is constitutive of the glyoxylate cycle. In addition, the same isoform was found to be active during pathogenic attack as well (hypocotyls). It might be assumed that in such a case the glyoxylate cycle is reinitiated as a part of a carbon reallocation system feeding on the diseased tissue cellular components.

Pathogenic attack and carbon reallocation in soybean leaves (Glycine max. L.): Reinitiation of the glyoxylate cycle as a defence reaction

Pathogenic attack by the fungus Botrytis cinerea (primary pathogen) on soybean leaves (Glycine max. L.; cv. Maple arrow) results in a hypersensitive response (necrotising infected leaves), in the establishment of local acquired resistance, as well as in the systemic induction of genes coding for pathogenesis-related proteins. It now appears that, concomitantly with these already well documented defence reactions, the pathogenic attack also induces the carbon reallocation mechanism based on the reinitiation of the glyoxylate cycle (pseudo-senescence of the infected leaves).

Thermoanalysis of soybean oil extracted by two methods

The thermal stability of vegetable oils is an important factor that affects their quality. In this study, we investigated the thermal stability of oil and lecithin extracted from soybeans by two distinct processes: mechanical extraction (pressing) and physical extraction (solvent). Thermal analysis was used to obtain information about different methodologies of extraction. The physically extracted products proved more stable than those extracted mechanically. Raman and UV-Vis techniques were applied to underpin the discussion of process differences.

Kinetics of oxidation of biodiesel from soybean oil mixed with TBHQ: determination of storage time

Synthetic antioxidants are an alternative to prevent or retard the degradation of biofuels made from vegetable oils. In this study, it was evaluated the oxidative stability of B100 soybean oil biodiesel, in the presence of tercbutylhydroquinone (TBHQ). The results showed that the induction period, that precedes the seeding process, was delayed in the presence of the antioxidant. Moreover, the obtained results suggest that the B100 biodiesel containing TBHQ can present a storage time at 25 ºC, three times longer than the estimated time for the pure B100.

Effect of continuous growing of resistant soybean genotypes on Meloidogyne arenaria race 1 reproduction

Even though resistance is the most promising tactic for root-knot nematode management on soybean (Glycine max), virulent biotypes may occur and be selected on specific resistant plant genotypes. In the present study, reproduction rate of Meloidogyne arenaria race 1 increased after four sequences of continuous culture of the parasite on resistant soybean genotypes.

Seleção de genótipos de soja resistentes a duas estirpes de Soybean mosaic virus

Soybean mosaic virus (SMV) is the most prevalent virus on soybeans (Glycine max) throughout the world. It causes mottling on seeds and has been associated with yield reduction. Recently, a new strain (SMV 95-1) was found infecting resistant cultivars, causing dwarfing and systemic necrosis. In a screening test carried out with the strains SMV 95-1 and SMV 95-2 on the germplasm collection, several resistant cvs. Embrapa 60, Embrapa 61, Embrapa 62, Embrapa 66 , Embrapa 133, Embrapa 134, Embrapa 135, and Embrapa 136 were identified as resistant to both strains. The resistant genotypes may serve in future soybean breeding programs in Brazil.

Cultural practices and genetic resistance as factors affecting soybean stem canker and plant yield in the Cerrado

Field experiments were conducted in the 1995-96 soybean (Glycine max) growing season to evaluate the effects of cultural practices and host genetic resistance on the intensity of soybean stem canker, caused by Diaporthe phaseolorum f.sp. meridionalis (Dpm). Experiments were conducted in a commercial field severely infected in the previous (1994-95) season. In one study, minimum tillage (MT) and no-tillage (NT) cropping systems were investigated for their effects on disease development and on plant yields in cvs. FT-Cristalina (susceptible) and FT-Seriema (moderately resistant). Another study evaluated the effects of plant densities (8, 15, 21 and 36 plants/m) on disease development in cvs. FT-Cristalina, FT-101 (moderately resistant) and FT-104 (resistant). Disease incidence and severity were consistently lower in NT than in MT, and plant yields were increased by 23% and 14% in the NT system for the susceptible and moderately resistant cultivars, respectively, compared to the yields in the MT system. The Gompertz and Logistic models described well the disease progress curves in all situations. For both susceptible and moderately resistant cultivars, disease severity increased proportionately to the increase in plant densities. At the end of the season, 100% of the plants of cv. FT-Cristalina were infected by Dpm, at all plant densities. Disease levels on cv. FT-101 were intermediate while only very low disease levels were recorded on cv. FT-104. There was a consistent negative correlation between stem canker severity and yield. Some practices demonstrated potential for direct application in disease control, and could be combined considering their additive effects.

Use of semi-seletive media for detection of Sclerotinia sclerotiorum on bean and soybean seeds

This work was aimed at evaluating the possibility of using bromophenol blue as an indicator for detecting the presence of Sclerotinia sclerotiorum in the seeds of dry-beans (Phaseolus vulgaris) and soybean (Glycine max), through incubation of the seeds on an agar medium and "blotter" substrates. The seeds were artificially inoculated with four S. sclerotiorum isolates, plated on the agar medium, named Neon, and on modified Neon agar media all incubated at 14 and 20 ºC for seven days in the dark. Half of the seeds inoculated were surface desinfested prior to plating on the medium. The seeds showing change of colour in the medium, from blue to light yellow, as well as formation of typical mycelium and sclerotia in some cases, were considered to be infected or contaminated by S. sclerotiorum. The two incubation temperatures compared did not show significant (P<0.05) differences in detection level for most of the isolates tested on the different media. According to results obtained in this study, the Neon agar medium with incubation at 14 or 20 ºC has proved to be a reliable and quick method for the detection of S. sclerotiorum mycelium in naturally infected seeds of bean and soybean.

Uptake and translocation of nitrogen, phosphorus and calcium in soybean infected with Meloidogyne incognita and M. javanica

Two soybean (Glycine max) cultivars were used in this study, Ocepar 4, rated as moderately resistant to Meloidogyne incognita race 3 but susceptible to M. javanica, and 'BR 16', susceptible to both nematodes. The effect of nematodes infection on the uptake and transport of N, P and Ca to the shoot was studied in plants growing in a split root system. The upper half was inoculated with 0, 3,000, 9,000 or 27,000 eggs/plant while the lower half received 15N, 32P or 45Ca. Infected plants showed an increase of root but a decrease of shoot mass with increasing inoculum levels. In general, total endogenous nutrients increased in the roots and tended to decrease in the shoots with increasing inoculum levels. When concentrations were calculated, there was an increase in the three nutrients in the roots, and an increase of Ca but no significant variation of N and P was observed in the shoots. The total amount of 15N in the roots increased at the highest inoculum levels but 32P and 45Ca decreased. In the shoots there was a reduction of 32P and 45Ca. The specific concentrations of the labelled nutrients (abundance or radioactivity/tissue mass) also showed a decrease of 32P and 45Ca in the shoots and roots of infected plants and an increase of 15N in the shoots. Considering that overall nutrient concentrations reflect cumulative nutrient uptake and the data from labelled elements gave information at a specific moment of the infection, thus nematodes do interfere with nutrient uptake and translocation.

Mosaic in Senna occidentalis in southern Brazil induced by a new strain of Soybean mosaic virus

Plants of Senna occidentalis (sin. Cassia occidentalis) with mosaic symptoms were collected near a soybean (Glycine max) field where some plants exhibited symptoms of mosaic and blistering. A preliminary examination of leaf tissue from diseased S. occidentalis by electron microscopy revealed the presence of pinwheel inclusions as well as long flexuous particles, indicating the presence of a potyvirus. Host range, serology, and amino acid sequence from this potyvirus were similar to those from other Brazilian isolates of Soybean mosaic virus (SMV). The 3'- terminal region of the genomic RNA was cloned and a cDNA sequence of 1.9 kb upstream of the poly (A) tract was determined. The sequence contains a single open reading frame and a 3'- non-translated region (NTR) of 259 bp. The nucleotide sequence of the CP gene of SMV-Soc was 98% identical to that of Brazilian isolates SMV-B, SMV-L, and SMV-FT10. The percentage of nucleotide identity of their 3'-NTR's was 91, 98, and 99% in relation to SMV-L, SMV-B, and SMV-FT10, respectively. In contrast to other Brazilian SMV isolates studied, SMV-Soc was able to infect sunflower (Helianthus annuus). Based on these results, the S. occidentalis isolate was identified as a new strain of SMV belonging to the SMV strain, group G5 and was named SMV-Soc. This is the first report of naturaly occurring SMV infecting plants of S. occidentalis in Brazil, adding this weed as a new source of SMV in the field.

Effect of population densities of Heterodera glycines race 3 on leaf area, photosynthesis and yield of soybean

The effect of Heterodera glycines on photosynthesis, leaf area and yield of soybean (Glycine max) was studied in two experiments carried out under greenhouse condition. Soybean seeds were sown in 1.5 l (Experiment 1) or 5.0 l (Experiment 2) clay pots filled with a mixture of field soil + sand (1:1) sterilized with methyl bromide. Eight days after sowing, seedlings were thinned to one per pot, and one day later inoculated with 0; 1.200; 3.600; 10.800; 32.400 or 97.200 J2 juveniles of H. glycines. Experiment 1 was carried out during the first 45 days of the inoculation while Experiment 2 was conducted during the whole cycle of the crop. Measurements of photosynthetic rate, stomatic conductance, chlorophyll fluorescence, leaf color, leaf area, and chlorophyll leaf content were taken at ten-day intervals throughout the experiments. Data on fresh root weight, top dry weight, grain yield, number of eggs/gram of roots, and nematode reproduction factor were obtained at the end of the trials. Each treatment was replicated ten times. There was a marked reduction in both photosynthetic rate and chlorophyll content, as well as an evident yellowing of the leaves of the infected plants. Even at the lowest Pi, the effects of H. glycines on the top dry weight or grain yield were quite severe. Despite the parasitism, soybean yield was highly correlated with the integrated leaf area and, accordingly, the use of this parameter was suggested for the design of potential damage prediction models that include physiological aspects of nematode-diseased plants.