Nematicidal effect of an Amaranthus hypochondriacus L. cystatin (AhCPI) on the root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood

The root-knot nematode Meloidogyne incognita is one of the most damaging plant parasitic nematodes in the world. In this study, the effect of cystatin from Amaranthus hypochondriacus (AhCPI) as a potential control agent for M. incognita was explored. In vitro bioassays demonstrated that AhCPI affects the growth and development of eggs and the infectivity of juveniles (J2) of M. incognita, such as mortality and slower development, showing characteristic tissue damage. Mortality levels were quantified by Probit analysis, estimating LC₅₀s of 1.4 mg/mL for eggs and 0.028 mg/mL for J2. In planta bioassays showed that infected tomato seedlings treated with 0.056 mg/mL of AhCPI showed a 60% reduction in the number of galls, as compared with untreated J2-inoculated seedlings. Under greenhouse conditions, three applications of 10 mL of AhCPI (1.4 mg/mL) in the soil around the stem of M. incognita-infected tomato plants, reduced the number of galls by 93 ± 8%, as compared to the control M. incognita-infected plants. The application of AhCPI to the infected plants increased the yield (10.7%) of harvested tomato fruits, as compared to infected plants. These results show the potential of AhCPI for the control of M. incognita in tomato plants.     

An immunological method for quantifying antibacterial activity in Salmo salar (Linnaeus, 1758) skin mucus

Antimicrobial peptides (AMPs) are a pivotal component of innate immunity in lower vertebrates. The aim of this study was to develop an immunological method for quantifying AMPs in Salmo salar skin mucus. A known antimicrobial peptide derived from histone H1 previously purified and described from S. salar skin mucus (SAMP H1) was chemically synthesized and used to obtain antibodies for the quantification of the molecule via ELISA. Using skin mucus samples, a correlation of bacterial growth inhibition versus SAMP H1 concentration (ELISA) was established. The results provide the first evidence for quantifying the presence of active AMPs in the skin mucus of S. salar through the use of an immunological method.     

A comparative analysis of the phytoplankton from six pampean lotic systems (Buenos Aires, Argentina)

Phytoplankton, as well as physical and chemical variables of six lotic systems of Buenos Aires province, tributaries of Río de la Plata river (Rodríguez, El Gato, El Pescado, Buñirrigo and J. Blanco streams, Samborombón river) were analysed and results were related to the degree of anthropic pollution. Surveys were carried out seasonally from May 1997 to June 1998. Sample sites were stablished at 2 or 3 stations depending on the length of the systems, from the headwaters to the mouths. Specific composition, species richness, dominance, diversity and eveness were assessed. Diatoms and clorophytes presented the highest number of species. Cyanobacteria were dominant in 60% of the samples and diatoms were subdominant in 42% of them. Samborombón river showed minimun and maximun phytoplankton densities among the six systems (50 cell.ml^–1 and 188475 cell.ml^–1, respectively). Indexes values showed a non-defined pattern, increasing or decreasing in the most polluted sites. The first axis of the PCA which included the physical and chemical variables is characterized by the nutrient concentrations and it is related to the pollution of these systems, separating samples of Rodríguez and El Gato streams, which presented high positive scores, from the rest of them. The second axis is represented by variables which determine the water conductivity and separates samples from the mouths of Samborombón river and Buñirrigo stream, which presented high positive scores, from the rest of them. The species PCA indicate that its distribution is related to the first two components of the chemical data set PCA. The first axis separates species found in high-conductivity sites, such as the mouths of Samborombón and Buñirrigo systems (i.e. Cocconeis placentula, Aulacoseira granulata, Melosira varians, Actinocyclus normanii, Dictyosphaerium ehrenbergianum) from those registered in low-conductivity sites, such as J. Blanco stream (i.e. Pseudanabaena catenata, Nitzschia gracilis, Mallomonas sp.). The second axis separates and mesosaprobic species from polluted sites -Rodríguez and El Gato streams- (i.e. Gomphonema parvulum, Navicula cryptocephala, Nitzschia palea, N. umbonata, Oscillatoria amphibia, Lyngbya limnetica, Euglena acus) from those found in oligosaprobic non-polluted sites – J. Blanco stream – (Nitzschia gracilis, Pseudanabaena catenata, Gomphonema clavatum).     

Serotonin Modulation of Low-Affinity Ouabain Binding in Rat Brain Determined by Quantitative Autoradiography

Previous results showed that Na⁺/K⁺-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na⁺/K⁺-ATPase activity and [³H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which isoform is involved in the Na⁺/K⁺-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [³H]ouabain binding to rat brain slices was employed at different [³H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [³H]ouabain binding was sensitive to serotonin 10^–3 M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [³H]ouabain concentrations, strongly suggests the participation of the 2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K⁺ handling by the glial 2 isoform of the pump.     

Isoenzymic forms of carbonic anhydrase in the red macroalga Porphyra leucosticta

Different isoenzymes of carbonic anhydrase (CA; EC 4.2.1.1) have been separated using thalli of the red macroalga Porphyra leucosticta Thuret in Le Jolis. Homogenates of the thallus were centrifuged in order to separate soluble and membrane proteins. The fraction containing membrane proteins was subdivided by centrifuging into two fractions: green and nongreen membrane proteins. CA activity was detected in all the fractions. Because external CA (measured on intact thallus) represented 15% of total activity, it was concluded that most of the CA (ca. 80%) was soluble and internal. Direct evidence regarding the different function of external and internal CA was obtained by determining the effects on photosynthesis of two specific CA inhibitors with different capacity for entering cell. It was concluded that internal CA was necessary to 'trap' the CO₂ entering the cell and thus maintain a favorable CO₂ gradient that permits its diffusive entry. Changes in the O₂ evolution rate at inorganic carbon ( C _i) concentration saturating for photosynthesis and on the photosynthetic conductance for C _i were found when external CA was inhibited. Based on these changes and the significant CA activity (ca. 9% of the total activity) found in nongreen membrane fraction, the presence of external CA associated with plasma membrane was postulated. The presence of CA associated with chloroplast membrane was also suggested.     

Biochemical and phenotypical characterization of transgenic tomato plants overexpressing a basic peroxidase

Tomato plants ( Lycopersicon esculentum Mill. cv. Pera) were transformed via Agrobacterium tumefaciens with the binary vector pKYLX71 containing a tomato basic peroxidase (EC 1.11.1.7) gene, tpx1 , under the control of the cauliflower mosaic virus (CaMV35S) promoter. Transgenic plants showed a 2–5-fold increase in the activity of the peroxidase ionically bound to the cell wall, whereas soluble peroxidase activity remained similar or even lower than wild-type plants. Isoelectric focusing showed the presence of a new isoperoxidase of pI ca 9 in the ionically bound extract. Western blot also showed the presence of a new band at 41 kDa that was absent in the wild-type extract. A 40–220% increment of lignin content of the leaf was found in transgenic plants. Shoot phenotype of transgenic plants was similar to wild type, although under stress, the plants appeared wilted and the new leaves had a reduced area and were thicker than wild-type or older transgenic leaves. The root system was underdeveloped in transgenic plants, but the rooting ability of the stem was not affected by the overexpression of peroxidase. Finally, the morphogenetic response of cotyledon and hypocotyl explants from transgenic plants was evaluated. In the case of cotyledons, the percentage of explants with shoot was not different from wild-type plants. For hypocotyl, one of the transgenic lines showed a 30% reduction in the percentage of shoot organogenesis. The results are discussed in relation to the role of tpx1 in lignin synthesis.     

Effect of simultaneous down-regulation of pectate lyase and endo-β-1,4-glucanase genes on strawberry fruit softening

Strawberry is a soft fruit with a short postharvest shelf-life. The loss of fruit firmness during ripening is mainly due to the disassembly of parenchyma cell walls mediated by the expression of genes encoding enzymes acting on pectins, such as pectate lyase, or hemicellulose, e.g. endo-β-1,4-glucanase. To determine if the simultaneous down-regulation of FaplC and FaEG3 genes, encoding a pectate lyase and a endo-β-1,4-glucanase, respectively, exerted an additive effect on strawberry softening, transgenic plants expressing tandem antisense sequences of both genes under the control of the constitutive promoter CaMV35S were generated. Fifteen independent transgenic lines were obtained and fruit yields and several quality parameters of transgenic ripe fruit were recorded during two consecutive years. Fruit yield was reduced in most of the lines, especially in the first evaluation period, and five out of 15 lines (33 %) did not set fruit. The expression of FaplC and FaEG3 genes was measured in ripe fruits from six selected lines showing the highest fruit yields. All selected lines showed a high level of FaplC gene silencing, ranging from 97 to 71 %; however, FaEG3 gene expression was only significantly down-regulated in two lines. Fruit colour and soluble solids contents were similar in control and transgenic ripe fruits, while fruit weight was slightly lower than control in some of the lines. In all lines, transgenic fruits were significantly firmer than control, with an increase in firmness ranging from 19 to 32 %. The reduction of fruit softening in transgenic fruits was not correlated with the suppression of FaEG3 gene expression, and lines with the highest simultaneous down-regulation of FaplC and FaEG3 showed similar fruit firmness to lines where only FaplC was suppressed. These results indicate that pectate lyase and endo-β-1,4-glucanase do not act in an additive or synergistic way during strawberry softening, and question the role of glucanases in this process.