Biostimulants Derived from Moroccan Seaweeds: Seed Germination Metabolomics and Growth Promotion of Tomato Plant

Polysaccharides extracted from seaweeds can function as plant biostimulants. The aim of this study was to assess the effects of Polysaccharide Enriched Extracts (PEEs) obtained from 17 Moroccan seaweeds, on tomato seed germination and plant growth. Three concentrations (0.02, 0.05 and 0.1 mg mL⁻¹) of PEEs were applied to tomato seeds to evaluate their effect on 3 germination parameters: germination percentage (GP), germination speed (GS) and mean germination time (MGT). Metabolomic analysis by GC–MS was subsequently performed on seedlings. In the second experiment, four PEEs concentrations (0.02, 0.05, 0.1 and 0.2 mg mL⁻¹) were used as foliar spray or as soil application to tomato plants. Their growth parameters (number of leaves, shoot length, fresh and dry weight of stem and roots) and biochemical parameters (chlorophyll a and b) were measured. Results indicated a significant increase of GP and GS associated with a significant reduction of MGT of tomato seeds treated with 0.02 mg mL⁻¹ of PEEs obtained from Gigartina sp., Gigartina pistillata, Chondracanthus acicularis, Gelidium crinale, Schizymenia dubyi, Cystoseira. foeniculacea and Fucus spiralis. Similar results were also obtained by application of higher PEEs concentration (0.1 mg mL⁻¹) extracted from Ulva rigida, Codium tomentosum, Codium decorticatum and Bifurcaria bifurcata. Metabolomic analysis on seedlings detected the presence of some metabolites which could possibly be involved in seed germination enhancement or inhibition. The results of the second experiment showed that the same PEEs cited above at the same concentrations enhanced plant dry weight and chlorophyll a content except Gigartina sp., C. foeniculacea and C. decorticatum. Furthermore, soil application of PEEs was more effective in improving plant growth parameters than foliar application. The study shows the potential of PEEs from Moroccan seaweed to be used as biostimulants for a sustainable agriculture.

     

Digestibility of extruded proteins and metabolic transit of N ε -carboxymethyllysine in rats

Milk proteins are frequently used as supplements in fortified foods. However, processing produces chemical changes which likely affect the nutritional advantage. This study was intended to explore the possible difference in digestibility between extruded and non-extruded caseins and how the dietary N ^ε -carboxymethyllysine (CML) is metabolised. Normal rats were randomized into either an extruded protein diet (EP) or the same with unextruded proteins (UEP), for two periods of 2 weeks at 7 to 9 and 11 to 13 weeks of age. However, no difference in protein digestibility was detected between the two diets, either in young or in adult animals, despite a 9.4-fold higher level of CML and an 8.5-fold higher level of lysinoalanine in the EP than in the UEP. No diet-related changes were observed in plasma CML, either protein bound or free. Amounts of 38 and 48 % of the orally absorbed CML were excreted in urine and faeces, respectively, in UEP-fed rats. Lower rates of excretion were found in the EP-fed rats (23 and 37 %, respectively). A second animal study using a single oral dose of free CML (400 μg/rat) was set up to measure the systemic concentration of CML every hour from 0 to 4 h. It revealed that protein-bound CML was not affected by the oral dose of CML, and the highest free CML level found in the circulation was 600 ng/mL. Extruded proteins, therefore, appear to be well digested, and CML rapidly eliminated. Since its elimination is, however, incomplete, the question of its biodistribution and metabolism remains open.     

Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy

Purpose

PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo.

Experimental Design

We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitonealy administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated.

Results

We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed.

Conclusion

Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.     

Biodiversity within Medicago truncatula genotypes toward response to iron deficiency: Investigation of main tolerance mechanisms

Iron (Fe) deficiency is one of the major environmental stresses affecting plant production in the world. The selection of tolerant genotypes is considered an effective remediation strategy for this stress. The present study was carried out in order to investigate the biodiversity within Medicago truncatula plants in response to Fe deficiency, to identify tolerant genotypes and to assess the main tolerance mechanisms. To do this, a screening test was performed on 20 M. truncatula genotypes cultivated in minimal medium. Biometric and physiological markers were analyzed, including plant biomass, chlorophyll and root architecture. Results showed a biodiversity among the 20 genotypes. Interestingly, Fe deficiency tolerance was highest in TN8.20 and A17 genotypes. However, the lowest tolerance behavior was observed in TN1.11 and TN6.18. In order to investigate the main tolerance mechanisms, an experiment was conducted in the hydroponic system on already selected genotypes. Assessment of Fe deficiency tolerance was performed mainly on plant growth parameters, Fe (III)-chelate-reductase activity, rhizosphere acidification and antioxidant system defense. The relative better tolerance of A17 and TN8.20 to Fe deficiency was positively correlated with their capacity to maintain higher Fe-acquisition efficiency in roots via rhizosphere acidification and the stimulation of Fe (III)-chelate-reductase activity. Moreover, tolerant genotypes showed the lowest decreases in chlorophyll content and photosynthetic activity (CO₂ assimilation) compared to the sensitive ones. The efficiency of antioxidant capacity of the tolerant genotypes was revealed in stimulation of catalase (CAT) and peroxidase (POX) activities as well as accumulation of polyphenols, leading to the maintenance of cell integrity under Fe deficiency.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.     

Effect of α-Amylase Degradation on Physicochemical Properties of Pre-High Hydrostatic Pressure-Treated Potato Starch

The effect of high hydrostatic pressure (HHP) on the susceptibility of potato starch (25%, w/v) suspended in water to degradation by exposure to bacterial α-amylase (0.02%, 0.04% and 0.06%, w/v) for 40 min at 25°C was investigated. Significant differences (p < 0.05) in the structure, morphology and physicochemical properties were observed. HHP-treated potato starch (PS) exposed to α-amylase (0.06%, w/v) showed a significantly greater degree of hydrolysis and amount of reducing sugar released compared to α-amylase at a concentration of 0.04% (w/v) or 0.02% (w/v). Native PS (NPS) granules have a spherical and elliptical form with a smooth surface, whereas the hydrolyzed NPS (hNPS) and hydrolyzed HHP-treated PS granules showed irregular and ruptured forms with several cracks and holes on the surface. Hydrolysis of HHP-treated PS by α-amylase could decrease the average granule size significantly (p <0.05) from 29.43 to 20.03 μm. Swelling power decreased and solubility increased with increasing enzyme concentration and increasing pressure from 200–600 MPa, with the exception of the solubility of HHP-treated PS at 600 MPa (HHP₆₀₀ PS). Fourier transform infrared spectroscopy (FTIR) showed extensive degradation of the starch in both the ordered and the amorphous structure, especially in hydrolyzed HHP₆₀₀ PS. The B-type of hydrolyzed HHP₆₀₀ PS with α-amylase at a concentration 0.06% (w/v) changed to a B+V type with an additional peak at 2θ = 19.36°. The HHP₆₀₀ starch with 0.06% (w/v) α-amylase displayed the lowest value of T _o (onset temperature), T _c (conclusion temperature) and ΔH _gel (enthalpies of gelatinization). These results indicate the pre-HHP treatment of NPS leads to increased susceptibility of the granules to enzymatic degradation and eventually changes of both the amorphous and the crystalline structures.     

Chelators enhanced biocide inhibition of planktonic sulfate-reducing bacterial growth

Biocides are currently the primary mitigation method to control sulfate-reducing bacteria (SRB) in biofouling, reservoir souring and microbiologically influenced corrosion. Increasingly restrictive environmental regulations and safety concerns on biocide uses demand more efficient dosing of biocides. Chelators have been known to enhance antibiotics because of their properties such as increasing the permeability of the outer cell membrane of Gram-negative bacteria. Two readily biodegradable chelators, ethylenediaminedisuccinate (EDDS) and N-(2-hydroxyethyl)iminodiacetic acid (HEIDA) disodium salts that are touted as potential replacements of ethylenediaminetetraacetic acid (EDTA), were evaluated as potential biocide enhancers for glutaraldehyde and tetrakis hydroxymethyl phosphonium sulfate (THPS) in their inhibition of planktonic SRB growth. Desulfovibrio vulgaris ATCC 7757 and Desulfovibrio desulfuricans ATCC 14563 were grown in modified ATCC 1249 medium and in enriched artificial seawater, respectively. Laboratory tests in 100 ml anaerobic vials showed that EDDS or HEIDA alone did not inhibit SRB growth. However, when EDDS or HEIDA was combined with glutaraldehyde or THPS, each of them enhanced the biocide inhibition of planktonic SRB growth.     

Thigmomorphogenesis in Solanum lycopersicum: Morphological and biochemical responses in stem after mechanical stimulation

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.Key words: Mechanical stimulation, PAL, CAD, POD, IAAIn their environment, plants are constantly submitted to several stimuli such as wind, rain and wounding. The growth response of plants to such stimuli was termed thigmomorphogenesis and was observed in a wide range of plants.^1–3 The most common thigmomorphogenetic response is a retardation of tissue elongation accompanied by an increase in thickness.⁴ The plant response to mechanical perturbation is mainly restricted to the young developing internode, since no influence can be detected when the internode has reached its final length.^5,6 These plant growth modifications, which characterize thigmomorphogenesis, are related to biochemical events associated with lignification process⁷ and ethylene production.^8,9In tomato plant the length of internodes 4 (N4) and 5 (N5) was measured 14 days after rubbing of the fourth internode. Results reported in Figure 1 show that rubbing led to a significant reduction of elongation of the stressed internode (N4) (decrease of N4 length from 4.3 cm in the control plant to 2.9 in the rubbed one). This effect was not limited to the rubbed area but affected also the elongation of the neighboring internodes (N5) that were shorter in rubbed plants than in control ones.Open in a separate windowFigure 1Internode lengths of control and rubbed plants measured 14 day after mechanical stress applied to the fourth internode. Standard errors are indicated by vertical bars.Results reported in Figure 2 show an increase in PAL activity in both internodes N4 and N5, 24 hours after mechanical stress application as compared with corresponding controls. CAD activity was also investigated in N4 and N5, 24 h after rubbing of the fourth internode. Results presented in Figure 3 show that mechanical stress application induces a strong increase of CAD activity in the rubbed internode N4 (5.3 nkatal μg-1 protein) with an approximately two-fold increase when compared to control tomato internodes (2.3 nkatal μg-1 protein). Further, CAD activity in N5 was also increased in the rubbed internode (5.538 nkatal μg-1 protein) as compared with the control one (3.256 nkatal μg-1 protein).Open in a separate windowFigure 2PAL activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Open in a separate windowFigure 3CAD activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Syringaldazine (S-POD) and gaïacol (G-POD) peroxidase activities were measured in tomato N4 and N5. Results reported in Figure 4 show an increase in soluble peroxidase activity with both substrates in the rubbed internode N4 as compared with control plant. Enhancement in peroxidase activities in N4 was more pronounced with gaïacol (80.7 U) as an electron donor than syringaldazine (33.8 U). Similar results were observed in internode 5 as compared with control one (Fig. 4).Open in a separate windowFigure 4(A) Syringaldazine-POD (Syr-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars. (B) Gaiacol-POD (G-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.IAA was quantified in control and rubbed plant internodes 24 h after rubbing of the fourth internode. Results reported in figure 5 show that in control sample and as expected, the content of IAA was found to be higher in the younger internode (N5) as compared to the older one (N4). Rubbing led to a significant decrease in IAA levels in N4 (5.06 nmol g⁻¹ MF⁻¹) as compared with corresponding controls (7.27 nmol g⁻¹ MF⁻¹). Similar results were observed in internode 5, where IAA content was reduced from 16.52 nmol g⁻¹ MF⁻¹ in control internode to 12.35 nmol g⁻¹ MF⁻¹ in the rubbed internode (Fig. 5).Open in a separate windowFigure 5IAA Level of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.The results reported here establish an evident correlation between growth limitation of the rubbed internode and their degree of lignification, the increase in lignification enzymes activities and auxin degradation after mechanical stress application.Auxin seems to be involved in thigmomorphogenesis.¹⁰ It was proposed that MIS (Mechanically-induced stress) has opposite effects on auxin levels in the two species studied to date, Phaseolus vulgaris¹⁰ and Bryonia dioica.^11,12 Auxin level as measured by bioassay, increased in Phaseolus vulgaris following rubbing of the stem.¹⁰ It was proposed that a build up of auxin may result from the reduced polar transport of IAA at the rubbed internode, causing a build up of IAA in the stem tissue. Exogenous IAA did not reverse the MIS inhibition of growth in Phaseolus vulgaris and high levels of IAA retarded growth in non-stressed plants.¹⁰ Thus, retardation of extension growth in Phaseolus vulgaris may have been caused by high levels of endogenous auxin and the increase in stem diameter by increased ethylene production.⁴ However, ethylene increases radial growth only if auxin is present.¹³Boyer¹¹ reported a decrease in auxinlike activity in Bryonia dioica following MIS and this was confirmed in the same species by Hofinger et al.¹² who reported a decrease in IAA using gas chromatography-mass spectrometry. Auxin catabolism was accompanied with changes in both soluble and ionically bound cell wall basic peroxidases¹⁴ and the appearance of an additional peroxidase. This can suggest that in Bryonia, auxin catabolism is hastened by mechanical stimulated peroxidase. In addition, Boyer et al.¹⁵ reported that lithium pre-treatment prevents both thigmomorphogenesis and appearance of specific cathodic isoperoxidase in Bryonia plants subjected to MIS. This is give further credence to the possibility that the peroxidase-auxin system is involved in Bryonia thigmomorphogenesis. In addition, ethylene increases peroxidase activity which reduces the auxin content in the tissue to a level low enough not to support normal growth. We have evidence that decrease of auxin level contribute to mechanism leading to tomato internode inhibition subjected to mechanical stress.Growth inhibition has been suggested to be the result of tissues lignification.⁶ As the initial enzyme in the monolignol biosynthesis pathway, PAL has a direct influence on lignin accumulation.¹⁶ The characteristics of lignin differ among cell wall tissues and plant organs.¹⁷ It comprises polyphenolic polymers derived from the oxidative polymerization of different monolignols, including p-coumaryl, coniferyl and sinapyl alcohols via a side pathway of phenylalanine metabolism leading to lignin synthesis.¹⁸ The increase in lignin content in the rubbed tomato internode could be a response mechanism to mechanical damage caused by rubbing.³ It is known that plants create a natural barrier that includes lignin and suberin synthesis, components directly linked to support systems.^19,20The increase in lignin content of rubbed tomato internode³ is paralleled by a rise in CAD activity and whilst such direct proportionality between CAD activity and lignin accumulation does not always agree with the results in the literature, it clearly is responding in ways similar to those of the other enzymes in the pathway.²¹Mechanical stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation.²² The degradation of cell membranes is suggested to bring about rapid changes in ionic flux, especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio and in leakage of solutes, among them electron donors such as ascorbic acid and phenolic substances. The increased intracellular relative calcium level activated secretion of basic peroxidases²³ into the free space where, in association with the electron donors and may be with the circulating IAA, they eliminate the peroxides, and facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production²⁴ would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase and peroxidases. The resulting lignification and cell wall rigidification determines the growth response of tomato internode to the mechanical stress.     

β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. K _m and V _max values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the β-glucosidase conjugate component were tested by the ELISA method.