Search

Key Message: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

Several studies have described the effects of seed exudates against microorganisms, but only few of them have investigated the proteins that have defensive activity particularly against nematode parasites. This study focused on the proteins released in the exudates of soybean seeds and evaluated their nematicidal properties against Meloidogyne incognita. A proteomic approach indicated the existence of 63 exuded proteins, including β-1,3-glucanase, chitinase, lectin, trypsin inhibitor, and lipoxygenase, all of which are related to plant defense. The presence of some of these proteins was confirmed by their in vitro activity. The soybean exudates were able to reduce the hatching of nematode eggs and to cause 100% mortality of second-stage juveniles (J2). The pretreatment of J2 with these exudates resulted in a 90% reduction of the gall number in tobacco plants. These findings suggest that the exuded proteins are directly involved in plant defense against soil pathogens, including nematodes, during seed germination.

Key Message: The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.

This study assessed whether chemical analyses are sufficient to guarantee the safety of heat processing of soybeans (SB) for human/animal consumption. The effects of extrusion and dry-toasting were analyzed upon seed composition and performance of broiler chicks. None of these induced appreciable changes in protein content and amino acid composition. Conversely, toasting reduced all antinutritional proteins by over 85%. Despite that, the animals fed on toasted SB demonstrated a low performance (feed efficiency 57.8 g/100 g). Extrusion gave place to higher contents of antinutrients, particularly of trypsin inhibitors (27.53 g/kg flour), but animal performance was significantly (p < 0.05) better (feed efficiency 63.2 g/100 g). Upon the basis of chemical analyses, dry-toasting represents the treatment of choice. However, considering the results of the feeding trials, extrusion appears to be the safest method. In conclusion, in order to evaluate the reliability of any processing method intended to improve nutritional value, the combination of chemical and animal studies is necessary.

Abstract: A novel toxic protein from Glycine max seeds, named Gm-TX, was purified by combination of differential precipitation with ammonium sulphate, DEAE-cellulose, anhydrotrypsin-sepharose 4B and Superdex 200 HR fast-protein liquid chromatography. Gm-TX is composed of one polypeptide chain of 28kDa (SDS-PAGE), with pI 5.1–5.2, contains <0.5% neutral sugar, has N-terminal sequence comprised of KTISSEDSPFFNCREK, maximum absorption spectrum at 280nm and molar extinction coefficient of 16.9. It exhibited ribonuclease activity (1821.42±3.34UA/h/mgP) toward yeast RNA and promoted rabbit erythrocyte agglutination (120.4HU/mgP) mediated by anti-Gm-TX IgG, which was inhibited by mannose. It was devoid of trypsin inhibitory, urease and chitinase activities. Gm-TX was lethal to mice by intraperitoneal route with LD50 of 4.5mg/kg. Moreover, it showed dose-dependent insecticidal activity on the major economically important pests Callosobruchus maculatus and Dysdercus peruvianus, producing ca. 50% and 56% mortality when incorporated into artificial diets at a level of 1.16% and 0.25%, respectively. These data indicate that Gm-TX is a candidate protein that could be expressed in genetically transformed plants for improved resistance to insect pests. [Copyright &y& Elsevier]