A Comprehensive Analysis Using Colorimetry, Liquid Chromatography-Tandem Mass Spectrometry and Bioassays for the Assessment of Indole Related Compounds Produced by Endophytes of Selected Wheat Cultivars.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS), colorimetry, and bioassays were employed for the evaluation of the ability of endophytic bacterial strains to synthesize indole-related compounds (IRCs) and in particular indole-3-acetic acid (IAA). A total of 54 endophytic strains belonging to seven bacterial genera isolated from tissues of common and spelt wheat cultivars were studied. The endophytic bacteria isolated from different tissues of the tested wheat types were capable of IRCs production, including IAA, which constituted from 1.75% to 52.68% of all IRCs, in in vitro conditions via the tryptophan dependent pathway. The selected post-culture medium was also examined using a plant bioassay. Substantial growth of wheat coleoptile segments treated with the bacterial post-culture medium was observed in several cases. Our data suggest that the studied endophytic bacteria produce auxin-type compounds to support plant development. Summarizing, our approach to use three complementary methods for estimation of IRCs in different endophytic strains provides a comprehensive picture of their effect on wheat growth.

New Insight into the Composition of Wheat Seed Microbiota.

Endophytes are associated with host plants throughout their life history from seed germination to fruit development. One of the most important plant organs colonized by endophytic microbiota is the seed. The aim of this study was to determine the structure of the seed core microbiome inhabiting the endosperms and embryos of eight wheat cultivars with the use of a culture-independent technique. The seeds of Triticum aestivum L. cv. Hondia, Wilejka, STH, Opcja, Tybalt, Euforia and Triticum spelta L. cv. Rokosz and Schwabencorn (producer: Plant Breeding Strzelce Sp. z o.o. Group IHAR) were studied. Rokosz and Hondia were cultured in vitro and in vivo to identify obligatory bacterial endophytes. A restrictive analysis of reads originating from the in vitro plants has demonstrated that the bacterial genera Paenibacillus and Propionibacterium inhabiting Rokosz and Hondia plants have a status of obligatory microorganisms. Greater biodiversity of seed-borne endophytes was found in the seed endosperms than in the embryos. The multiple comparison analysis of the OTU abundance indicated that the seed part significantly influenced the relative abundance. The seed-born microbiome is not statistically significantly dependent on the wheat cultivars; however, it cannot be claimed that every wheat seed is the same.

Bacterial Endophytes of Spring Wheat Grains and the Potential to Acquire Fe, Cu, and Zn under Their Low Soil Bioavailability.

Wheat grains are usually low in essential micronutrients. In resolving the problem of grain micronutritional quality, microbe-based technologies, including bacterial endophytes, seem to be promising. Thus, we aimed to (1) isolate and identify grain endophytic bacteria from selected spring wheat varieties (bread Oksamyt myronivs'kyi, Struna myronivs'ka, Dubravka, and emmer Holikovs'ka), which were all grown in field conditions with low bioavailability of microelements, and (2) evaluate the relationship between endophytes' abilities to synthesize auxins and the concentration of Fe, Zn, and Cu in grains. The calculated biological accumulation factor (BAF) allowed for comparing the varietal ability to uptake and transport micronutrients to the grains. For the first time, bacterial endophytes were isolated from grains of emmer wheat T. turgidum subsp. dicoccum. Generally, the 12 different isolates identified in the four varieties belonged to the genera Staphylococcus, Pantoea, Sphingobium, Bacillus, Kosakonia, and Micrococcus (NCBI accession numbers: MT302194-MT302204, MT312840). All the studied strains were able to synthesize the indole-related compounds (IRCs; max: 16.57 µg∙mL-1) detected using the Salkowski reagent. The IRCs produced by the bacterial genera Pantoea spp. and Bacillus spp. isolated from high-yielding Oksamyt myronivs'kyi and Holikovs'ka grains may be considered as one of the determinants of the yield of wheat and its nutritional characteristics.

Effect of BSO-supplemented heavy metals on antioxidant enzymes in Arabidopsis thaliana.

Activities of the ascorbate-glutathione cycle enzymes were investigated in leaves of Arabidopsis thaliana plants grown for 7 and 14 days in modified Hoagland nutrient solution containing Cd and Cu alone or supplemented with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. In A. thaliana treated with Cd+BSO, the modifying BSO effect involved dehydroascorbate reductase (DHAR) activity after 7 days of treatment and ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activities after 14 days. DHAR activity increased, while activities of APX, MDHAR and GR decreased in comparison to that found with Cd alone. BSO supplied in combination with Cu modified the metal effect on APX activity after 7 days of exposure and on MDHAR activity after 14 days. Cu+BSO enhanced APX activity, but decreased MDHAR activity as compared to that with Cu alone. Similarities and varieties in the modifying BSO effect, depending on the metal, have been discussed. The modifying BSO effect was more pronounced in the plants exposed to Cd than Cu, and was opposite after 7 and 14 days. However, differences between the effects of the individual metals on the enzymes were greater after 7 days of plant exposure.

Role of polyisoprenoids in tobacco resistance against biotic stresses.

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria (P. syringae) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.

The redox state and activity of superoxide dismutase classes in Arabidopsis thaliana under cadmium or copper stress.

The redox state of glutathione and ascorbate as well as the activity of superoxide dismutase classes were determined in leaves of Arabidopsis thaliana grown for seven days in the nutrient solution containing 0, 5 and 50 microM Cd or Cu excess. A decrease in GSH/GSSG ratio was found in plants under Cd and Cu stress. In the plants exposed to Cu stress the activity of all SOD classes increased. However, in the plants treated with Cd the activity of FeSOD and MnSOD was elevated, but CuZnSOD activity was diminished in comparison with control. In these plants the activity of SOD classes was dependent on both the GSH/GSSG and AA/DHA ratios, while in those exposed to Cu excess - on the GSH/GSSG ratio. Differences were shown in the changes both in redox state and activity of SOD classes caused by the metals differing in physiochemical properties. Moreover, relationships between changes in SOD class activities and ROS levels were discussed.

The activity of lipoxygenase in Arabidopsis thaliana (L.) Heynh -- a preliminary study.

The activity of lipoxygenase (EC 1.13.11.12) in Arabidopsis thaliana (L.) Heynh seedlings and mature plants was estimated spectrophotometrically at 234 nm. Linoleic acid was used as a substrate. Lipoxygenase activity showed two pH optima: at 7.0 and 10.0 in seedlings, and at pH 8.0 and 10.0 in leaves of mature plants. Seven-week-old plants were transferred to a hydroponic system and treated with different concentrations of Cd(2+) or Cu(2+) [in microM]: 0, 5, 25, 50, 100 for 7 days. The lipoxygenase activities at pH 8.0 and 10.0 depended on the metal that was added to the nutrient solution. The main change in lipoxygenase activity was under Cd(2+)stress at pH 8.0 and under Cu(2+)excess at pH 10.0.

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana.

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.