No abstract

Changes in the metabolome of germinating seeds and seedlings caused by metal nanoparticles are poorly understood. In the present study, the effects of bio-synthesized silver nanoparticles ((Bio)Ag NPs) on grains germination, early seedlings development, and metabolic profiles of roots, coleoptile, and endosperm of wheat were analyzed. Grains germinated well in (Bio)Ag NPs suspensions at the concentration in the range 10–40 mg/L. However, the growth of coleoptile was inhibited by 25%, regardless of (Bio)Ag NPs concentration tested, whereas the growth of roots gradually slowed down along with the increasing concentration of (Bio)Ag NPs. The deleterious effect of Ag NPs on roots was manifested by their shortening, thickening, browning of roots tips, epidermal cell death, progression from apical meristem up to root hairs zone, and the inhibition of root hair development. (Bio)Ag NPs stimulated ROS production in roots and affected the metabolic profiles of all tissues. Roots accumulated sucrose, maltose, 1-kestose, phosphoric acid, and some amino acids (i.e., proline, aspartate/asparagine, hydroxyproline, and branched-chain amino acids). In coleoptile and endosperm, contrary to roots, the concentration of most metabolites decreased. Moreover, coleoptile accumulated galactose. Changes in the concentration of polar metabolites in seedlings revealed the affection of primary metabolism, disturbances in the mobilization of storage materials, and a translocation of sugars and amino acids from the endosperm to growing seedlings.

The metabolic re-arrangements of peas (Pisum sativum L.) under soil drought and re-watering are still not fully explained. The search for metabolic markers of the stress response is important in breeding programs, to allow for the selection drought-resistant cultivars. During the present study, changes in the polar metabolite content in pea plant shoots were measured under repeated short-term soil drought and subsequent re-watering. A gas chromatograph, equipped with a mass spectrometer (GC-MS), was used for the metabolite profiling of pea plants during their middle stage of vegetation (14–34 days after sowing, DAS). The major changes occurred in the concentration of amino acids and some soluble carbohydrates. Among them, proline, γ-aminobutyric acid (GABA), branched-chain amino acids, hydroxyproline, serine, myo-inositol, and raffinose were accumulated under each soil drought and decreased after re-watering. Besides, the obtained results show that the first drought/re-watering cycle increased the ability of pea plants to restore a metabolic profile similar to the control after the second similar stress. The accumulation of proline seems to be an important part of drought memory in pea plants. However, confirmation of this suggestion requires metabolite profiling studies on a broader spectrum of pea cultivars.

Methylated inositol, D-pinitol (3-O-methyl-D-chiro-inositol), is a common constituent in legumes. It is synthesized from myo-inositol in two reactions: the first reaction, catalyzed by myo-inositol-O-methyltransferase (IMT), consists of a transfer of a methyl group from S-adenosylmethionine to myo-inositol with the formation of D-ononitol, while the second reaction, catalyzed by D-ononitol epimerase (OEP), involves epimerization of D-ononitol to D-pinitol. To identify the genes involved in D-pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co-expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo-keto reductase and MtOEPB, encoding a short-chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo-inositol to D-ononitol and showed that MtOEPA enzyme has NAD + -dependent D-ononitol dehydrogenase activity, while MtOEPB enzyme has NADP + -dependent D-pinitol dehydrogenase activity. Both enzymes are required for epimerization of D-ononitol to D-pinitol, which occurs in the presence of NAD + and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of D-ononitol and D-pinitol in transformants. As this two-step pathway of D-ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD + , we speculate that one of the functions of this pathway might be regeneration of NADP + during drought stress.Keywords: carbohydrate metabolism, drought, legumes, Medicago truncatula, metabolic adaptation to stress, metabolic pathways. D-pinitol and drought resistance was also found by Guo and Oosterhuis (1997), who reported that soybean cultivars that showed increased resistance to soil drought accumulated larger quantities of D-pinitol than sensitive varieties.

The use of silver nanoparticles (Ag NPs) on plants is accompanied by the occurrence of Ag+ ions, so the research of the effects of both on plants should be related. Therefore, in our study, the effects of Ag NPs suspension (containing Ag0 at 20 mg/L) and AgNO3 solutions (with the concentration of Ag+ ions at 20 and 50 mg/L) on the seed germination and early seedling growth (4 days) of pea (Pisum sativum L.) were compared. Both Ag NPs and AgNO3 did not decrease seed germination, and even stimulated seedling growth. In seedlings developing in the Ag NPs suspension, an increase in monosaccharides, homoserine and malate was noted. In the next experiment, the effect of short-term seed imbibition (8 h) in AgNO3 at elevated concentrations, ranging from 100 to 1000 mg/L, on the further seed germination, seedling growth (in absence of AgNO3) and their polar metabolic profiles were evaluated. The seed imbibition in AgNO3 solutions at 500 and 1000 mg/L reduced seed germination, inhibited seedlings’ growth and caused morphological deformations (twisting and folding of root). The above phytotoxic effects were accompanied by changes in amino acids and soluble carbohydrates profiles, in both sprouts and cotyledons. In deformed sprouts, the content of homoserine and asparagine (major amino acids) decreased, while alanine, glutamic acid, glutamine, proline, GABA (γ-aminobutyric acid) and sucrose increased. The increase in sucrose coincided with a decrease in glucose and fructose. Sprouts, but not cotyledons, also accumulated malic acid and phosphoric acid. Additionally, cotyledons developed from seeds imbibed with AgNO3 contained raffinose and stachyose, which were not detectable in sprouts and cotyledons of control seedlings. The obtained results suggest the possible disturbances in the mobilization of primary (oligosaccharides) and presumably major storage materials (starch, proteins) as well as in the primary metabolism of developing seedlings.

The effects of elicitors on broccoli (Brassica oleracea L. var. Italica) and radish (Raphanus sativus L.) sprouts were evaluated. Seeds and then sprouts were soaked daily for 30 min over 6 days in water (control) or a mixture of FeEDTA and sodium silicate or sodium silicate alone. The contents of the flavonoids and phenolic acids (free, esters, and glycosides) were determined using HPLC-ESI-MS/MS. Phenolic compounds were released from the esters after acid hydrolysis and from the glycosides using alkaline hydrolysis. Quercetin, kaempferol, (‒)-epicatechin, naringenin, apigenin, and luteolin derivatives were found in broccoli and radish sprouts, while derivatives of iso-rhamnetin, orientin, and vitexin were not present at measurable levels. The flavonoid contents, especially derivatives of quercetin, were considerably higher in the broccoli sprouts than in the radish sprouts. The quantitatively major phenolic acid content in the sprouts of both species was found to be p-hydroxybenzoic acid. Its content in the radish sprouts was several times higher than in the broccoli sprouts. The total flavonoid content of broccoli sprouts was 507–734 µg/g DW, while that of the radish sprouts ranged from 155 µg/g DW to 211 µg/g DW. In contrast, total phenolic acids were higher in radish sprouts, ranging from 11,548 to 13,789 µg/g DW, while in broccoli sprouts, they ranged from 2652 to 4527 µg/g DW, respectively. These differences resulted radish sprouts having higher antioxidant activity compared to broccoli sprouts. The applied elicitors increased the content of the total phenolic acids and the antioxidant activity of radish and broccoli sprouts, while they decreased the level of the total flavonoids in broccoli sprouts.

The conditions for determining the antioxidant properties of cyclitols (d-pinitol, l-quebrachitol, myo-, l-chiro-, and d-chiro-inositol), selected flavanones (hesperetin, naringenin, eriodictyol, and liquiritigenin) and glutathione by spectrophotometric methods—CUPRAC and with DPPH radical, and by a chromatographic method DPPH-UHPLC-UV, have been identified. Interactions of the tested compounds and their impact on the ox-red properties were investigated. The RSA (%) of the compounds tested was determined. Very low antioxidative properties of cyclitols, compared with flavanones and glutathione alone, were revealed. However, a significant increase in the determined antioxidative properties of glutathione by methyl-ether derivatives of cyclitols (d-pinitol and l-quebrachitol) was demonstrated for the first time. Thus, cyclitols seem to be a good candidate for creating drugs for the treatment of many diseases associated with reactive oxygen species (ROS) generation.

The present study clarified changes in the contents of polar metabolites (amino acids, organic acids, saccharides, cyclitols, and phosphoric acid) in leaf senescence in Ginkgo biloba with or without the application of methyl jasmonate (JA-Me) in comparison with those in naturally senescent leaf blades and petioles. The contents of most amino acids and citric and malic acids were significantly higher in abaxially, and that of myo-inositol was lower in abaxially JA-Me-treated leaves than in adaxially JA-Me-treated and naturally senescent leaves. The levels of succinic and fumaric acids in leaves treated adaxially substantially high, but not in naturally senescent leaves. In contrast, sucrose, glucose, and fructose contents were much lower in leaf blades and petioles treated abaxially with JA-Me than those treated adaxially. The levels of these saccharides were also lower compared with those in naturally senescent leaves. Shikimic acid and quinic acid were present at high levels in leaf blades and petioles of G. biloba. In leaves naturally senescent, their levels were higher compared to green leaves. The shikimic acid content was also higher in the organs of naturally yellow leaves than in those treated with JA-Me. These results strongly suggest that JA-Me applied abaxially significantly enhanced processes of primary metabolism during senescence of G. biloba compared with those applied adaxially. The changes in polar metabolites in relation to natural senescence were also discussed.