Zinc Oxide Nanoparticles Affect Early Seedlings' Growth and Polar Metabolite Profiles of Pea (Pisum sativum L.) and Wheat (Triticum aestivum L.).

The growing interest in the use of zinc oxide nanoparticles (ZnO NPs) in agriculture creates a risk of soil contamination with ZnO NPs, which can lead to phytotoxic effects on germinating seeds and seedlings. In the present study, the susceptibility of germinating seeds/seedlings of pea and wheat to ZnO NPs of various sizes (≤50 and ≤100 nm) applied at concentrations in the range of 100-1000 mg/L was compared. Changes in metabolic profiles in seedlings were analyzed by GC and GC-MS methods. The size-dependent harmful effect of ZnO NPs on the seedling's growth was revealed. The more toxic ZnO NPs (50 nm) at the lowest concentration (100 mg/L) caused a 2-fold decrease in the length of the wheat roots. In peas, the root elongation was slowed down by 20-30% only at 1000 mg/L ZnO NPs. The metabolic response to ZnO NPs, common for all tested cultivars of pea and wheat, was a significant increase in sucrose (in roots and shoots) and GABA (in roots). In pea seedlings, an increased content of metabolites involved in the aspartate-glutamate pathway and the TCA cycle (citrate, malate) was found, while in wheat, the content of total amino acids (in all tissues) and malate (in roots) decreased. Moreover, a decrease in products of starch hydrolysis (maltose and glucose) in wheat endosperm indicates the disturbances in starch mobilization.

Changes in the Carbohydrate Profile in Common Buckwheat (Fagopyrum esculentum Moench) Seedlings Induced by Cold Stress and Dehydration.

Plant species are sensitive to stresses, especially at the seedling stage, and they respond to these conditions by making metabolic changes to counteract the negative effects of this. The objectives of this study were to determine carbohydrate profile in particular organs (roots, hypocotyl, and cotyledons) of common buckwheat seedlings and to verify whether carbohydrate accumulation is similar or not in the organs in response to cold stress and dehydration. Roots, hypocotyl, and cotyledons of common buckwheat seedlings have various saccharide compositions. The highest concentrations of cyclitols, raffinose, and stachyose were found in the hypocotyl, indicating that they may be transported from cotyledons, although this needs further studies. Accumulation of raffinose and stachyose is a strong indicator of the response of all buckwheat organs to introduced cold stress. Besides, cold conditions reduced d-chiro-inositol content, but did not affect d-pinitol level. Enhanced accumulation of raffinose and stachyose were also a distinct response of all organs against dehydration at ambient temperature. The process causes also a large decrease in the content of d-pinitol in buckwheat hypocotyl, which may indicate its transformation to d-chiro-inositol whose content increased at that time. In general, the sucrose and its galactosides in hypocotyl tissues were subject to the highest changes to the applied cold and dehydration conditions compared to the cotyledons and roots. This may indicate tissue differences in the functioning of the protective system(s) against such threats.

Exogenously Applied Cyclitols and Biosynthesized Silver Nanoparticles Affect the Soluble Carbohydrate Profiles of Wheat (Triticum aestivum L.) Seedling.

Cyclitols, such as myo-inositol and its isomers and methyl derivatives (i.e., d-chiro-inositol and d-pinitol (3-O-methyl-chiro-inositol)), are classified as osmolytes and osmoprotectants and are significantly involved in plant responses to abiotic stresses, such as drought, salinity and cold. Moreover, d-pinitol demonstrates a synergistic effect with glutathione (GSH), increasing its antioxidant properties. However, the role of cyclitols in plant protection against stresses caused by metal nanoparticles is not yet known. Therefore, the present study examined the effects of myo-inositol, d-chiro-inositol and d-pinitol on wheat germination, seedling growth and changes in the profile of soluble carbohydrates in response to biologically synthesized silver nanoparticles ((Bio)Ag NPs). It was found that cyclitols were absorbed by germinating grains and transported within the growing seedlings but this process was disrupted by (Bio)Ag NPs. Cyclitols applied alone induced sucrose and 1-kestose accumulation in seedlings slightly, while (Bio)Ag NP doubled the concentrations of both sugars. This coincided with a decrease in monosaccharides; i.e., fructose and glucose. Cyclitols and (Bio)Ag NPs present in the endosperm resulted in reductions in monosaccharides, maltose and maltotriose, with no effect on sucrose and 1-kestose. Similar changes occurred in seedlings developing from primed grains. Cyclitols that accumulated in grain and seedlings during grain priming with d-pinitol and glutathione did not prevent the phytotoxic effects of (Bio)Ag NPs.

Accumulation of Anthocyanins in Detached Leaves of Kalanchoë blossfeldiana: Relevance to the Effect of Methyl Jasmonate on This Process.

Accumulation of anthocyanins in detached leaves and in excised stems of Kalanchoë blossfeldiana kept under natural light conditions in the presence or absence of methyl jasmonate (JA-Me) was investigated. When the abaxial surface of detached leaves was held lower than the adaxial surface (the normal or natural position) under natural light conditions, anthocyanins were not accumulated on the abaxial side of the leaves. In contrast, when the adaxial surface of detached leaves was held lower than the abaxial surface (inverted position), anthocyanins were highly accumulated on the abaxial side of the leaves. These phenomena were independent of the growth stage of K. blossfeldiana as well as photoperiod. Application of JA-Me in lanolin paste significantly inhibited anthocyanin accumulation induced on the abaxial side of detached leaves held in an inverted position in a dose-dependent manner. Anthocyanin accumulation in the excised stem in response to natural light was also significantly inhibited by JA-Me in lanolin paste. Possible mechanisms of anthocyanin accumulation on the abaxial side of detached K. blossfeldiana leaves held in an inverted position under natural light conditions and the inhibitory effect of JA-Me on this process are described. The accompanying changes in the content of primary metabolites and histological analyses were also described.

Analyses of Antioxidative Properties of Selected Cyclitols and Their Mixtures with Flavanones and Glutathione.

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.

Metabolite profiling of semi-leafless pea (Pisum sativum L.) under progressive soil drought and subsequent re-watering.

Four semi-leafless pea (Pisum sativum L.) cultivars at the vegetative stage of growth were exposed to progressive soil drought, which lasted for 18 days until the plants began to wilt, after which a 7-day period of the recovery from stress followed, when plant watering was resumed. The soil drought negatively affected plant growth, slowing down the rate of shoot elongation, decreasing the accumulation of fresh and dry weight, inhibiting the development of new leaves, and delaying the flowering of plants. Changes in the levels of 41 polar metabolites (identified by GC-MS) were established by the GC-FID method in the shoot tip, stem, stipules and tendrils, separately. Drought caused re-arrangement in the metabolism in all parts of the pea shoot, leading to a significant increase in the content of total polar metabolites. Although changes in most metabolites in the same parts of shoot were not identical among the pea cultivars studied, some metabolites were uniformly accumulated until 18th day of drought and decreased after recovery. They were i) proline and malate in all, while myo-inositol in most parts of shoot (of all the pea cultivars), ii) sucrose and glycine in the shoot tip, iii) homoserine in the stem and iv) GABA in stipules. These findings signify that the pea adjustment to progressive soil drought includes both accumulation of osmolytes and osmoprotectants and translocation of some of them (proline, sucrose, myo-inositol) to the shoot tip, thereby protecting the youngest tissues from damage caused by water deficit.

Two-step d-ononitol epimerization pathway in Medicago truncatula.

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.

The acquisition of desiccation tolerance in developing Vicia hirsuta seeds coincides with an increase in galactinol synthase expression and soluble α-D-galactosides accumulation.

Galactinol is the galactosyl donor for the biosynthesis of both the raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C). Its synthesis by galactinol synthase (GolS, EC 2.4.1.123) is the first committed step of the soluble α-D-galactosides biosynthetic pathway in orthodox seeds. The deposition of galactosides in seeds is suggested to be associated with desiccation tolerance (DT). In this work, for the first time, we cloned and characterized two Vicia hirsuta (L.) S.F. Gray galactinol synthase genes (VhGolS1, VhGolS2), analyzed galactinol synthase activity and measured the accumulation of galactosides of both sucrose and D-pinitol in relation to the acquisition of DT in developing seeds of this wild species. A developmentally induced increase of VhGolS1 expression preceded the rise of GolS activity in crude protein extract from maturing seeds, while the expression of the VhGolS2 gene remained low. GolS activity peaked just after the beginning of the maturation drying phase. The increase of GolS activity was not followed by galactinol accumulation, instead the high enzyme activity was related to high levels of galactose bound in soluble galactosides of the RFO and galactosyl pinitol series. Acquisition of DT coincided with an increase of VhGolS1 expression, high galactinol synthase activity and the accumulation of oligogalactosides in seeds. DT was positively correlated with the high content of soluble α-D-galactosides of both the RFO and galactosyl pinitol series as well as with the amount of galactose bound in these galactosides.

Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

D-chiro-inositol affects accumulation of raffinose family oligosaccharides in developing embryos of Pisum sativum.

Developing garden pea embryos are able to take up exogenously applied cyclitols: myo-inositol, which naturally occurs in pea, and two cyclitols absent in pea plants: d-chiro-inositol and d-pinitol. The competition in the uptake of cyclitols by pea embryo, insensitivity to glucose and sucrose, and susceptibility to inhibitor(s) of H(+)-symporters (e.g. CCCP and antimycin A) suggest that a common cyclitol transporter is involved. Both d-chiro-inositol and d-pinitol can be translocated through the pea plant to developing embryos. During seed maturation drying, they are used for synthesis of mainly mono-galactosides, such as fagopyritol B1 and galactosyl pinitol A. Accumulation of d-chiro-inositol (and formation of fagopyritols), but not d-pinitol, strongly reduces accumulation of verbascose, the main raffinose oligosaccharide in pea seeds. The reasons for the observed changes are discussed.

Effect of jasmonic acid-methyl ester on the composition of carbohydrates and germination of yellow lupine (Lupinus luteus L.) seeds.

Mature seeds of yellow lupine contained sucrose, raffinose family oligosaccharides (RFOs), and galactosyl cyclitols as major soluble carbohydrates. The study showed that RFOs dominated in lupine seeds (16% DW). The disappearance of both types of alpha-d-galactosides in germinating lupine seeds was strongly inhibited by the presence of jasmonic acid-methyl ester (JA-Me) at a concentration of 10(-3)M in the incubation medium. JA-Me inhibited the activity of alpha-D-galactosidase (fraction I) during seed germination. Anatomical studies of lupine roots have shown certain cell structure differences between control and JA-Me-treated seedlings. The cross-sections of plant roots treated with JA-Me showed a characteristic folding of the cell walls in all root tissues, starting from the rhyzodermis, cortex and vascular cylinder. In water-treated (control) plants, the cell walls were rounded with no folding.

Molecular structure of lathyritol, a galactosylbornesitol from Lathyrus odoratus seeds, by NMR.

The molecular structure of galactosyl-D-(-)-bornesitol, a novel compound isolated from sweet pea seeds, was determined to be alpha-D-galactopyranosyl-(1-->3)-1-O-methyl-1D-myo-inositol by 1D and 2D NMR spectroscopy and is assigned the trivial name lathyritol.