Epitranscriptome insights into Riccia fluitans L. (Marchantiophyta) aquatic transition using nanopore direct RNA sequencing.

BACKGROUND: Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition. RESULTS: A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions. CONCLUSIONS: These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

Antifungal Properties of Bio-AgNPs against D. pinodes and F. avenaceum Infection of Pea (Pisum sativum L.) Seedlings.

Ascochyta blight and Fusarium root rot are the most serious fungal diseases of pea, caused by D. pinodes and F. avenaceum, respectively. Due to the lack of fully resistant cultivars, we proposed the use of biologically synthesized silver nanoparticles (bio-AgNPs) as a novel protecting agent. In this study, we evaluated the antifungal properties and effectiveness of bio-AgNPs, in in vitro (poisoned food technique; resazurin assay) and in vivo (seedlings infection) experiments, against D. pinodes and F. avenaceum. Moreover, the effects of diseases on changes in the seedlings' metabolic profiles were analyzed. The MIC for spores of both fungi was 125 mg/L, and bio-AgNPs at 200 mg/L most effectively inhibited the mycelium growth of D. pinodes and F. avenaceum (by 45 and 26%, respectively, measured on the 14th day of incubation). The treatment of seedlings with bio-AgNPs or fungicides before inoculation prevented the development of infection. Bio-AgNPs at concentrations of 200 mg/L for D. pinodes and 100 mg/L for F. avenaceum effectively inhibited infections' spread. The comparison of changes in polar metabolites' profiles revealed disturbances in carbon and nitrogen metabolism in pea seedlings by both pathogenic fungi. The involvement of bio-AgNPs in the mobilization of plant metabolism in response to fungal infection is also discussed.

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.

Polar Metabolites Profiling of Wheat Shoots (Triticum aestivum L.) under Repeated Short-Term Soil Drought and Rewatering.

The response of wheat (Triticum aestivum L.) plants to the soil drought at the metabolome level is still not fully explained. In addition, research focuses mainly on single periods of drought, and there is still a lack of data on the response of plants to short-term cyclical periods of drought. The key to this research was to find out whether wheat shoots are able to resume metabolism after the stress subsides and if the reaction to subsequent stress is the same. Gas chromatography coupled with mass spectrometry (GC-MS) is one of the most valuable and fast methods to discover changes in the primary metabolism of plants. The targeted GC-MS analyses of whole shoots of wheat plants exposed (at the juvenile stage of development) to short-term (five days) mild soil drought/rewatering cycles (until the start of shoot wilting) enabled us to identify 32 polar metabolites. The obtained results revealed an accumulation of sugars (sucrose, fructose, glucose, and 1-kestose), proline, and malic acid. During five days of recovery, shoots regained full turgor and continued to grow, and the levels of accumulated metabolites decreased. Similar changes in metabolic profiles were found during the second drought/rewatering cycle. However, the concentrations of glucose, proline, and malic acid were higher after the second drought than after the first one. Additionally, the concentration of total polar metabolites after each plant rewatering was elevated compared to control samples. Although our results confirm the participation of proline in wheat responses to drought, they also highlight the responsiveness of soluble carbohydrate metabolism to stress/recovery.

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.

The comparison of polymorphism among Avena species revealed by retrotransposon-based DNA markers and soluble carbohydrates in seeds.

Here, we compared the polymorphism among 13 Avena species revealed by the iPBS markers and soluble carbohydrate profiles in seeds. The application of seven iPBS markers generated 83 bands, out of which 20.5% were polymorphic. No species-specific bands were scored. Shannon's information index (I) and expected heterozygosity (He) revealed low genetic diversity, with the highest values observed for A. nuda (I = 0.099; He = 0.068). UPGMA clustering of studied Avena accessions and PCoA results showed that the polyploidy level is the main grouping criterion. High-resolution gas chromatography revealed that the studied Avena accessions share the same composition of soluble carbohydrates, but significant differences in the content of total (5.30-22.38 mg g-1 of dry weight) and particular sugars among studied samples were observed. Sucrose appeared as the most abundant sugar (mean 61.52% of total soluble carbohydrates), followed by raffinose family oligosaccharides (31.23%), myo-inositol and its galactosides (6.16%), and monosaccharides (1.09%). The pattern of interspecific variation in soluble carbohydrates, showed by PCA, was convergent to that revealed by iPBS markers. Thus, both methods appeared as a source of valuable data useful in the characterization of Avena resources or in the discussion on the evolution of this genus.

The Size-Dependent Effects of Silver Nanoparticles on Germination, Early Seedling Development and Polar Metabolite Profile of Wheat (Triticum aestivum L.).

The phytotoxicity of silver nanoparticles (Ag NPs) to plant seeds germination and seedlings development depends on nanoparticles properties and concentration, as well as plant species and stress tolerance degrees. In the present study, the effect of citrate-stabilized spherical Ag NPs (20 mg/L) in sizes of 10, 20, 40, 60, and 100 nm, on wheat grain germination, early seedlings development, and polar metabolite profile in 3-day-old seedlings were analyzed. Ag NPs, regardless of their sizes, did not affect the germination of wheat grains. However, the smaller nanoparticles (10 and 20 nm in size) decreased the growth of seedling roots. Although the concentrations of total polar metabolites in roots, coleoptile, and endosperm of seedlings were not affected by Ag NPs, significant re-arrangements of carbohydrates profiles in seedlings were noted. In roots and coleoptile of 3-day-old seedlings, the concentration of sucrose increased, which was accompanied by a decrease in glucose and fructose. The concentrations of most other polar metabolites (amino acids, organic acids, and phosphate) were not affected by Ag NPs. Thus, an unknown signal is released by small-sized Ag NPs that triggers affection of sugars metabolism and/or distribution.

Pharmacokinetics of Myo-Inositol in a Wistar Rat Animal Model.

Myo-inositol is the most popular inositol in living organisms, where it is present as a sugar alcohol, in a free form, and can also be described as a lipid. The aim of this study was to check the concentration change of a myo-inositol solution from the time of oral administration and over a 48 h period in Wistar-type rats. All rats received 2 g/kg of inositol as a solution in distilled water by oral gavage. Estimated parameters were based on the serum concentration of myo-inositol observed in nine individual rats with regard to time. Observations were described as a one-compartment pharmacokinetic model with first-order absorption and zero-order endogenous input of checked inositol. The highest myo-inositol concentration was observed in the first hour after oral administration in the serum of all tested rats. Then, the concentration began decreasing immediately after the maximal peak. The inositol concentration continued to decrease, but after 24 h its level was still higher than before the administration. The plasma profile of the myo-inositol concentration showed a rapid decline over time, possibly due to the metabolism of this compound.

The Imbibition of Pea (Pisum sativum L.) Seeds in Silver Nitrate Reduces Seed Germination, Seedlings Development and Their Metabolic Profile.

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 Effect of Bio-Synthesized Silver Nanoparticles on Germination, Early Seedling Development, and Metabolome of Wheat (Triticum aestivum L.).

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.

Beneficial In Vitro Effects of a Low Myo-Inositol Dose in the Regulation of Vascular Resistance and Protein Peroxidation under Inflammatory Conditions.

Oxidative stress induces functional changes in arteries. Therefore, the effect of myo-inositol, a possible anti-inflammatory/antioxidant agent was studied on human plasma and rat thoracic arteries. Aortic rings from male Wistar rats (3 months of age) were incubated with myo-inositol (1, 10 and 100 µM, 120 min) and analyzed using the gas chromatography (GC) method. In another experiment, aortic rings were protected first with myo-inositol (1 µM, 60 min) and then subjected to a thromboxane receptor agonist (U-46619, 0.1 nM, 60 min). Therefore, these four groups under the following conditions were studied: (i) the control in the vehicle; (ii) myo-inositol; (iii) the vehicle plus U-46619; (iv) myo-inositol plus U-46619. The hemostatic parameters of human plasma and an H2O2/Fe2+ challenge for lipid and protein peroxidation were also performed. Myo-inositol was not absorbed into the pre-incubated aortic rings as measured by the GC method (0.040 µg/mg, p ≥ 0.8688). The effect of myo-inositol was more significant in the impaired arteries due to U-46619 incubation, which resulted in an improved response to acetylcholine (% Emax: 58.47 vs. 86.69), sodium nitroprusside (logEC50: −7.478 vs. −8.076), CORM-2 (% Emax: 44.08 vs. 83.29), pinacidil (logEC50: −6.489 vs. −6.988) and noradrenaline (logEC50: −7.264 vs. −6.525). This was most likely a possible response to increased nitric oxide release (×2.6-fold, p < 0001), and decreased hydrogen peroxide production (×0.7-fold, p = 0.0012). KCl-induced membrane depolarization was not modified (p ≥ 0.4768). Both the plasma protein carbonylation (×0.7-fold, p = 0.0006), and the level of thiol groups (×3.2-fold, p = 0.0462) were also improved, which was not significant for TBARS (×0.8-fold, p = 0.0872). The hemostatic parameters were also not modified (p ≥ 0.8171). A protective effect of myo-inositol was demonstrated against prooxidant damage to human plasma and rat thoracic arteries, suggesting a strong role of this nutraceutical agent on vasculature which may be of benefit against harmful environmental effects.

Changes in Metabolic Profiles of Pea (Pisum sativum L.) as a Result of Repeated Short-Term Soil Drought and Subsequent Re-Watering.

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.

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.

The Application of Fe-EDTA and Sodium Silicate Affects the Polyphenols Content in Broccoli and Radish Sprouts.

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.

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.

Changes in Polar Metabolites Content during Natural and Methyl-Jasmonate-Promoted Senescence of Ginkgo biloba Leaves.

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.

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.