Plant Heterotrophic Cultures: No Food, No Growth.

Plant cells are capable of uptaking exogenous organic substances. This inherited trait allows the development of heterotrophic cell cultures in various plants. The most common of them are Nicotiana tabacum and Arabidopsis thaliana. Plant cells are widely used in academic studies and as factories for valuable substance production. The repertoire of compounds supporting the heterotrophic growth of plant cells is limited. The best growth of cultures is ensured by oligosaccharides and their cleavage products. Primarily, these are sucrose, raffinose, glucose and fructose. Other molecules such as glycerol, carbonic acids, starch, and mannitol have the ability to support growth occasionally, or in combination with another substrate. Culture growth is accompanied by processes of specialization, such as elongation growth. This determines the pattern of the carbon budget. Culture ageing is closely linked to substrate depletion, changes in medium composition, and cell physiological rearrangements. A lack of substrate leads to starvation, which results in a decrease in physiological activity and the mobilization of resources, and finally in the loss of viability. The cause of the instability of cultivated cells may be the non-optimal metabolism under cultural conditions or the insufficiency of internal regulation.

Large area structural color printing based on dot-matrix laser interference patterning.

Optically Variable Devices (OVDs) are widely used as security features in anti-counterfeiting efforts. OVDs enable the display of color dynamic effects that are easily interpreted by the user. However, obtaining these elements over large areas poses certain challenges in terms of efficiency. The paper presents a modified approach for manufacturing plasmonic type OVDs through dot-matrix technology, which is a standard origination step of security holograms. By adjusting the spatial filters in the optical scheme, it is possible to double the resolution of the recorded quasi-sinusoidal diffraction gratings. The experiments confirm the creation of diffraction gratings with frequencies from 1600 to 3500 lines per mm, which facilitates the production of plasmonic zero-order spectral filters. The paper shows how the transmission characteristics of the studied elements are affected by the geometric parameters of the diffraction grating, silver layer thickness, angle of incidence, and polarization of light. The results have shown that using the proposed method it is possible to obtain 1D or 2D structural color OVD-image on a large area - several square centimeters and more. High speed recording of such elements is provided: the exposure time was from 120 to 400 ms depending on the grating resolution for a 0.05 mm2 frame, the total printing time for the size of the 25×25 mm2 OVD was about 2.5 hours for a 1D element, and less than 3.5 hours for a 2D element. Thus, the proposed method and the OVD elements produced by it can be useful to designers of optical security elements as a simpler and faster alternative to electron-beam lithographic technologies.

Plant Life with and without Oxygen: A Metabolomics Approach.

Oxygen deficiency is an environmental challenge which affects plant growth, the development and distribution in land and aquatic ecosystems, as well as crop yield losses worldwide. The capacity to exist in the conditions of deficiency or the complete lack of oxygen depends on a number of anatomic, developmental and molecular adaptations. The lack of molecular oxygen leads to an inhibition of aerobic respiration, which causes energy starvation and the acceleration of glycolysis passing into fermentations. We focus on systemic metabolic alterations revealed with the different approaches of metabolomics. Oxygen deprivation stimulates the accumulation of glucose, pyruvate and lactate, indicating the acceleration of the sugar metabolism, glycolysis and lactic fermentation, respectively. Among the Krebs-cycle metabolites, only the succinate level increases. Amino acids related to glycolysis, including the phosphoglycerate family (Ser and Gly), shikimate family (Phe, Tyr and Trp) and pyruvate family (Ala, Leu and Val), are greatly elevated. Members of the Asp family (Asn, Lys, Met, Thr and Ile), as well as the Glu family (Glu, Pro, Arg and GABA), accumulate as well. These metabolites are important members of the metabolic signature of oxygen deficiency in plants, linking glycolysis with an altered Krebs cycle and allowing alternative pathways of NAD(P)H reoxidation to avoid the excessive accumulation of toxic fermentation products (lactate, acetaldehyde, ethanol). Reoxygenation induces the downregulation of the levels of major anaerobically induced metabolites, including lactate, succinate and amino acids, especially members of the pyruvate family (Ala, Leu and Val), Tyr and Glu family (GABA and Glu) and Asp family (Asn, Met, Thr and Ile). The metabolic profiles during native and environmental hypoxia are rather similar, consisting in the accumulation of fermentation products, succinate, fumarate and amino acids, particularly Ala, Gly and GABA. The most intriguing fact is that metabolic alterations during oxidative stress are very much similar, with plant response to oxygen deprivation but not to reoxygenation.

The Effects of Rhizophagus irregularis Inoculation on Transcriptome of Medicago lupulina Leaves at Early Vegetative and Flowering Stages of Plant Development.

The study is aimed at revealing the effects of Rhizophagus irregularis inoculation on the transcriptome of Medicago lupulina leaves at the early (second leaf formation) and later (flowering) stages of plant development. A pot experiment was conducted under conditions of low phosphorus (P) level in the substrate. M. lupulina plants were characterized by high mycorrhizal growth response and mycorrhization parameters. Library sequencing was performed on the Illumina HiseqXTen platform. Significant changes in the expression of 4863 (padj < 0.01) genes from 34049 functionally annotated genes were shown by Massive Analysis of cDNA Ends (MACE-Seq). GO enrichment analysis using the Kolmogorov-Smirnov test was performed, and 244 functional GO groups were identified, including genes contributing to the development of effective AM symbiosis. The Mercator online tool was used to assign functional classes of differentially expressed genes (DEGs). The early stage was characterized by the presence of six functional classes that included only upregulated GO groups, such as genes of carbohydrate metabolism, cellular respiration, nutrient uptake, photosynthesis, protein biosynthesis, and solute transport. At the later stage (flowering), the number of stimulated GO groups was reduced to photosynthesis and protein biosynthesis. All DEGs of the GO:0016036 group were downregulated because AM plants had higher resistance to phosphate starvation. For the first time, the upregulation of genes encoding thioredoxin in AM plant leaves was shown. It was supposed to reduce ROS level and thus, consequently, enhance the mechanisms of antioxidant protection in M. lupulina plants under conditions of low phosphorus level. Taken together, the obtained results indicate genes that are the most important for the effective symbiosis with M. lupulina and might be engaged in other plant species.

Effects of Trophic Acclimation on Growth and Expression Profiles of Genes Encoding Enzymes of Primary Metabolism and Plastid Transporters of Chlamydomonas reinhardtii.

In this paper, the effect of prolonged trophic acclimation on the subsequent growth of Chlamydomonas reinhardtii batch cultures was studied. The mixotrophic (light + acetate) acclimation stimulated subsequent growth at both mixotrophy and autotrophy conditions and altered the expression profile of genes encoding enzymes of primary metabolism and plastid transporters. Besides the trophic effect, the influence of Chlamydomonas culture growth stage on gene expression was determined. Under mixotrophic conditions, this effect was most pronounced in the first half of the exponential growth with partial retention of the previous acclimation period traits. The autotrophy acclimation effect was more complex and its significance was enhanced at the end of the growth and in the stationary phase.

Diversity of Arbuscular Mycorrhizal Fungi in Distinct Ecosystems of the North Caucasus, a Temperate Biodiversity Hotspot.

BACKGROUND: Investigations that are focused on arbuscular mycorrhizal fungus (AMF) biodiversity is still limited. The analysis of the AMF taxa in the North Caucasus, a temperate biodiversity hotspot, used to be limited to the genus level. This study aimed to define the AMF biodiversity at the species level in the North Caucasus biotopes. METHODS: The molecular genetic identification of fungi was carried out with ITS1 and ITS2 regions as barcodes via sequencing using Illumina MiSeq, the analysis of phylogenetic trees for individual genera, and searches for operational taxonomic units (OTUs) with identification at the species level. Sequences from MaarjAM and NCBI GenBank were used as references. RESULTS: We analyzed >10 million reads in soil samples for three biotopes to estimate fungal biodiversity. Briefly, 50 AMF species belonging to 20 genera were registered. The total number of the AM fungus OTUs for the "Subalpine Meadow" biotope was 171/131, that for "Forest" was 117/60, and that for "River Valley" was 296/221 based on ITS1/ITS2 data. The total number of the AM fungus species (except for virtual taxa) for the "Subalpine Meadow" biotope was 24/19, that for "Forest" was 22/13, and that for "River Valley" was 28/24 based on ITS1/ITS2 data. Greater AMF diversity, as well as number of OTUs and species, in comparison with that of forest biotopes, characterized valley biotopes (disturbed ecosystems; grasslands). The correlation coefficient between "Percentage of annual plants" and "Glomeromycota total reads" r = 0.76 and 0.81 for ITS1 and ITS2, respectively, and the correlation coefficient between "Percentage of annual plants" and "OTUs number (for total species)" was r = 0.67 and 0.77 for ITS1 and ITS2, respectively. CONCLUSION: High AMF biodiversity for the river valley can be associated with a higher percentage of annual plants in these biotopes and the active development of restorative successional processes.

The Role of Aquaporins in Plant Growth under Conditions of Oxygen Deficiency.

Plants frequently experience hypoxia due to flooding caused by intensive rainfall or irrigation, when they are partially or completely submerged under a layer of water. In the latter case, some resistant plants implement a hypoxia avoidance strategy by accelerating shoot elongation, which allows lifting their leaves above the water surface. This strategy is achieved due to increased water uptake by shoot cells through water channels (aquaporins, AQPs). It remains a puzzle how an increased flow of water through aquaporins into the cells of submerged shoots can be achieved, while it is well known that hypoxia inhibits the activity of aquaporins. In this review, we summarize the literature data on the mechanisms that are likely to compensate for the decline in aquaporin activity under hypoxic conditions, providing increased water entry into cells and accelerated shoot elongation. These mechanisms include changes in the expression of genes encoding aquaporins, as well as processes that occur at the post-transcriptional level. We also discuss the involvement of hormones, whose concentration changes in submerged plants, in the control of aquaporin activity.

The Role of Medicago lupulina Interaction with Rhizophagus irregularis in the Determination of Root Metabolome at Early Stages of AM Symbiosis.

The nature of plant-fungi interaction at early stages of arbuscular mycorrhiza (AM) development is still a puzzling problem. To investigate the processes behind this interaction, we used the Medicago lupulina MlS-1 line that forms high-efficient AM symbiosis with Rhizophagus irregularis. AM fungus actively colonizes the root system of the host plant and contributes to the formation of effective AM as characterized by a high mycorrhizal growth response (MGR) in the host plant. The present study is aimed at distinguishing the alterations in the M. lupulina root metabolic profile as an indicative marker of effective symbiosis. We examined the root metabolome at the 14th and 24th day after sowing and inoculation (DAS) with low substrate phosphorus levels. A GS-MS analysis detected 316 metabolites. Results indicated that profiles of M. lupulina root metabolites differed from those in leaves previously detected. The roots contained fewer sugars and organic acids. Hence, compounds supporting the growth of mycorrhizal fungus (especially amino acids, specific lipids, and carbohydrates) accumulated, and their presence coincided with intensive development of AM structures. Mycorrhization determined the root metabolite profile to a greater extent than host plant development. The obtained data highlight the importance of active plant-fungi metabolic interaction at early stages of host plant development for the determination of symbiotic efficiency.

Evolution of 14-3-3 Proteins in Angiosperm Plants: Recurring Gene Duplication and Loss.

14-3-3 proteins are key regulatory factors in plants and are involved in a broad range of physiological processes. We addressed the evolutionary history of 14-3-3s from 46 angiosperm species, including basal angiosperm Amborella and major lineage of monocotyledons and eudicotyledons. Orthologs of Arabidopsis isoforms were detected. There were several rounds of duplication events in the evolutionary history of the 14-3-3 protein family in plants. At least four subfamilies (iota, epsilon, kappa, and psi) formed as a result of ancient duplication in a common ancestor of angiosperm plants. Recent duplication events followed by gene loss in plant lineage, among others Brassicaceae, Fabaceae, and Poaceae, further shaped the high diversity of 14-3-3 isoforms in plants. Coexpression data showed that 14-3-3 proteins formed different functional groups in different species. In some species, evolutionarily related groups of 14-3-3 proteins had coexpressed together under certain physiological conditions, whereas in other species, closely related isoforms expressed in the opposite manner. A possible explanation is that gene duplication and loss is accompanied by functional plasticity of 14-3-3 proteins.

Mycorrhiza-Induced Alterations in Metabolome of Medicago lupulina Leaves during Symbiosis Development.

The present study is aimed at disclosing metabolic profile alterations in the leaves of the Medicago lupulina MlS-1 line that result from high-efficiency arbuscular mycorrhiza (AM) symbiosis formed with Rhizophagus irregularis under condition of a low phosphorus level in the substrate. A highly effective AM symbiosis was established in the period from the stooling to the shoot branching initiation stage (the efficiency in stem height exceeded 200%). Mycorrhization led to a more intensive accumulation of phosphates (glycerophosphoglycerol and inorganic phosphate) in M. lupulina leaves. Metabolic spectra were detected with GS-MS analysis. The application of complex mathematical analyses made it possible to identify the clustering of various groups of 320 metabolites and thus demonstrate the central importance of the carbohydrate and carboxylate-amino acid clusters. The results obtained indicate a delay in the metabolic development of mycorrhized plants. Thus, AM not only accelerates the transition between plant developmental stages but delays biochemical "maturation" mainly in the form of a lag of sugar accumulation in comparison with non-mycorrhized plants. Several methods of statistical modeling proved that, at least with respect to determining the metabolic status of host-plant leaves, stages of phenological development have priority over calendar age.

Involvement of Reactive Oxygen Species in ABA-Induced Increase in Hydraulic Conductivity and Aquaporin Abundance.

The role of reactive oxygen species (ROS) in ABA-induced increase in hydraulic conductivity was hypothesized to be dependent on an increase in aquaporin water channel (AQP) abundance. Single ABA application or its combination with ROS manipulators (ROS scavenger ascorbic acid and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI)) were studied on detached roots of barley plants. We measured the osmotically driven flow rate of xylem sap and calculated root hydraulic conductivity. In parallel, immunolocalization of ABA and HvPIP2;2 AQPs was performed with corresponding specific antibodies. ABA treatment increased the flow rate of xylem, root hydraulic conductivity and immunostaining for ABA and HvPIP2;2, while the addition of antioxidants prevented the effects of this hormone. The obtained results confirmed the involvement of ROS in ABA effect on hydraulic conductivity, in particular, the importance of H2O2 production by ABA-treated plants for the effect of this hormone on AQP abundance.

Metabolic Alterations in Pisum sativum Roots during Plant Growth and Arbuscular Mycorrhiza Development.

Intensive exchange of nutrients is a crucial part of the complex interaction between a host plant and fungi within arbuscular mycorrhizal (AM) symbiosis. For the first time, the present study demonstrates how inoculation with AMF Rhizophagus irregularis affects the pea (Pisum sativum L.) root metabolism at key stages of plant development. These correspond to days 21 (vegetation), 42 (flowering initiation), and 56 (fruiting-green pod). Metabolome profiling was carried out by means of a state-of-the-art GC-MS technique. The content shifts revealed include lipophilic compounds, sugars, carboxylates, and amino acids. The metabolic alterations were principally dependent on the stage of plant development but were also affected by the development of AM fungi, a fact which highlights interaction between symbiotic partners. The comparison of the present data with the results of leaf metabolome profiling earlier obtained did not reveal common signatures of metabolic response to mycorrhization in leaves and roots. We supposed that the feedback for the development and symbiotic interaction on the part of the supraorganismic system (root + AM fungi) was the cause of the difference between the metabolic profile shift in leaf and root cells that our examination revealed. New investigations are required to expand our knowledge of metabolome plasticity of the whole organism and/or system of organisms, and such results might be put to use for the intensification of sustainable agriculture.

Potassium Efflux and Cytosol Acidification as Primary Anoxia-Induced Events in Wheat and Rice Seedlings.

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K+ concentration, [K+]cyt, and cytosolic pH, pHcyt, in rice and wheat, plants with different tolerances to hypoxia. The [K+]cyt and pHcyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K+ from protoplasts of both plants after a lag-period of 300-450 s. The [K+]cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K+ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K+]cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pHcyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K+ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.

AM-Induced Alteration in the Expression of Genes, Encoding Phosphorus Transporters and Enzymes of Carbohydrate Metabolism in Medicago lupulina.

Plant-microbe interactions, including those of arbuscular mycorrhiza (AM), have been investigated for a wide spectrum of model plants. The present study focuses on an analysis of gene expression that encodes phosphate and sugar transporters and carbohydrate metabolic enzymes in a new model plant, the highly mycotrophic Medicago lupulina MLS-1 line under conditions of phosphorus deficiency and inoculation with Rhizophagus irregularis. Expression profiles were detected by RT-PCR at six plant stages of development (second leaf, third leaf, shooting, axillary shoot branching initiation, axillary shoot branching, flowering initiation). In comparison to control (without AM), the variant with AM inoculation exhibited a significant elevation of transcription levels of carbohydrate metabolic enzymes (MlSUS, MlHXK1) and sucrose transporters (MlSUC4) in M. lupulina leaves at the shooting stage. We suggest that this leads to a significant increase in the frequency of AM infection, an abundance of mycelium in roots and an increase in AM efficiency (which is calculated by the fresh weight of aerial parts and roots at the axillary shoot branching initiation stage). In roots, the specificity of MlPT4 and MlATP1 gene expressions were revealed for effective AM symbiosis. The level of MlPT4 transcripts in AM roots increased more than tenfold in comparison to that of non-specific MlPT1 and MlPT2. For the first time, MlPT1 expression was shown to increase sharply against MlPT2 in M. lupulina roots without AM at the shooting initiation stage. A significant increase in MlRUB expression was revealed at late stages in the host plant's development, during axillary shoot branching and flowering initiation. The opposite changes characterized MlHXK1 expression. Alteration in MlHXK1 gene transcription was the same, but was more pronounced in roots. The obtained results indicate the importance of genes that encode phosphate transporters and the enzymes of carbohydrate metabolism for effective AM development at the shooting stage in the host plant.

Phase Imbalance Optimization in Interference Linear Displacement Sensor with Surface Gratings.

In interferential linear displacement sensors, accurate information about the position of the reading head is calculated out of a pair of quadrature (sine and cosine) signals. In double grating interference schemes, diffraction gratings combine the function of beam splitters and phase retardation devices. Specifically, the reference diffraction grating is located in the reading head and regulates the phase shifts in diffraction orders. Measurement diffraction grating moves along with the object and provides correspondence to the displacement coordinate. To stabilize the phase imbalance in the output quadrature signals of the sensor, we propose to calculate and optimize the parameters of these gratings, based not only on the energetic analysis, but along with phase relationships in diffraction orders. The optimization method is based on rigorous coupled-wave analysis simulation of the phase shifts of light in diffraction orders in the optical system. The phase properties of the reference diffraction grating in the interferential sensor are studied. It is confirmed that the possibility of quadrature modulation depends on parameters of static reference scale. The implemented optimization criteria are formulated in accordance with the signal generation process in the optical branch. Phase imbalance and amplification coefficients are derived from Heydemann elliptic correction and expressed through the diffraction efficiencies and phase retardations of the reference scale. The phase imbalance of the obtained quadrature signals is estimated in ellipticity correction terms depending on the uncertainties of influencing parameters.

Indoleacetic Acid Levels in Wheat and Rice Seedlings under Oxygen Deficiency and Subsequent Reoxygenation.

The lack of oxygen and post-anoxic reactions cause significant alterations of plant growth and metabolism. Plant hormones are active participants in these alterations. This study focuses on auxin-a phytohormone with a wide spectrum of effects on plant growth and stress tolerance. The indoleacetic acid (IAA) content in plants was measured by ELISA. The obtained data revealed anoxia-induced accumulation of IAA in wheat and rice seedlings related to their tolerance of oxygen deprivation. The highest IAA accumulation was detected in rice roots. Subsequent reoxygenation was accompanied with a fast auxin reduction to the control level. A major difference was reported for shoots: wheat seedlings contained less than one-third of normoxic level of auxin during post-anoxia, while IAA level in rice seedlings rapidly recovered to normoxic level. It is likely that the mechanisms of auxin dynamics resulted from oxygen-induced shift in auxin degradation and transport. Exogenous IAA treatment enhanced plant survival under anoxia by decreased electrolyte leakage, production of hydrogen peroxide and lipid peroxidation. The positive effect of external IAA application coincided with improvement of tolerance to oxygen deprivation in the 35S:iaaM × 35S:iaaH lines of transgene tobacco due to its IAA overproduction.

Metabolic alterations in pea leaves during arbuscular mycorrhiza development.

Arbuscular mycorrhiza (AM) is known to be a mutually beneficial plant-fungal symbiosis; however, the effect of mycorrhization is heavily dependent on multiple biotic and abiotic factors. Therefore, for the proper employment of such plant-fungal symbiotic systems in agriculture, a detailed understanding of the molecular basis of the plant developmental response to mycorrhization is needed. The aim of this work was to uncover the physiological and metabolic alterations in pea (Pisum sativum L.) leaves associated with mycorrhization at key plant developmental stages. Plants of pea cv. Finale were grown in constant environmental conditions under phosphate deficiency. The plants were analyzed at six distinct time points, which corresponded to certain developmental stages of the pea: I: 7 days post inoculation (DPI) when the second leaf is fully unfolded with one pair of leaflets and a simple tendril; II: 21 DPI at first leaf with two pairs of leaflets and a complex tendril; III: 32 DPI when the floral bud is enclosed; IV: 42 DPI at the first open flower; V: 56 DPI when the pod is filled with green seeds; and VI: 90-110 DPI at the dry harvest stage. Inoculation with Rhizophagus irregularis had no effect on the fresh or dry shoot weight, the leaf photochemical activity, accumulation of chlorophyll a, b or carotenoids. However, at stage III (corresponding to the most active phase of mycorrhiza development), the number of internodes between cotyledons and the youngest completely developed leaf was lower in the inoculated plants than in those without inoculation. Moreover, inoculation extended the vegetation period of the host plants, and resulted in increase of the average dry weight per seed at stage VI. The leaf metabolome, as analyzed with GC-MS, included about three hundred distinct metabolites and showed a strong correlation with plant age, and, to a lesser extent, was influenced by mycorrhization. Metabolic shifts influenced the levels of sugars, amino acids and other intermediates of nitrogen and phosphorus metabolism. The use of unsupervised dimension reduction methods showed that (i) at stage II, the metabolite spectra of inoculated plants were similar to those of the control, and (ii) at stages IV and V, the leaf metabolic profiles of inoculated plants shifted towards the profiles of the control plants at earlier developmental stages. At stage IV the inoculated plants exhibited a higher level of metabolism of nitrogen, organic acids, and lipophilic compounds in comparison to control plants. Thus, mycorrhization led to the retardation of plant development, which was also associated with higher seed biomass accumulation in plants with an extended vegetation period. The symbiotic crosstalk between host plant and AM fungi leads to alterations in several biochemical pathways the details of which need to be elucidated in further studies.

Phosphatidic acids mediate transport of Ca2+ and H+ through plant cell membranes.

Phosphatidic acids (PAs) are a key intermediate in phospholipid biosynthesis, and a central element in numerous signalling pathways. Functions of PAs are related to their fundamental role in molecular interactions within cell membranes modifying membrane bending, budding, fission and fusion. Here we tested the hypothesis that PAs are capable of direct transport of ions across bio-membranes. We have demonstrated that PAs added to the maize plasma membrane vesicles induced ionophore-like transmembrane transport of Ca2+, H+ and Mg2+. PA-induced Ca2+ fluxes increased with an increasing PAs acyl chain unsaturation. For all the PAs analysed, the effect on Ca2+ permeability increased with increasing pH (pH 8.0>pH 7.2>pH 6.0). The PA-induced Ca2+, Mg2+ and H+ permeability was also more pronounced in the endomembrane vesicles as compared with the plasma membrane vesicles. Addition of PA to protoplasts from Arabidopsis thaliana (L.) Heynh. roots constitutively expressing aequorin triggered elevation of the cytosolic Ca2+ activity, indicating that the observed PA-dependent Ca2+ transport occurs in intact plants.

Metabolic Alterations in Male-Sterile Potato as Compared to Male-Fertile.

The common potato, Solanum tuberosum L., is the fourth most important agricultural crop worldwide. Until recently, vegetative propagation by tubers has been the main method of potato cultivation. A shift of interest to sexual potato reproduction by true botanical seeds is due to the appearance of a new hybrid seed breeding strategy whose successful application for many crop species has been supported by male sterility. This investigation was focused on the study of differences in the metabolite profiles of anthers at the mature pollen stage from male-fertile and male-sterile genotypes of S. tuberosum. Application of gas chromatography coupled with a mass spectrometry method allowed detection of metabolic profiles for 192 compounds. Further data analysis with several libraries fully identified 75 metabolites; a similar amount was defined up to the classes. Metabolic profiles in the anthers of fertile genotypes were significantly distinguished from male-sterile ones by the accumulation of carbohydrates, while the anthers of sterile genotypes contained a higher amount of amino acids. In comparison with male-fertile plants, male-sterile genotypes had undeveloped pollen grain characters; i.e., smaller grain size, a thicker exine, "permanent tetrads" that failed to disintegrate into microspores, and the absence of pollen apertures that might be due to a disorder in the metabolism of carbohydrates and fatty acids.

The levels of peroxisomal catalase protein and activity modulate the onset of cell death in tobacco BY-2 cells via reactive oxygen species levels and autophagy.

In plant cells, peroxisomes participate in the metabolism of reactive oxygen species (ROS). One of the major regulators of cellular ROS levels - catalase (CAT) - occurs exclusively in peroxisomes. CAT activity is required for immunity-triggered autophagic programmed cell death (PCD). Autophagy has been recently demonstrated to represent a route for degradation of peroxisomes in plant cells. In the present study, the dynamics of the cellular peroxisome pool in tobacco BY-2 cell suspension cultures were used to analyse the effects of inhibition of basal autophagy with special attention to CAT activity. Numbers of peroxisomes per cell, levels of CAT protein and activity, cell viability, ROS levels and expression levels of genes encoding components of antioxidant system were analysed upon application of 3-methyladenine (3-MA), an inhibitor of autophagy, and/or aminotriazole (AT), an inhibitor of CAT. When applied separately, 3-MA and AT led to an increase in cell death, but this effect was attenuated by their simultaneous application. The obtained data suggest that both the levels of CAT protein in peroxisomes as well as CAT activity modulate the onset of cell death in tobacco BY-2 cells via ROS levels and autophagy.