Anastasia Kirpichnikova

The ability of plants to survive oxygen deficiency is associated with significant changes in metabolism. Metabolic profiling of wheat seedlings under anoxia and subsequent reoxygenation conditions was performed using GC-MS. A total of 374 and 298 compounds were detected in root and shoot metabolomes, respectively. All intermediates of central metabolism were identified. Early anoxic responses of root and shoot metabolomes showed similarity, leading to the accumulation of amino acids (Ala, GABA and Tyr), carboxylates (lactate and succinate), nucleotides and amines, together with a decrease in sugars. The metabolic response to long-term anoxia varied significantly in the roots and shoots of wheat seedlings and was related to the redistribution of carbon flux from glycolysis predominantly to lipids in the roots, while it was directed to carboxylates and GABA in the shoots. Imposition of 24 h of reaeration after short-term anoxia (6 h) switched the metabolome toward a normoxic profile, predominantly in roots. Anaerobically down-regulated metabolites were accumulated, while anaerobic intermediates were depleted post-anoxia. The effects of more prolonged anoxia on wheat seedling metabolomes were less reversible, particularly in shoots. Interestingly, several metabolites with not fully understood roles (e.g., hydroxyl carboxylates, α,ω-dicarboxylic acids, polyols) were detected under anoxic conditions in wheat seedlings, which could potentially serve as markers of plant sensitivity to oxygen deficiency.

ДИНАМИКА СОДЕРЖАНИЯ АКВАПОРИНОВ ПЛАЗМАЛЕММЫ И ТОНОПЛАСТА ПОДСЕМЕЙСТВ PIP И TIP В ХОДЕ РОСТА РАСТЯЖЕНИЕМ КЛЕТОК КОЛЕОПТИЛЕЙ ПРОРОСТКОВ РИСА, РАЗЛИЧАЮЩИХСЯ СКОРОСТЬЮ РОСТА В УСЛОВИЯХ НОРМОКСИИ И ЗАТОПЛЕНИЯ А.А. Кирпичникова, П.Д. Смирнов, Г.В. Данелия, В.В. Емельянов, М.Ф. Шишова

THE PLASMA MEMBRANE AND TONOPLAST PIP AND TIP AQUAPORINS ABUNDANCE DURING THE ELONGATION GROWTH OF COLEOPTILE CELLS OF RICE SEEDLINGS WITH DIFFERENT GROWTH RATES UNDER NORMOXIA AND SUBMERGENCE А.А. Kirpichnikova, P.D. Smirnov, G.V. Daneliia, V.V. Yemelyanov, M.F. Shishova

BACKGROUND: Rice coleoptiles were used to investigate the importance of V H+-ATPase in vacuolization during elongation growth under normoxic and hypoxic conditions. AIM of the study was to find out a link between growth intensity, protein amount of subunits B and E and transcription of genes encoding those proteins. MATERIALS AND METHODS: The investigation was carried out on two rice varieties of domestic selection, fast-growing Kuban 3 and slow-growing Amethyst. Seedlings were grown in etiolated conditions at normoxia and submergence. Western-blot analysis was employed to evaluate amount of subunits B and E in microsomal fraction. qRT-PCR was used to distinguish differences in expression of genes encoding subunits B and E of V H+-ATPase. RESULTS: The growth under aerobic conditions was more consistent with the changes in subunits B and E of V H+-ATPase which was determined at the proteomic level, while the hypoxic growth had a stronger correspondence with changes in OsVHAs gene expression. Varietal differences were revealed only when comparing the transcription intensity, which did not affect the growth dynamics of coleoptiles. Obtained data suggested the existence of differences in the regulation of the enzyme at the transcriptional and proteomic levels during coleoptile elongation. CONCLUSIONS: The importance of the B and E subunits of V-ATPase involvement in vacuolization during the growth process of rice coleoptiles under different oxygen level was demonstrated.

Plants known as obligate aerobes developed different mechanisms to overcome the damage incurred under oxygen limitation. One of the survival strategies to have commonly appeared in hydrophytic plants is the escape strategy, which accelerates plant axial organs’ growth in order to escape hypoxic conditions as soon as possible. The present study aimed to distinguish the alterations in coleoptile elongation, viability and metabolic profiles in coleoptiles of slow- and fast-growing rice varieties. All the parameters were tested at 3, 5 and 7 days after sowing, to highlight changes during seedling development in normal and submerged conditions. The obtained results indicated that coleoptile elongation correlated with higher resistance to oxygen deprivation. GS-MS-based metabolic profiling indicated that coleoptiles of the fast-growing cultivar accumulated higher amounts of sugar phosphates, disaccharides, fatty acid derivatives and sterols, which are important for maintaining growth, membrane stability and viability. The slow-growing variety was characterized by a greater abundance of carboxylates, including lactate and phosphoric acid, indicating an energy crisis and cytosol acidification, leading to cell damage and low tolerance. Therefore, a metabolomics approach could be used for phenotyping (chemotyping) in the large-scale screening of newly developed varieties with higher tolerance to oxygen deprivation.

Tobacco BY-2 cell culture is one of the most widely used models in plant biology. The main advantage of BY-2 suspension cultures is the synchronization of cell development and the appearance of polar elongation. In batch culture, BY-2 cells passed through the lag, proliferation, elongation, and stationary phases. During this process, the composition of the growth medium changed dramatically. Sucrose was rapidly eliminated; hexose first accumulated and then depleted. The medium’s pH initially decreased and then rose with aging. As a result of the crosstalk between the internal and external stimuli, cells pass through complicated systemic rearrangements, which cause metabolomic alterations. The early stages were characterized by high levels of amino acids and sterols, which could be interpreted as the result of synthetic activity. The most intense rearrangements occurred between the proliferation and active elongation stages, including repression of amino acid accumulation and up-regulation of sugar metabolism. Later stages were distinguished by higher levels of secondary metabolites, which may be a non-specific response to deteriorating conditions. Senescence was followed by some increase in fatty acids and sterols as well as amino acids, and probably led to self-destructive processes. A correlation analysis revealed relationships between metabolites’ covariation, their biochemical ratio, and the growth phase.

BACKGROUND: Heterotrophic cell cultures are widely used as a model in plant biology. During a culture cycle the composition of the medium changes: the sucrose and other substrates are depleted, metabolism products are accumulated and the density increases. Finally, arrest of a growth is followed by cell death in a short time. These processes are accompanied with physiological alterations, corresponding to senescence. AIM: To resolve metabolic features of tobacco cells in growing and stationary senescent suspension cultures VBI-0. MATERIALS AND METHODS: Nicotiana tabacum VBI-0 cells were cultured in suspension MS medium supplied with 3% sucrose. Cells were sampled at 7th day, during intensive growth, and at 28th day, when the culture was in the stationary phase. The GC-MS method was used to profile the metabolites. RESULTS: Sucrose depletion in media caused starvation of heterotrophic tobacco cell culture and was associated with a decrease in the accumulation of free amino acids. At the same time, the level of pentoses and complex sugars, including sucrose, increased, while the levels of glucose and fructose were not changed significantly and levels of hexose phosphates decreased. During culture senescence cells showed higher levels of accumulation of malate, pyruvate and some other carboxylates. CONCLUSIONS: The metabolomic data indicate that culture senescence was associated with a drop in amino acids metabolism, a decrease in the activity of the upper part of glycolysis, and the accumulation of complex sugars, pentoses and carboxylates.

The review examines modern knowledge on the mechanisms of the early stages of plant cell elongation growth. Coleoptiles are used as a model object representing juvenile organs of cereal seedlings. Elongation growth is considered to be a protective morphophysiological stage of seedling development during hypogeal germination. The molecular mechanisms of elongation growth include: changes in the properties of the cell wall, activation of proton pumps, as well as aquaporins of plasma membrane and tonoplast. Particular attention is paid to the hormonal system of regulation, including auxin and ethylene. Coleoptiles of rice, a semi-aquatic plant tolerant to oxygen deficiency, demonstrate that the mechanisms of elongation growth are changing intensively under submergence, but they completely ensure cell growth. There is also a redistribution of importance and abundance between phytohormones. The data presented in the review indicate the necessity to continue investigations on the mechanisms of elongation growth under normal and stress conditions.

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.

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.