RESUMEN
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.
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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.
Asunto(s)
Ecosistema , Oxígeno , Humanos , Aminoácidos , Hipoxia , Lactatos , Piruvatos , Succinatos , Ácido gamma-Aminobutírico , Fragmentos de Péptidos , TripsinaRESUMEN
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.
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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.
Asunto(s)
Acuaporinas , Acuaporinas/genética , Acuaporinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Hipoxia , Desarrollo de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Agua/metabolismoRESUMEN
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.
RESUMEN
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.
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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.
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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.
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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.
Asunto(s)
Ácidos Indolacéticos/análisis , Ácidos Indolacéticos/farmacología , Oryza/química , Oxígeno/farmacología , Plantones/química , Triticum/química , Electrólitos/análisis , Electrólitos/metabolismo , Peróxido de Hidrógeno/metabolismo , Ácidos Indolacéticos/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Oryza/efectos de los fármacos , Oryza/metabolismo , Estrés Oxidativo , Reguladores del Crecimiento de las Plantas/análisis , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Brotes de la Planta/química , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plantones/efectos de los fármacos , Plantones/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Triticum/efectos de los fármacos , Triticum/metabolismoRESUMEN
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.
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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.
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Auxin is one of the crucial regulators of plant growth and development. The discovered auxin cytosolic receptor (TIR1) is not involved in the perception of the hormone signal at the plasma membrane. Instead, another receptor, related to the ABP1, auxin binding protein1, is supposed to be responsible for the perception at the plasma membrane. One of the fast and sensitive auxin-induced reactions is an increase of Ca(2+) cytosolic concentration, which is suggested to be dependent on the activation of Ca(2+) influx through the plasma membrane. This investigation was carried out with a plasmalemma enriched vesicle fraction, obtained from etiolated maize coleoptiles. The magnitude of Ca(2+) efflux through the membrane vesicles was estimated according to the shift of potential dependent fluorescent dye diS-C3-(5). The obtained results showed that during coleoptiles ageing (3rd, 4th and 5th days of seedling etiolated growth) the magnitude of Ca(2+) efflux from inside-out vesicles was decreased. Addition of ABP1 led to a recovery of Ca(2+) efflux to the level of the youngest and most sensitive cells. Moreover, the efflux was more sensitive, responding from 10(-8) to 10(-6) M 1-NAA, in vesicles containing ABP1, whereas native vesicles showed the highest efflux at 10(-6) M 1-NAA. We suggest that auxin increases plasma membrane permeability to Ca(2+) and that ABP1 is involved in modulation of this reaction.
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The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1 mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Oryza/metabolismo , Protoplastos/metabolismo , Triticum/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Hipoxia de la Célula , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quelantes/farmacología , Citosol/efectos de los fármacos , Ácido Egtácico/farmacología , Colorantes Fluorescentes , Fura-2/análogos & derivados , Fura-2/metabolismo , Lantano/farmacología , Células del Mesófilo/efectos de los fármacos , Células del Mesófilo/metabolismo , Oryza/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Protoplastos/efectos de los fármacos , Plantones/efectos de los fármacos , Plantones/metabolismo , Transducción de Señal , Triticum/efectos de los fármacos , Verapamilo/farmacologíaRESUMEN
The auxin-induced changes in cytosolic concentrations of Ca(2+) and H(+) ions were investigated in protoplasts from maize coleoptile cells at 3rd, 4th and 5th day of development of etiolated seedlings. The shifts in [Ca(2+)](cyt) and [H(+)](cyt) were detected by use of fluorescence microscopy in single protoplasts loaded with the tetra[acetoxymethyl]esters of the fluorescent calcium binding Fura 2, or pH-sensitive carboxyfluorescein, BCECF, respectively. Both the auxin-induced shifts in the ion concentrations were specific to the physiologically active synthetic auxin, naphthalene-1-acetic acid (1-NAA), and not to the non-active naphthalene-2-acetic acid (2-NAA). Regardless of the age of the seedlings, the rise in [Ca(2+)](cyt) was prior to the acidification in all investigated cases. The maximal acidification coincided with the highest amplitude of [Ca(2+)](cyt) change, but not directly depended on the concentration of 1-NAA. Within aging of the seedlings the amplitude of auxin-induced [Ca(2+)](cyt) elevation decreased. The shift in auxin-induced acidification was almost equal at 3rd and 4th day, but largely dropped at 5th day of development. The acidification was related to changes in the plasma membrane H(+)-ATPase activity, detected as phosphate release. The decrement in amplitude of both the tested auxin-triggered reactions well coincided with the end of the physiological function of the coleoptile. Hence the primary auxin-induced increase in [Ca(2+)](cyt), which is supposed to be an important element of hormone signal perception and transduction, can be used as a test for elucidation of plant cell sensitivity to auxin.