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1.
Plant J ; 116(6): 1696-1716, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37713307

RESUMO

We investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH-isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress-related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non-stressed plants contained twofold elevated fructose-2,6-bisphosphate (F2,6P2 ) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose-to-sucrose ratios, but also more lipids. Above-ground biomass lay in between susceptible Xanthi and resistant SNN, with photo-assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid-to-carbohydrate ratios, resulting in increased harvest yields - also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non-stressed plants. In leaves of salt-watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non-stressed plants showed enhanced PSII-induction kinetics (upon dark-light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids - especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi-cP2 lines. Apparently, metabolic priming by F2,6P2 -combined with sustained NADP(H) turnover-bypasses the genetically fixed growth-defence trade-off, rendering tobacco plants more stress-resilient and productive.


Assuntos
Isoenzimas , Nicotiana , Isoenzimas/metabolismo , Nicotiana/genética , NADP/metabolismo , Sementes/genética , Sementes/metabolismo , Sacarose/metabolismo , Ácidos Graxos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Folhas de Planta/metabolismo
2.
Plant Physiol ; 156(3): 1464-80, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21571669

RESUMO

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B'γ (B'γ) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b'γ plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b'γ cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b'γ mutant shows constitutive activation of both salicylic acid- and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b'γ leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b'γ leaves. We suggest that the specific B'γ subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Luz , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Southern Blotting , Metilação de DNA/genética , Metilação de DNA/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células do Mesofilo/citologia , Células do Mesofilo/efeitos da radiação , Células do Mesofilo/ultraestrutura , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Proteína Fosfatase 2/genética , Subunidades Proteicas/genética , Proteômica , Espécies Reativas de Oxigênio/metabolismo
3.
Plant Methods ; 6: 14, 2010 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-20534155

RESUMO

BACKGROUND: Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection. RESULTS: Infrared laser desorption ionization orthogonal time-of-flight mass spectrometry allows rapid and simultaneous detection in both negative and positive ion mode of a wide range of naturally occurring primary and secondary metabolites. An unsupervised principal component analysis was employed to identify correlations between changes in metabolite expression (obtained at different times and sample treatment conditions) and the overall defence response.A one-dimensional projection of the principal components 1 and 2 obtained from positive ion mode spectra was used to generate a Biological Response Index (BRI). The BRI obtained for each sample treatment was compared with the number of dead cells found in the respective tissue. The high correlation between these two values suggested that the BRI provides a rapid assessment of the plant response against the pathogen infection. Evaluation of the loading plots of the principal components (1 and 2) reveals a correlation among three metabolic cascades and the defence response generated in infected leaves. Analysis of selected phytohormones by liquid chromatography electrospray ionization mass spectrometry verified our findings. CONCLUSION: The described methodology allows for rapid assessment of infection-specific changes in the plant metabolism, in particular of phenolics, alkaloids, oxylipins, and carbohydrates. Moreover, potential novel biomarkers can be detected and used to predict the quality of plant infections.

4.
Proc Natl Acad Sci U S A ; 106(19): 8061-6, 2009 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-19416911

RESUMO

In source leaves of resistant tobacco, oxidative burst and subsequent formation of hypersensitive lesions after infection with Phytophthora nicotianae was prevented by inhibition of glucose-6-phosphate dehydrogenase (G6PDH) or NADPH oxidases. This observation indicated that plant defense could benefit from improved NADPH availability due to increased G6PDH activity in the cytosol. A plastidic isoform of the G6PDH-encoding gene, G6PD, displaying high NADPH tolerance was engineered for cytosolic expression (cP2), and introduced into a susceptible cultivar. After infection, transgenic (previously susceptible) lines overexpressing cP2 showed early oxidative bursts, callose deposition, and changes in metabolic parameters. These responses resulted in timely formation of hypersensitive lesions similar to resistant plants, although their extent varied considerably between different transgenic lines. Additional RNAi suppression of endogenous cytosolic G6PD isoforms resulted in highly uniform defense responses and also enhanced drought tolerance and flowering. Cytosolic G6PDH seems to be a crucial factor for the outcome of plant defense responses; thus, representing an important target for modulation of stress resistance. Because isoenzyme replacement of G6PDH in the cytosol was beneficial under various kinds of cues, we propose this strategy as a tool to enhance stress tolerance in general.


Assuntos
Citosol/metabolismo , Glucosefosfato Desidrogenase/química , Isoenzimas/química , Desidratação , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Cinética , Modelos Biológicos , NADPH Oxidases/metabolismo , Estresse Oxidativo , Folhas de Planta/metabolismo , Fenômenos Fisiológicos Vegetais , Interferência de RNA , Explosão Respiratória , Nicotiana/fisiologia
5.
Plant Physiol ; 147(3): 1288-99, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18502974

RESUMO

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild-type tobacco (Nicotiana tabacum) Samsun NN (SNN) with plants with RNA interference (RNAi)-mediated repression of cwINV (SNNcwINV). In source leaves of SNNcwINV, the activity of cwINV was repressed by about 90%. Sucrose export and apoplastic carbohydrate levels were significantly reduced, while photosynthesis and dark respiration exhibited little or no change. Activities of sucrose synthase and phosphofructokinase were depressed moderately, while ADP-glucose pyrophosphorylase was diminished greatly. Yet, the content of cytosolic/vacuolar carbohydrates was not significantly lower, which correlated with the absence of phenotypic effects in SNNcwINV under normal growing conditions. By contrast, defense-related processes in primary metabolism and hypersensitive cell death were impaired and delayed in correlation with repression of cwINV. The increase in cwINV observed in source leaves of the resistant wild type following infection with Phytophthora nicotianae was absent in SNNcwINV. Also, defense-related callose deposition at cell-to-cell interfaces, the related decline in sugar export, and accumulation of apoplastic carbohydrates were reduced and delayed. Expression of pathogenesis-related proteins and increase in phenylalanine ammonia-lyase and glucose-6-phosphate dehydrogenase activities were alleviated. Formation of hydrogen peroxide and development of hypersensitive lesions were weak and heterogeneous, and the pathogen was able to sporulate. We conclude that in photosynthetically active leaves of the apoplastic phloem loader, tobacco cwINV plays an essential role for acquisition of carbohydrates during plant-pathogen interactions and that the availability of these carbohydrates supports the onset of the hypersensitive reaction and ensures successful defense.


Assuntos
Parede Celular/metabolismo , Interações Hospedeiro-Parasita , Nicotiana/fisiologia , Phytophthora/fisiologia , beta-Frutofuranosidase/metabolismo , Metabolismo dos Carboidratos , Morte Celular , Glucanos/metabolismo , Hexoses/metabolismo , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Interferência de RNA , Sacarose/metabolismo , Nicotiana/enzimologia , Nicotiana/microbiologia
6.
Plant Signal Behav ; 3(10): 885-7, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19704530

RESUMO

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.

7.
Plant J ; 45(6): 968-81, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16507087

RESUMO

Peroxiredoxin Q (Prx Q) is one out of 10 peroxiredoxins encoded in the genome of Arabidopsis thaliana, and one out of four that are targeted to plastids. Peroxiredoxin Q functions as a monomeric protein and represents about 0.3% of chloroplast proteins. It attaches to the thylakoid membrane and is detected in preparations enriched in photosystem II complexes. Peroxiredoxin Q decomposes peroxides using thioredoxin as an electron donor with a substrate preference of H(2)O(2) > cumene hydroperoxide >> butyl hydroperoxide >> linoleoyl hydroperoxide and insignificant affinity towards complex phospholipid hydroperoxide. Plants with decreased levels of Prx Q did not have an apparently different phenotype from wildtype at the plant level. However, similar to antisense 2-cysteine (2-Cys) Prx plants [Baier, M. et al. (2000)Plant Physiol., 124, 823-832], Prx Q-deficient plants had a decreased sensitivity to oxidants in a leaf slice test as indicated by chlorophyll a fluorescence measurements. Increased fluorescence ratios of photosystem II to I at 77 K and modified transcript levels of plastid- and nuclear-encoded proteins show that regulatory mechanisms are at work to compensate for the lack of Prx Q. Apparently Prx Q attaches to photosystem II and has a specific function distinct from 2-Cys peroxiredoxin in protecting photosynthesis. Its absence causes metabolic changes that are sensed and trigger appropriate compensatory responses.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Peroxidases/fisiologia , Fotossíntese/fisiologia , Tilacoides/enzimologia , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA Bacteriano/genética , Fluorescência , Membranas Intracelulares/enzimologia , Mutagênese Insercional , Oxirredução , Peroxidases/análise , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas , Fenótipo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plastídeos/metabolismo , RNA Mensageiro/metabolismo
8.
Plant Physiol ; 136(4): 4265-74, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15563617

RESUMO

We investigated adaptive responses of the photosynthetic electron transport to a decline in the carbon assimilation capacity. Leaves of different ages from wild-type tobacco (Nicotiana tabacum) L. var Samsun NN and young mature leaves of tobacco transformants with impaired photoassimilate export were used. The assimilation rate decreased from 280 in young mature wild-type leaves to below 50 mmol electrons mol chlorophyll(-1) s(-1) in older wild-type leaves or in transformants. The electron transport capacity, measured in thylakoids isolated from the different leaves, closely matched the leaf assimilation rate. The numbers of cytochrome (cyt)-bf complexes and plastocyanin (PC) decreased with the electron transport and assimilation capacity, while the numbers of photosystem I (PSI), photosystem II, and plastoquinone remained constant. The PC to PSI ratio decreased from five in leaves with high assimilation rates, to values below one in leaves with low assimilation rates, and the PC versus flux correlation was strictly proportional. Redox kinetics of cyt-f, PC, and P700 suggest that in leaves with low electron fluxes, PC is out of the equilibrium with P700 and cyt-f and the cyt-f reoxidation rate is restricted. It is concluded that the electron flux is sensitive to variations in the number of PC, relative to PSI and cyt-bf, and PC, in concert with cyt-bf, is a key component that adjusts to control the electron transport rate. PC dependent flux control may serve to adjust the electron transport rate under conditions where the carbon assimilation is diminished and thereby protects PSI against over-reduction and reactive oxygen production.


Assuntos
Carbono/metabolismo , Transporte de Elétrons/fisiologia , Nicotiana/metabolismo , Fotossíntese/fisiologia , Plastocianina/fisiologia , Clorofila/metabolismo , Clorofila A , Luz , Modelos Logísticos , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Folhas de Planta/metabolismo , Tilacoides/metabolismo , Fatores de Tempo
9.
Biochim Biophys Acta ; 1659(1): 63-72, 2004 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-15511528

RESUMO

Reduction kinetics of cytochrome f, plastocyanin (PC) and P(700) ('high-potential chain') in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810-860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P(700). In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P(700). In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the 'high-potential chain' does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the 'high potential chain'. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.


Assuntos
Clorofila/metabolismo , Citocromos f/metabolismo , Modelos Biológicos , Folhas de Planta/fisiologia , Plastocianina/metabolismo , Spinacia oleracea/metabolismo , Tilacoides/fisiologia , Cloroplastos/fisiologia , Cloroplastos/efeitos da radiação , Simulação por Computador , Homeostase/fisiologia , Luz , Oxirredução , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/efeitos da radiação , Folhas de Planta/efeitos da radiação , Spinacia oleracea/efeitos da radiação , Tilacoides/efeitos da radiação
10.
Funct Plant Biol ; 29(6): 697-705, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32689516

RESUMO

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. We investigated photosynthetic electron transport in leaves of the facultative crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum L. After CAM induction, electron transport exhibited variable redox kinetics during the diurnal CAM cycle. In CAM Phase IV, most of PSI (P700) and chlorophyll a fluorescence relaxed with a halftime of 20 ms after a saturating light pulse. This time-constant may reflect the overall linear electron flux from PSII to PSI in saturating light. Comparable relaxation kinetics were also determined for C3 plants. At the end of CAM Phase I and during Phase II, slow components (> 50% of signal amplitude) appeared in both P700 reduction and fluorescence relaxation. They displayed halftimes > 250 ms and > 1 s, suggesting a strong restriction of the linear electron flux from H2O to NADP. The appearance of the slow redox components was accompanied by a decrease in the Fv/Fm ratio of chlorophyll a fluorescence, suggesting a reversible detachment of light-harvesting complex (LHC) II from PSII. The slow redox fractions and the depression of Fv/Fm disappeared again in parallel to malate decarboxylation during CAM Phase III. We discuss a reversible downregulation of linear electron flux during CAM Phases II and III, due to a reversible deprivation of cytochrome-b6f complexes (cyt-bfs) and PSI from the linear system. In parallel, a redistribution of some LHCIIs could also occur. This could be an adaptive response to a reduced metabolic demand for NADPH due to a limited carbon flux through the Calvin cycle, resulting from low Rubisco activation. Furthermore, the cyt-bfs and PSIs deprived of linear electron transport could support cyclic electron flux to cover an increased ATP demand during gluconeogenesis in CAM Phase III.

11.
New Phytol ; 151(3): 585-595, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33853257

RESUMO

• The sink-source transition of developing Nicotiana tabacum (tobacco) leaves was studied here using chlorophyll fluorescence imaging. • In accordance with leaf development, the quantum efficiency of PSII, showed a steep gradient across the leaf with increasing values towards the tip. • The linear electron transport rate (ETR) saturated at higher CO2 concentrations in the younger, than in the mature, part of the leaf, probably due to a lower Rubisco activity or a higher CO2 diffusion resistance. • The induction of ETR at CO2 concentrations near the compensation point after long-term dark adaptation of the young leaf, showed distinct responses; ETR rose rapidly in the basal but more slowly in the apical regions. There was a correlation between fast induction and carbohydrate import, as measured by 14 C-translocation. In the basal regions, larger pools of metabolic intermediates are expected due to imported carbohydrates. These might be used in the Calvin cycle directly after dark-light transition providing the electron acceptors for the faster induction of ETR. Additionally, a higher mitochondrial respiration can provide CO2 for the Calvin cycle in these regions.

12.
Oecologia ; 69(1): 134-139, 1986 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28311696

RESUMO

The effect of high temperatures on the photosynthetic apparatus of Preissia quadrata (Scop.) Nees, Conocephalum conicum (L.) Dum. and Marchantia polymorpha L. were investigated. changes in the activities of various photosynthetic reactions were followed by measuring light-dependent oxygen evolution, chlorophyll a fluorescence and light-induced absorbance changes at 518 nm.Mild heat treatment of the thalli led to reversible depression of photosynthesis; the period necessary for complete recovery depended on the extent of heat damage. Irreversible inactivation of photosynthesis after more severe heat stress was caused by damage of photosystem II. On principle, the pattern of reversible and irreversible heat inactivation of photosynthetic reactions in liverwort thalli resembles that observed in leaves of higher plants. However, in contrast to a number of Spermatophyta, exposure of liverwort thalli to high sublethal temperatures did not result in a significant increase in the heat stability of the photosynthetic apparatus indicating that the heat hardening capacity of hygrophytic hepatics is extremely low.

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