Ethylene signaling may be involved in the regulation of tocopherol biosynthesis in Arabidopsis thaliana

AbstractTocopherol biosynthesis was investigated in ein3-1, etr1-1 and eto1-1 mutants of Arabidopsis thaliana, which show a defect in ethylene signaling, perception and over-produce ethylene, respectively. A mutation in the EIN3 gene delayed the water-stress related increase in α-tocopherol and caused a reduction in the levels of this antioxidant by ca. 30% compared to the wild type. In contrast to the wild type and ein3-1 mutants, both etr1-1 and eto1-1 mutants showed a sharp (up to 5-fold) increase in α-tocopherol levels during leaf aging. It is concluded that ethylene perception and signaling may be involved in the regulation of tocopherol biosynthesis during water stress and leaf aging

Regulación de la biosíntesis de la vitamina E y su función en señalización celular en condiciones de estrés abiótico

[spa] La vitamina E incluye una serie de compuestos amfipáticos esenciales en la dieta tanto para humanos como para animales y que únicamente puede ser sintetizada por organismos fotosintéticos, los tocoferoles y los tocotrienoles. Descubierta a principios del siglo XX, no fue hasta los años 60 que se asoció a propiedades antioxidantes. En plantas, se sabe que los niveles de tocoferol (la forma mayoritaria en hojas) aumenta en respuesta al estrés y que está implicado en el control de los niveles de las especies reactivas del oxígeno, la síntesis de las cuáles suele aumentar drásticamente en condiciones de estrés y pueden ir asociadas a graves daños celulares si no se regulan adecuadamente sus niveles. También interviene en la prevención de la propagación de la peroxidación lipídica en condiciones de estrés oxidativo, además de otras nuevas funciones que poco a poco se van descubriendo, como la importancia durante la germinación de semillas, la implicación en el transporte de fotoasimilados o un posible papel en señalización celular. En esta tesis se estudió la regulación de la biosíntesis de α- y γ-tocoferol en condiciones de estrés abiótico (déficit hídrico y estrés salino), así como su posible implicación en señalización celular en la planta modelo Arabidopsis thaliana y en la planta con metabolismo CAM, Aptenia cordifolia. Pero la vitamina E no actúa sola. Como antioxidante, puede trabajar coordinadamente con el ascorbato o con carotenoides además de con diversas fitohormonas, por lo que además, se estudió el papel como mecanismo fotoprotector, juntamente con los carotenoides y los niveles hormonales, tanto en condiciones de estrés como influenciados por la edad de la hoja. Los estudios en A. thaliana se llevaron a cabo utilizando diversos mutantes ya fueran de etileno o de tocoferol como aproximaciones a sistemas deficientes de ambos compuestos. Se observó que el etileno promueve la síntesis de tocoferol en A. thaliana, de forma especifica a través de las proteínas EIN2, EIN3 y EIL1. Además, niveles elevados de etileno promueven aumentos de tocoferol en hojas viejas y mutaciones en el gen que codifica para la proteína EIN3 es capaz de resistir dosis moderadas de salinidad, cambiando el balance hormonal foliar y aumentando el estrés oxidativo. Sorprendentemente, la biosíntesis de tocoferol no resultó estar regulada a nivel transcripcional. En el modelo de planta CAM, la A. cordifolia, el ABA jugó un papel importante en la regulación de la biosíntesis de tocoferol en condiciones de déficit hídrico. Juntamente con el β-caroteno y el ciclo de las xantofilas, los tocoferoles ayudan a la protección del PSII en condiciones de déficit hídrico y estrés salino cuando la demanda de fotoprotección es elevada, pero cuando los carotenoides se empiezan a degradar, el tocoferol es incapaz de substituirlos. Además, el γ-tocoferol parece tener funciones específicas además de precursor del α-tocoferol influenciando de forma específica los niveles de expresión de genes implicados en la biosíntesis, percepción y señalización del etileno en A. thaliana. Finalmente, cabe destacar que el tocoferol interviene en la comunicación cloroplasto-núcleo a través de regular los niveles de expresión de genes relacionados con el etileno, formando parte de la intrincada red de señalización retrógrada.[eng] Vitamin E includes different amphipathic compounds, tocopherols and tocotrienols, essentials in the diet of humans and animals. It can be synthesized only by photosynthetic organisms and it was discovered in the early twentieth century but it was not until the 60‘s when it was associated with its antioxidant properties. In plants, it is known that the levels of α-tocopherol (the major form in leaves) increase in response to stress and they are involved in the control of the levels of reactive oxygen species (ROS). Under stress conditions the levels of ROS tend to increase dramatically and they could be associated with serious cell damage if their levels are not properly regulated. Moreover tocopherols are involved in prevention of the lipidic peroxidation propagation under oxidative stress conditions. Furthermore, new functions are gradually being discovered, like their importance in the process of seed germination, their role on the transport of the photoassimilates or their possible function in cell signalling. During this PhD I have studied the regulation of the biosynthesis of α- and γ-tocopherol under abiotic stress conditions (water and salt stress) and their possible role in cell signalling in the model plant Arabidopsis thaliana and in the CAM plant, Aptenia cordifolia. As an antioxidant, it might work in coordination with ascorbate or carotenoids as well as with some phytohormones. That is why we have studied their role as photoprotective mechanism together with carotenoids and the hormone levels in both stress conditions and the influence of leave age. The studies in A. thaliana were carried out using tocopherol or ethylene mutants as approximations to deficient systems of both compounds. It was observed that ethylene promotes synthesis of tocopherol in A. thaliana, specifically through EIN2, EIN3 and EIL1 proteins. In addition, elevated levels of ethylene induced tocopherol increase in old leaves. ein3-1 mutant plants were able to withstand moderate doses of salinity, changing leaf hormonal balance and increasing oxidative stress. Surprisingly, the biosynthesis of tocopherol was not being regulated at the transcriptional level. In the model CAM plant, A. cordifolia, the ABA played an important role in the regulation of tocopherol biosynthesis under water stress conditions. Together with β-carotene and the xanthophyll cycle, tocopherols help in the protection of PSII under both water and salt stress conditions when the demand for photoprotection is high. When carotenoids were beginning to degrade, tocopherols were unable to replace them. In addition, the γ-tocopherol seemed to have specific functions not only as α-tocopherol precursor. It can specifically influence the expression levels of genes involved in ethylene biosynthesis, perception and signalling in A. thaliana. Finally, in this PhD thesis it is demonstrated that tocopherol is involved in the chloroplast-nucleus communication by regulating the expression levels of genes related to ethylene as part of the intricate network of retrograde signalling

An altered tocopherol composition in chloroplasts reduces plant resistance to <i>Botrytis cinerea</i>

Tocopherols are lipid-soluble antioxidants that contribute to plant resistance to abiotic stresses. However, it is still unknown to what extent alterations in tocopherol composition can affect the plant response to biotic stresses. The response to bacterial and fungal attack of the vte1 mutant of Arabidopsis thaliana, which lacks both α- and γ-tocopherol, was compared to that of the vte4 mutant (which lacks α- but accumulates γ-tocopherol) and the wild type (with accumulates α-tocopherol in leaves). Both mutants exhibited similar kinetics of cell death and resistance in response to Pseudomonas syringae. In contrast, both mutants exhibited delayed resistance when infected with Botrytis cinerea. Lipid and hormonal profiling was employed with the aim of assessing the underlying cause of this differential phenotype. Although an altered tocopherol composition in both mutants strongly influenced fatty acid composition, and strongly altered jasmonic acid and cytokinin contents upon infection with B. cinerea, differences between genotypes in these phytohormones were observed during late stages of infection only. By contrast, genotype-related effects on lipid peroxidation, as indicated by malondialdehyde accumulation, were observed early upon infection with B. cinerea. We conclude that an altered tocopherol composition in chloroplasts may negatively influence the plant response to biotic stress in Arabidopsis thaliana through changes in the membrane fatty acid composition, enhanced lipid peroxidation and delayed defence activation when challenged with B. cinerea

Application of a rapid and sensitive method for hormonal and Vitamin E profiling reveals crucial regulatory mechanisms in flower senescence and fruit ripening

Knowledge of ripeness and regulation of postharvest processes is an important tool to prevent loss of commercial value in both fruit and cut flower markets. The joint analysis of hormones and vitamin E levels can reveal complex interactions between hormones and oxidative stress as key regulators of postharvest processes. Profiling of both groups of metabolic compounds was performed during the ripening of non-climacteric fruits (red raspberry, Rubus idaeus L.) and senescence of ethylene-insensitive flowers (Dutch Iris, Iris x hollandica L.). After an initial extraction of the sample, without further purification steps, the hormonal profile was analyzed by UPLC-MS/MS and vitamin E levels were measured by HPLC. This methodological approach was very fast and had enough sensitivity for the analysis of small samples. Raspberry fruit maturation was characterized by a decline of cytokinin levels [zeatin, zeatin riboside, 2-isopentenyl adenine, and isopentenyl adenosine (Z, ZR, 2-iP, and IPA, respectively)] and gibberellins (GA1 in particular). Exogenous application of ABA prevented δ-tocopherol loss during fruit ripening. Iris floral senescence was also under strict hormonal control, also mediated by cytokinins and gibberellins. Z, ZR, 2-iP, GA9, and GA24 levels decreased in inner tepals, whereas the level of IPA decreased in style-merged-to-stigma tissues, thus suggesting tissue-specific roles for different hormones. α-Tocopherol levels decreased during senescence of inner tepals, hence suggesting enhanced oxidative stress. In conclusion, the rapid and sensitive hormonal and vitamin E profiling presented here can help in understanding the key physiological processes underlying fruit ripening and floral senescence

Glutathione and transpiration as key factors conditioning oxidative stress in Arabidopsis thaliana exposed to uranium

Although oxidative stress has been previously described in plants exposed to uranium (U), some uncertainty remains about the role of glutathione and tocopherol availability in the different responsiveness of plants to photo-oxidative damage. Moreover, in most cases, little consideration is given to the role of water transport in shoot heavy metal accumulation. Here, we investigated the effect of uranyl nitrate exposure (50 μM) on PSII and parameters involved in water transport (leaf transpiration and aquaporin gene expression) of Arabidopsis wild type (WT) and mutant plants that are deficient in tocopherol (vte1: null α/γ-tocopherol and vte4: null α-tocopherol) and glutathione biosynthesis (high content: cad1.3 and low content: cad2.1). We show how U exposure induced photosynthetic inhibition that entailed an electron sink/source imbalance that caused PSII photoinhibition in the mutants. The WT was the only line where U did not damage PSII. The increase in energy thermal dissipation observed in all the plants exposed to U did not avoid photo-oxidative damage of mutants. The maintenance of control of glutathione and malondialdehyde contents probed to be target points for the overcoming of photoinhibition in the WT. The relationship between leaf U content and leaf transpiration confirmed the relevance of water transport in heavy metals partitioning and accumulation in leaves, with the consequent implication of susceptibility to oxidative stress. © 2014 Springer-Verlag Berlin Heidelberg.RA and RP were supported by Ministry of Economy and Competitiveness of Spain (AGL2011-25403 project). The authors would like to thank Serge Berthet for his help with the ICP-MS measurements.Peer Reviewe