The RPN12a proteasome subunit is essential for the multiple hormonal homeostasis controlling the progression of leaf senescence

The 26S proteasome is a conserved multi-subunit machinery in eukaryotes. It selectively degrades ubiquitinated proteins, which in turn provides an efficient molecular mechanism to regulate numerous cellular functions and developmental processes. Here, we studied a new loss-of-function allele of RPN12a, a plant ortholog of the yeast and human structural component of the 19S proteasome RPN12. Combining a set of biochemical and molecular approaches, we confirmed that a rpn12a knock-out had exacerbated 20S and impaired 26S activities. The altered proteasomal activity led to a pleiotropic phenotype affecting both the vegetative growth and reproductive phase of the plant, including a striking repression of leaf senescence associate cell-death. Further investigation demonstrated that RPN12a is involved in the regulation of several conjugates associated with the auxin, cytokinin, ethylene and jasmonic acid homeostasis. Such enhanced aptitude of plant cells for survival in rpn12a contrasts with reports on animals, where 26S proteasome mutants generally show an accelerated cell death phenotype.A loss-of-function mutation in the RPN12a proteasome subunit in Arabidopsis thaliana shows that this protein is essential for hormonal homeostasis, thereby modulating plant's development and the progression of leaf senescence

Chitosan-Modified Polyethyleneimine Nanoparticles for Enhancing the Carboxylation Reaction and Plants' CO2 Uptake

Increasing plants' photosynthetic efficienc y is a major challenge that must be addressed in order to cover the food demands of the growing population in the changing climate. Photosynthes i s is greatly limited at the initial carboxylation reaction, where CO2 is converted to the organic acid 3-PGA, catalyzed by the RuBisCO enzyme. RuBisCO has poor affinity for CO2, but also the CO2 concentration at the RuBisCO site is limited by the diffusion of atmospheric CO2 through the various leaf compartments to the reaction site. Beyond genetic engineer-ing, nanotechnology can offer a materials-based approach for enhancing photosynthesis, and yet, it has mostly been explored for the light-dependent reactions. In this work, we developed polyethyleneimine-based nanoparticl e s for enhancing the carbox-ylation reaction. We demonstrate that the nanoparticles can capture CO2 in the form of bicarbonate and increase the CO2 that reacts with the RuBisCO enzyme, enhancing the 3-PGA production in in vitro assays by 20%. The nanoparticles can be introduced to the plant via leaf infiltration and, because of the functionalization with chitosan oligomers, they do not induce any toxic effect to the plant. In the leaves, the nanoparticles localize in the apoplastic space but also spontaneously reach the chloroplasts where photosynthetic activity takes place. Their CO2 loading-dependent fluorescence verifies that, in vivo, they maintain their abi l i t y to capture CO2 and can be therefore reloaded with atmospheric CO2 while in planta. Our results contribute to the development of a nanomaterials-based CO2-concentrating mechanism in plants t h a t can potentially increase photosynthetic efficiency and overall plants' CO2 storage

Metabolic control of arginine and ornithine levels paces the progression of leaf senescence

Pools of arginine and ornithine generated during protein degradation can pace the progression of leaf senescence by affecting the TCA cycle, polyamine biosynthesis and the ethylene signaling pathway.Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival

Edat cronològica, edat fisiològica i sexe: factors determinants de l'estrès oxidatiu en plantes

[cat] Els processos metabòlics en plantes, com ara la fotosíntesi, la fotorespiració i la respiració, comporten l’inevitable producció d’espècies reactives de l’oxigen (ROS) en cloroplasts, peroxisomes i mitocondris. En determinades concentracions les ROS poden actuar com a molècules implicades en la senyalització cel•lular, però degut a la seva elevada reactivitat, un augment de les ROS provoca l’oxidació de components cel•lulars alterant-ne la seva funció biològica i provocant dany oxidatiu a la planta. En condicions d’estrès, tant biòtic com abiòtic, les plantes experimenten un increment dels nivells de ROS. Per tal de mantenir l’homeòstasi redox, aquestes han desenvolupat un seguit de mecanismes antioxidants capaços de reduir els nivells de ROS evitant un possible dany oxidatiu. La recerca per desxifrar les respostes de les plantes a l’estrès ha anat en augment en els últims anys, però encara avui en dia és poc el coneixement que tenim sobre els mecanismes implicats en el cas de les plantes perennes, tot i que constitueixen una part molt important del regne vegetal. Per altra banda, tot i l’evident importància de l’estrès oxidatiu, són escassos els estudis que s’han plantejat com aquest es pot veure afectat per factors intrínsecs de la planta, com l’edat o el sexe. L’objectiu principal d’aquesta tesi ha estat determinar com l’edat de la planta, tant la cronològica com la fisiològica, i el sexe poden influir en l’estrès oxidatiu en plantes perennes. L’anàlisi del nivells d’estrès oxidatiu es va dur a terme mitjançant les mesures de diferents mecanismes antioxidants com els carotenoides, l’alfa-tocoferol i els antocians, però centrant-nos sobretot en el paper de l’àcid malondialdehid, un subproducte de la peroxidació lipídica. Els estudis es van realitzar en fulles i plantes juvenils de Pistacia lentiscus L., una espècie dioica i perenne típica del clima mediterrani, i en arbres moribunds de Fagus sylvatica L., proporcionant un bon model a causa de la seva avançada edat, tant cronològica com fisiològica. Els resultats obtinguts revelen que l’augment dels nivells de peroxidació lipídica com a indicador d’estrès oxidatiu pot significar un dany o un possible mecanisme de senyalització interna; per això, la consideració conjunta de l’edat cronològica i els nivells d’estrès oxidatiu és un bon indicador de l’edat fisiològica, tant a nivell de fulla com de planta sencera. L’esforç reproductiu en plantes dioiques ocasiona canvis en els mecanismes fotoprotectors en les femelles respecte als mascles en condicions ambientals adverses. Tot i que les femelles presenten uns nivells d’estrès oxidatiu superiors als dels mascles, no es veuen afectades negativament, el que suggereix un possible rol en senyalització. Així mateix, l’estudi a nivell modular mitjançant la comparació entre brots reproductius i no reproductius en femelles va revelar una major fotoprotecció en els brots reproductius, com indicaven els nivells d’antioxidants i la major dissipació d’excés d’energia en forma de calor, emfatitzant la importància de la diferenciació entre mòduls en l’estudi de les diferències entre sexes en plantes dioiques.[eng] Metabolic processes in plants such as photosynthesis, photorespiration and respiration, produce reactive oxygen species (ROS). ROS are highly toxic molecules but besides of its damaging nature they are implicated in cell signaling in different cellular processes. However, under stress conditions plants can suffer an increase of ROS levels. When ROS concentration becomes high enough to overwhelm antioxidant systems, plant suffer oxidative stress as a consequence of the unbalanced cellular redox status. Despite the importance to unravel plant stress responses, little is known about the mechanisms implicated in perennial plants. Furthermore, the possible effect of plant intrinsic factors, as plant age or reproductive effort, in oxidative stress levels is still poorly understood. The main objective of this thesis was to determine how plant age, both chronological and physiological, as well as reproductive effort may influence oxidative stress levels in perennial plants. With this purpose levels of antioxidants as carotenoids, anthocyanins and alpha-tocopherol together with endogenous contents of stress hormones were measured, but with special emphasis in malondialdehyde acid levels, a byproduct of lipid peroxidation. To better understand the plant age effect we used leaves and juvenile plants of Pistacia lentiscus as well as moribund beech trees. Increases in lipid peroxidation not only could mean an oxidative damage but play a signaling role. Therefore, chronolorgical age concomitantly with the measure of oxidative stress levels is a good indicator of plant physiological age. Sex-related changes in photoprotection mechanisms between female and male plants of Pistacia lentiscus, a dioecious plant, where observed under climatological adverse conditions. Although females phowed higher oxidative stress levels compared to males, females were not affected negatively, suggesting a role in signaling. In addition, photoprotection capacity was higher in reproductive shoots relative to non-reproductive shoots in females, thus suggesting that females prioritized protection to fruit-bearing shoots

Edat cronològica, edat fisiològica i sexe : factors determinants de l'estrèes oxidatiu en plantes. /

Thesis submitted in fulfillment of the requirements for the degree of doctor (PhD) in Biological Science

The RPN12a proteasome subunit is essential for the multiple hormonal homeostasis controlling the progression of leaf senescence

The 26S proteasome is a conserved multi-subunit machinery in eukaryotes. It selectively degrades ubiquitinated proteins, which in turn provides an efficient molecular mechanism to regulate numerous cellular functions and developmental processes. Here, we studied a new loss-of-function allele of RPN12a, a plant ortholog of the yeast and human structural component of the 19S proteasome RPN12. Combining a set of biochemical and molecular approaches, we confirmed that a rpn12a knock-out had exacerbated 20S and impaired 26S activities. The altered proteasomal activity led to a pleiotropic phenotype affecting both the vegetative growth and reproductive phase of the plant, including a striking repression of leaf senescence associate cell-death. Further investigation demonstrated that RPN12a is involved in the regulation of several conjugates associated with the auxin, cytokinin, ethylene and jasmonic acid homeostasis. Such enhanced aptitude of plant cells for survival in rpn12a contrasts with reports on animals, where 26S proteasome mutants generally show an accelerated cell death phenotype

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

Darkened Leaves Use Different Metabolic Strategies for Senescence and Survival

In plants, an individually darkened leaf initiates senescence much more rapidly than a leaf from a whole darkened plant. Combining transcriptomic and metabolomic approaches in Arabidopsis (Arabidopsis thaliana), we present an overview of the metabolic strategies that are employed in response to different darkening treatments. Under darkened plant conditions, the perception of carbon starvation drove a profound metabolic readjustment in which branched-chain amino acids and potentially monosaccharides released from cell wall loosening became important substrates for maintaining minimal ATP production. Concomitantly, the increased accumulation of amino acids with a high nitrogen-carbon ratio may provide a safety mechanism for the storage of metabolically derived cytotoxic ammonium and a pool of nitrogen for use upon returning to typical growth conditions. Conversely, in individually darkened leaf, the metabolic profiling that followed our 13C-enrichment assays revealed a temporal and differential exchange of metabolites, including sugars and amino acids, between the darkened leaf and the rest of the plant. This active transport could be the basis for a progressive metabolic shift in the substrates fueling mitochondrial activities, which are central to the catabolic reactions facilitating the retrieval of nutrients from the senescing leaf. We propose a model illustrating the specific metabolic strategies employed by leaves in response to these two darkening treatments, which support either rapid senescence or a strong capacity for survival