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From green to yellow: a leaf story
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).ORCID iD: 0000-0002-0546-7721
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

När ett blad gulnar genomgår det både morfologiska och metaboliska förändringar. Denna process benämns senescence och en förbättrad förståelse av dess mekanismer är viktiga både ur ett grundvetenskapligt perspektiv och för potentiella bioteknologiska applikationer. Denna avhandling rapporterar om flera viktiga aspekter relaterade till de cellulära och metaboliska mekanismer som sker under senescencen med tonvikt på mitokondriernas bidrag till denna process.

I ett första steg utvecklade vi metoder för att isolera antingen mycket funktionella mitokondrier eller mycket rena mitokondrier från blad av Arabidopsis thaliana. Dessa metoder användes sedan till för att studera mitokondriella bidrag till cellens redox balans och att uppskatta mitokondriernas kapacitet under senescence-processen. Framför allt jämfördes induktionen av senescencen berodende på olika mörkerbehandlingar av Arabidopsis. Jämförelse mellan individuellt mörklagda blad med hela mörklagda växter visade en betydande skillnad i metabolisk strategi mellan de två mörkerbehandlingarna. Genom att integrera data från mätningar av fotosyntes, respiration och konfokal mikroskopi med transcriptomics- och metabolomics-profiler föreslår vi att metabolismen hos blad från helt mörklagda växter antar ett ”stand-by läge” för att kunna bibehålla fotosynteskapaciteten så länge som möjligt. I kontrast till detta visar mitokondrier från individuellt mörklagda blad en hög aktivitet och kan därmed producera energi och kolskelett för degraderingen av cellkomponenter, vilket möjliggör återvinning av näringsämnen. Vi har även studerat dynamiken av det mikrotubulibaserade cytoskelettet under mörkerindicerad senescence. Mitokondriernas rörlighet påverkades av en tidig nedbrytning av mikrotubuli hos individuellt mörklagda blad men inte hos blad där hela växten mörkerbehandlats. Dessutom verkade ett flertal mikrotubuliassocierade proteiner (MAP’s) att vara involverade i buntningen av mikrotubuli runt kloroplasterna.

Sammanfattningsvis belyser det arbete som presenteras i denna avhandling ett flertal viktiga steg med avseende på metabolisk anpassning och andra cellulär mekanismer i Arabidopsisblad som utsätts för långvarig mörkerbehendling. Specifikt föreslår vi att mitokondrierna bidrar med speciella och viktiga funktioner under bladens senescence eftersom mitokondriernas roll under långvarig mörkerbehandling av blad verkar bero på den totala statusen av metabolismen hos växten.

Abstract [en]

When switching from green to yellow, a leaf undergoes both morphological and metabolic changes. This process is known as senescence and improved understanding of its mechanisms is important both from a fundamental scientific perspective but also for biotechnological applications. The present thesis reports on several important aspects regarding the cellular and metabolic mechanisms occurring during leaf senescence with an emphasis on the mitochondrial contribution to this process.

As a first step, we developed methods to isolate either highly functional crude mitochondria or highly purified mitochondria from leaves of Arabidopsis thaliana. These methods were further used to study mitochondrial contributions to cellular redox homeostasis and to estimate the mitochondrial capacities in leaves undergoing senescence. In particular, we compared the induction of senescence by different dark treatments in Arabidopsis. The comparison between individually darkened leaves and leaves from whole darkened plants revealed different metabolic strategies in response to darkness. Integrating data from measurements of photosynthesis, respiration and confocal laser microscopy with transcriptomics and metabolomics profiling, we suggested that metabolism in leaves of the whole darkened plants enter a “stand-by mode” with low mitochondrial activity in order to maintain the photosynthetic machinery for as long as possible. In contrast, mitochondria from individually darkened leaves are more active and may provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. We also investigated the dynamic of the microtubular cytoskeleton during dark-induced senescence. Mitochondrial mobility was affected by an early disruption of the microtubules in individually darkened leaves but not in whole darkened plants. In addition, several microtubules associated proteins (MAPs) seemed to be involved in the bundling of the microtubules around the chloroplasts.

Altogether, the work presented in this thesis highlights several important steps regarding the metabolic adjustments and the cellular mechanisms in Arabidopsis leaves submitted to prolonged darkness. In particular, we suggest the mitochondria to fulfill specific and important functions during leaf senescence since the role of mitochondria in leaves experiencing prolonged darkness appears very dependant on the whole metabolic status of the plant.

Place, publisher, year, edition, pages
Umeå: Umeå Plant Science Centre , 2007. , 54 p.
Keyword [en]
Arabidopsis thaliana, chloroplasts, cytoskeleton, darkness, metabolism, microscopy, mitochondria, microtubules, senescence, system redox
National Category
Botany
Identifiers
URN: urn:nbn:se:umu:diva-1362ISBN: 978-91-7264-400-7 (print)OAI: oai:DiVA.org:umu-1362DiVA: diva2:140766
Public defence
2007-10-12, KB3A9, KBC, KBC-huset, 10:00 (English)
Opponent
Supervisors
Available from: 2007-09-20 Created: 2007-09-20 Last updated: 2015-04-29Bibliographically approved
List of papers
1. Preparation of leaf mitochondria from Arabidopsis thaliana
Open this publication in new window or tab >>Preparation of leaf mitochondria from Arabidopsis thaliana
2005 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 124, no 4, 403-409 p.Article in journal (Refereed) Published
Abstract [en]

Arabidopsis thaliana is, perhaps, the most important model species in modern plant biology. However, the isolation of organelles from leaves of this plant has been difficult. Here, we present two different protocols for the isolation of mitochondria, yielding either highly functional crude mitochondria or highly purified mitochondria. The crude mitochondria were well coupled with the substrates tested (malate + glutamate, glycine and NADH), exhibiting respiratory control ratios of 2.1–3.9. Purified mitochondria with very low levels of chlorophyll contamination were obtained by Percoll gradient centrifugation, yielding 1.2 mg of mitochondrial protein from 50 g of leaves.

Identifiers
urn:nbn:se:umu:diva-15943 (URN)doi:10.1111/j.1399-3054.2005.00521.x (DOI)
Available from: 2007-08-06 Created: 2007-08-06 Last updated: 2017-12-14Bibliographically approved
2. Identification of plant glutaredoxin targets
Open this publication in new window or tab >>Identification of plant glutaredoxin targets
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2005 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 7, no 7-8, 919-929 p.Article in journal (Refereed) Published
Abstract [en]

Glutaredoxins (Grxs) are small ubiquitous proteins of the thioredoxin (Trx) family, which catalyze dithiol–disulfide exchange reactions or reduce protein-mixed glutathione disulfides. In plants, several Trx-interacting proteins have been isolated from different compartments, whereas very few Grx-interacting proteins are known. We describe here the determination of Grx target proteins using a mutated poplar Grx, various tissular and subcellular plant extracts, and liquid chromatography coupled to tandem mass spectrometry detection. We have identified 94 putative targets, involved in many processes, including oxidative stress response [peroxiredoxins (Prxs), ascorbate peroxidase, catalase], nitrogen, sulfur, and carbon metabolisms (methionine synthase, alanine aminotransferase, phosphoglycerate kinase), translation (elongation factors E and Tu), or protein folding (heat shock protein 70). Some of these proteins were previously found to interact with Trx or to be glutathiolated in other organisms, but others could be more specific partners of Grx. To substantiate further these data, Grx was shown to support catalysis of the stroma β-type carbonic anhydrase and Prx IIF of Arabidopsis thaliana, but not of poplar 2-Cys Prx. Overall, these data suggest that the interaction could occur randomly either with exposed cysteinyl disulfide bonds formed within or between target proteins or with mixed disulfides between a protein thiol and glutathione.

Place, publisher, year, edition, pages
Larchmont, NY: Mary Ann Liebert, 2005
Keyword
Adenosine Triphosphate/metabolism, Arabidopsis/enzymology/*metabolism, Carbon Dioxide/metabolism, Cell Respiration/radiation effects, Citric Acid/metabolism, Electron Transport, Molecular Chaperones/metabolism, Oxidative Stress, Oxidoreductases/*metabolism, Oxygen/metabolism, Peroxidases/metabolism, Photochemistry, Polysaccharides/biosynthesis/metabolism, Protein Biosynthesis, Sulfur/metabolism
Identifiers
urn:nbn:se:umu:diva-2568 (URN)doi:10.1089/ars.2005.7.919 (DOI)
Available from: 2007-09-20 Created: 2007-09-20 Last updated: 2017-12-14Bibliographically approved
3. The mitochondrial type II peroxiredoxin from poplar
Open this publication in new window or tab >>The mitochondrial type II peroxiredoxin from poplar
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2007 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 129, no 1, 196-206 p.Article in journal (Refereed) Published
Abstract [en]

Mitochondria are a major site of reactive oxygen species production and controlling the peroxide levels in this compartment is essential. Peroxiredoxins (Prx) are heme-free peroxidases, which use reactive cysteines for their catalysis and reducing systems for their regeneration. One of the two Prxs present in poplar mitochondria, Prx IIF, expressed as a recombinant protein, was found to reduce a broad range of peroxides with electrons provided preferentially by glutaredoxin and to a lesser extent by glutathione, all the thioredoxins tested being inefficient. This protein is constitutively expressed because it is found in all tissues analyzed. Its expression is modified during a biotic interaction between poplar and the rust fungus Melampsora laricii populina. On the other hand, Prx IIF expression does not substantially vary under abiotic stress conditions. Nevertheless, water deficit or chilling and probably induced senescence, but not photooxidative conditions or heavy metal treatment, also led to a small increase in PrxIIF abundance in Arabidopsis thaliana plants.

Place, publisher, year, edition, pages
Copenhagen: Munksgaard, 2007
Identifiers
urn:nbn:se:umu:diva-2569 (URN)doi:10.1111/j.1399-3054.2006.00785.x (DOI)
Available from: 2007-09-20 Created: 2007-09-20 Last updated: 2017-12-14Bibliographically approved
4. The different fate of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves
Open this publication in new window or tab >>The different fate of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves
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2007 (English)In: Plant, Cell and Environment, ISSN 0140-7791, E-ISSN 1365-3040, Vol. 30, no 12, 1523-1534 p.Article in journal (Refereed) Published
Abstract [en]

Senescence is an active process allowing the reallocation of valuable nutrients from the senescing organ towards storage and/or growing tissues. Using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs), we investigated the fate of mitochondria and chloroplasts during dark-induced leaf senescence. Combining in vivo visualization of fates of the two organelles by three-dimensional reconstructions of abaxial parts of leaves with functional measurements of photosynthesis and respiration, we showed that the two experimental systems displayed major differences during 6 d of dark treatment. In whole DPs, organelles were largely retained in both epidermal and mesophyll cells. However, while the photosynthetic capacity was maintained, the capacity of mitochondrial respiration decreased. In contrast, IDLs showed a rapid decline in photosynthetic capacity while maintaining a high capacity for mitochondrial respiration throughout the treatment. In addition, we noticed an unequal degradation of organelles in the different cell types of the senescing leaf. From these data, we suggest that metabolism in leaves of the whole DPs enters a ‘stand-by mode’ to preserve the photosynthetic machinery for as long as possible. However, in IDLs, mitochondria actively provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. Finally, the heterogeneity of the degradation processes involved during senescence is discussed with regard to the fate of mitochondria and chloroplasts in the different cell types.

Place, publisher, year, edition, pages
Oxford: Blackwell Scientific Publications Ltd, 2007
Keyword
microscopy, photosynthesis, respiration
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-2570 (URN)10.1111/j.1365-3040.2007.01724.x (DOI)
Available from: 2007-09-20 Created: 2007-09-20 Last updated: 2017-12-14Bibliographically approved
5. Leaf metabolism during dark induced senescence in Arabidopsis: integrating metabolomics and transcriptomics
Open this publication in new window or tab >>Leaf metabolism during dark induced senescence in Arabidopsis: integrating metabolomics and transcriptomics
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2571 (URN)
Available from: 2007-09-20 Created: 2007-09-20 Last updated: 2010-01-13Bibliographically approved
6. Disruption of the microtubules during dark-induced senescence
Open this publication in new window or tab >>Disruption of the microtubules during dark-induced senescence
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(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-2572 (URN)
Available from: 2007-09-20 Created: 2007-09-20 Last updated: 2015-04-29Bibliographically approved

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