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To “leaf” or not to “leaf”: Understanding the metabolic adjustments associated with leaf senescence
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).ORCID iD: 0000-0002-4842-7690
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The adequate execution of the final developmental stage of a leaf, leaf senescence, is crucial to the long-term survival of the plant. During senescence cellular structures like membranes, proteins, lipids and macromolecules are degraded and released nutrients are relocated to developing parts of the plant, such as young leaves, stems, flowers, siliques and ultimately seeds that are dependent on this nutrient remobilization. The first visible sign of senescence is the yellowing of leaves indicating the degradation of chlorophyll and the dismantling of chloroplasts. As a consequence, senescing leaves cannot perform photosynthesis anymore and the delivery of energy from the chloroplast is compromised. As chloroplasts lose their function, the course of the senescence program requires a stable alternative energy sources that support nutrient remobilization while simultaneously ensuring a basic metabolism.

To study leaf senescence I used the model plant Arabidopsis thaliana and applied different experimental approaches: Developmental Leaf Senescence (DLS), individual darkened leaves (IDL), completely darkened plants (DP) and a stay-green mutant which displays a delayed senescence phenotype during IDL. Using a combination of physiological, microscopic, transcriptomic and metabolomic analyses similarities and differences between these experimental setups were investigated with focus on the functions of mitochondria during leaf senescence.

The catabolism of amino acids and the subsequent release of glutamate into the mitochondrial matrix seem to play an important role for nitrogen remobilization during DLS and IDL. Glutamate is then transported to the cytoplasm and transformed into glutamine, which can serve as long distance nitrogen export metabolite in the plant. Furthermore, senescing leaves in IDL are not only source tissues for nutrient remobilization in the plant, but we also detected labelled carbon in the darkened leaves, indicating a communication between the IDL and leaves in light. In contrary to the senescence inducing systems of DLS and IDL, in DP and the stay-green mutant investigated here, senescence is not induced by dark treatment. In both experimental setups we measured an accumulation of amino acids in the darkened leaves, in particular those with high N content. This could make reduced carbon available as alternative energy source during darkness. In this thesis we observed that mitochondria play an important role in nutrient reallocation processes during leaf senescence. The overall energy status of senescing tissues depends on mitochondria and especially amino acid metabolism seems to have a vital role during the senescence processes both for energy supply and nutrient reallocation.

Abstract [sv]

Den process som sker när blad gulnar kallas senescens och är den sista fasen i dess utveckling som slutar med att bladet dör. Ett ändamålsenligt förlopp för denna process är avgörande för en växts långsiktiga överlevnad då viktiga näringsämnen, främst kväve, tas tillvara och återanvänds. Under senescesen degraderas cellulära strukturer och makromolekyler och de näringsämnen som frigörs omfördelas till lagring eller till utvecklande delar av växten. Speciellt för produktion av livskraftiga frön är denna remobilisering av resurser ytterst viktig. Att bladen gulnar under denna process beror på att kloroplasterna med deras gröna pigment, klorofyll, bryts ner. Som en konsekvens av detta tappar de gulnande bladen sin kapacitet till fotosyntes när kloroplasternas förmåga att omvandla ljusenergi till kemisk energi avtar. För att processen ska bli effektiv behövs en annan stabil energikälla både för att driva basal metabolism och omfördelning av näringsämnen. Det är här som min favoritorganell, mitokondrien, kommer in i bilden. Mitokondrierna står för cellandningen där reducerade föreningar kan brytas ner för att producera energi och ärden viktigaste komponenten i cellens energi metabolism vid sidan av kloroplasterna.

För att studera vad som händer när blad gulnar och hur mitokondrierna bidrar till denna process har jag använt modellväxten Arabidopsis thaliana, ett litet ogräs som på svenska heter backtrav. Senescensen kan induceras både av ålder och av yttre stimuli (t.ex. långvarigt mörker) och detta har jag utnyttjat i mina experiment. Speciellt snabbt går gulnandet om bara ett blad mörkläggs medan de andra får fortsätta att vara i ljus. Om däremot hela växten ställs i mörker behåller bladen sin gröna färg mycket längre. Vi har även isolerat en mutant där inte heller mörkläggning av individuella blad inducerar gulnande på samma sätt som i kontrollväxter. Genom att använda en kombination av fysiologiska metoder, mikroskopi, mätning av genuttryck och mätning av metabolitinnehåll analyserades likheter och skillnader mellan de olika experimentella angreppssätten med fokus på mitokondriella funktioner. Ända till den allra sista fasen av senescensen var mitokondrierna intakta och funktionella och energinåvån behölls hög i de gulnande bladen. Många mitokondriella reaktioner kopplade till nedbrytning av aminosyror visade sig resultera i produktion av aminosyran glutamat. Efter transport ut ur mitokondrien till cytoplasman kan denna ta upp ytterligare ett kväve och ge glutamin, en aminosyra med hög N/C kvot som anses viktig för transport av kväve till andra delar av växten. Resultat med inmärkt kol indikerade även att transport av kolhydrater eller andra föreningar kan ske från blad i ljus till de mörklagda bladen och att detta kan bidra till att effektivisera återvinningen av kväve. Till skillnad från åldrande blad och individuellt mörklagda blad gulnade inte blad från helt mörklagda växter eller den mutant som studerades. I båda dessa fall kunde vi i stället mäta en ackumulering av aminosyror i de mörklagda bladen, speciellt gällde detta för aminosyror med högt kväveinnehåll i förhållande till kol. På detta sätt skulle reducerat kol kunna frigöras som alternativ energikälla under mörkerbehandlingen tillsammans med andra nedbrytningsprodukter från den nedmontering av cellerna som sker. Sammanfattningsvis har jag alltså visat att mitokondrierna spelar en central roll för återvinningen och omfördelningen av näringsämnen kopplat till att bladen gulnar. Aktiva mitokondrier bidrar till denna process både genom att tillhandahålla den energi som krävs och de metaboliska reaktioner som behövs för att processen ska fungera optimalt. En detaljerad kunskap om den process som växten genomgår under senescensen kan på sikt få praktiska tillämpningar t.ex. för produktion av biomassa samt för ökad hållbarhet av t.ex. grönsaker vid lagring.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2018. , p. 114
Keywords [en]
Arabidopsis thaliana, metabolism, mitochondria, senescence
National Category
Botany
Research subject
biology
Identifiers
URN: urn:nbn:se:umu:diva-147700ISBN: 978-91-7601-900-9 (print)OAI: oai:DiVA.org:umu-147700DiVA, id: diva2:1205667
Public defence
2018-06-08, Lilla hörsalen, KB.E3.01, KBC-huset, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2018-05-17 Created: 2018-05-14 Last updated: 2018-06-09Bibliographically approved
List of papers
1. Dissecting the Metabolic Role of Mitochondria during Developmental Leaf Senescence
Open this publication in new window or tab >>Dissecting the Metabolic Role of Mitochondria during Developmental Leaf Senescence
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2016 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 172, no 4, p. 2132-2153Article in journal (Refereed) Published
Abstract [en]

The functions of mitochondria during leaf senescence, a type of programmed cell death aimed at the massive retrieval of nutrients from the senescing organ to the rest of the plant, remain elusive. Here, combining experimental and analytical approaches, we showed that mitochondrial integrity in Arabidopsis (Arabidopsis thaliana) is conserved until the latest stages of leaf senescence, while their number drops by 30%. Adenylate phosphorylation state assays and mitochondrial respiratory measurements indicated that the leaf energy status also is maintained during this time period. Furthermore, after establishing a curated list of genes coding for products targeted to mitochondria, we analyzed in isolation their transcript profiles, focusing on several key mitochondrial functions, such as the tricarboxylic acid cycle, mitochondrial electron transfer chain, iron-sulfur cluster biosynthesis, transporters, as well as catabolic pathways. In tandem with a metabolomic approach, our data indicated that mitochondrial metabolism was reorganized to support the selective catabolism of both amino acids and fatty acids. Such adjustments would ensure the replenishment of alpha-ketoglutarate and glutamate, which provide the carbon backbones for nitrogen remobilization. Glutamate, being the substrate of the strongly up-regulated cytosolic glutamine synthase, is likely to become a metabolically limiting factor in the latest stages of developmental leaf senescence. Finally, an evolutionary age analysis revealed that, while branched-chain amino acid and proline catabolism are very old mitochondrial functions particularly enriched at the latest stages of leaf senescence, auxin metabolism appears to be rather newly acquired. In summation, our work shows that, during developmental leaf senescence, mitochondria orchestrate catabolic processes by becoming increasingly central energy and metabolic hubs.

National Category
Botany
Identifiers
urn:nbn:se:umu:diva-131100 (URN)10.1104/pp.16.01463 (DOI)000391173400006 ()27744300 (PubMedID)
Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2018-06-09Bibliographically approved
2. Darkened leaves use different metabolic strategies for senescence and survival
Open this publication in new window or tab >>Darkened leaves use different metabolic strategies for senescence and survival
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2018 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 177, no 1, p. 132-150Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Arabidopsis thaliana, senescence, metabolism, dark induced senescence, survival
National Category
Botany
Research subject
biology
Identifiers
urn:nbn:se:umu:diva-147675 (URN)10.1104/pp.18.00062 (DOI)000431347500015 ()29523713 (PubMedID)
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2018-06-09Bibliographically approved
3. Metabolic adjustments required for extended leaf longevity under prolonged darkness revealed by a new loss of function allele of PIF5
Open this publication in new window or tab >>Metabolic adjustments required for extended leaf longevity under prolonged darkness revealed by a new loss of function allele of PIF5
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2018 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Senescence is regulated by a complex interplay of factors and regulatory circuits, which may be accelerated or delayed depending on the integrated signals. Using a forward genetic screen in Arabidopsis thaliana, we identified a mutant strongly delayed in its induction of senescence in response to prolonged darkness. This mutant, which corresponds to a novel loss-of-function allele of PIF5 (PHYTOCHROME-INTERACTING FACTOR 5), exhibits even slightly more extended survival of leaves in darkness than the previously reported pif5-3 TDNA knock-out line. In the present study, we additionally aimed at deciphering the metabolic and regulatory processes conferring this enhanced capacity for survival in pif5 mutants. We combined physiological, metabolomic and transcriptomic analyses, and discovered that the extended survival of mutant leaves in darkness was associated with reduced protein degradation, slight differences in amino acid catabolism related gene expression as well as strong reduction of amino acid transporter expression, which coincided with enhanced amino acid accumulation. Our findings suggest that enhanced survival in darkness could be mediated by moderate levels of protein degradation allowing build up and slow usage of amino acids as alternative respiratory substrates, while during irreversible senescence, strong degradative processes, together with enhanced amino acid transport either to the site of their metabolization inside the leaf, or to other organs in the plant, could promote the fast progression of senescence and antagonize survival. Comparative metabolomics and gene expression analyses suggested that the senescence regulatory network downstream of PIF5 organizes these irreversible stages of leaf senescence, promoting autophagy and amino acid export, possibly by direct binding of important senescence promoting factors like ORE1 to the promoters of some of the involved genes. The failure to induce these later stages may prolong the reversible phase of darkening, thus potentially leading to drastically increased viability of individually darkened leaves under darkness for over 2 weeks.

Publisher
p. 45
National Category
Developmental Biology
Identifiers
urn:nbn:se:umu:diva-147698 (URN)
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2018-06-09

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