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Significance of hydrolytic enzymes expressed during xylem cell death
Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Betydelsen av hydrolytiska enzymer uttryckta under xylemcelldöd (Swedish)
Abstract [en]

Xylem is an inherent feature of all vascular plants and functions in water transport and mechanical support. In order to efficiently transport water, xylem cells are reinforced by secondary walls before they undergo programmed cell death and their cell contents are removed by autolysis to create a hollow tube. During their differentiation, xylem cells express various hydrolytic enzymes, such as proteases, nucleases and lipases, but only in a few examples has their role in xylem cell death been characterized. This thesis focuses on the regulatory aspects of xylem cell death and the autolytic cell clearance in vessel elements and fibers of hybrid aspen (Populus tremula L. x tremuloides Michx.) and in vessel elements of Arabidopsis thaliana. Using comparative transcriptomic analysis, candidate genes for fiber-specific cell death processes were identified. Further, a hypothesis is presented on the regulation of thermospermine levels in the vasculature by a negative feedback-loop involving auxin and the class III Homeodomain-Leucine Zipper (HD-ZIP III) transcription factor HOMEOBOX8 (PtHB8). The role of the Arabidopsis METACASPASE9 (AtMC9) in xylem cell death was characterized using molecular tools, such as reporter lines and fluorescent fusion proteins, and electron microscopy (TEM). This showed that cell death initiation is not controlled by AtMC9. Instead, evidence is presented for the involvement of AtMC9 in the post mortem autolysis of vessel elements that follows tonoplast rupture and leads to the formation of the hollow conduit. Cell death-associated genes were further observed to be expressed during the emergence of lateral roots in Arabidopsis thaliana. This led to the discovery that cells overlying a lateral root primordium undergo cell death, which was demonstrated by detection of DNA degradation and TEM analysis. It is concluded that cell death facilitates emergence of lateral roots through the overlying tissues in a concerted manner with cell wall remodelling. Together, these findings show that although individual hydrolytic enzymes may be dispensable for plant growth and development, their common regulators are the tool for understanding their function and importance.

Abstract [sv]

Xylem är en karakteristisk vävnad i alla kärlväxter som leder vatten och mineraler samt har mekanisk stödfunktion. För att effektivt kunna transportera vatten förstärks xylemceller med sekundära cellväggar innan de dör genom programmerad celldöd. Deras cellinnehåll bryts ner genom autolys för att skapa ett ihåligt rör. Xylemceller uttrycker under sin differentiering olika hydrolytiska enzymer, såsom proteaser, lipaser och nukleaser, men bara för ett fåtal av dessa har funktionen under xylemcelldöd kartlagts. Denna avhandling fokuserar på reglering av xylemcelldöden och den autolytiska nedbrytningen av cellen, i såväl kärlelement och fibrer av hybridasp (Populus tremula L. x tremuloides Michx.) som i kärlelement av backtrav (Arabidopsis thaliana). Med hjälp av jämförande transkriptomanalys identifierades kandidatgener för fiber-specifika celldödsprocesser i hybridasp. Vidare utvecklades en hypotes om reglering av termosperminnivåer i vaskulaturen genom en negativ feedback-loop, som omfattar auxin reglering och klass III homeodomän-leucinzipper (HD-ZIP III) transkriptionsfaktorn HOMEOBOX8 (PtHB8). Funktionen av Arabidopsis METACASPASE9 (AtMC9) under xylemcelldöd karakteriserades med molekylära verktyg, såsom reporterlinjer och fluorescerande fusionsproteiner och elektronmikroskopi (TEM). Dessa analyser visade att celldödens initiering inte styrs av AtMC9. Istället presenteras bevis för en roll av AtMC9 i autolysen av kärlelement som sker post mortem efter att vakuolen har gått sönder och som slutför bildandet av det tomma kärlet. Genuttryck som associeras med celldöd observerades också under utvecklingen av laterala rötter i Arabidopsis thaliana. Detta ledde till upptäckten att celler som ligger ovanför ett lateralrotprimordium dör en programmerad celldöd och visar tecken på DNA-nedbrytning och autolys i TEM-analyser. Slutsatsen av denna studie är att celldöd i samspel med cellväggsmodifiering underlättar utväxten av laterala rötter genom de överliggande cellagren. Sammantaget tyder dessa upptäckter på att även om enstaka hydrolyserande enzymer inte är nödvändiga för växternas tillväxt och utveckling, så kan deras gemensamma reglering nyttjas för att förstå deras funktion och betydelse.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 49 p.
Keyword [en]
Arabidopsis, Populus, xylem cell death, metacaspase, fiber, lateral root emergence
National Category
Botany
Research subject
Molecular Biology; Physiological Botany; Molecular Cellbiology
Identifiers
URN: urn:nbn:se:umu:diva-81340ISBN: 978-91-7459-739-4 (print)OAI: oai:DiVA.org:umu-81340DiVA: diva2:653961
Public defence
2013-11-08, Hörsal F, Humanisthuset, Umeå universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2013-10-16 Created: 2013-10-07 Last updated: 2015-04-29Bibliographically approved
List of papers
1. A unique program for cell death in xylem fibers of Populus stem
Open this publication in new window or tab >>A unique program for cell death in xylem fibers of Populus stem
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2009 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 58, no 2, 260-274 p.Article in journal (Refereed) Published
Abstract [en]

Maturation of the xylem elements involves extensive deposition of secondary cell-wall material and autolytic processes resulting in cell death. We describe here a unique type of cell-death program in xylem fibers of hybrid aspen (Populus tremula x P. tremuloides) stems, including gradual degradative processes in both the nucleus and cytoplasm concurrently with the phase of active cell-wall deposition. Nuclear DNA integrity, as determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and Comet (single-cell gel electrophoresis) assays, was compromised early during fiber maturation. In addition, degradation of the cytoplasmic contents, as detected by electron microscopy of samples fixed by high-pressure freezing/freeze substitution (HPF-FS), was gradual and resulted in complete loss of the cytoplasmic contents well before the loss of vacuolar integrity, which is considered to be the moment of death. This type of cell death differs significantly from that seen in xylem vessels. The loss of vacuolar integrity, which is thought to initiate cell degradative processes in the xylem vessels, is one of the last processes to occur before the final autolysis of the remaining cell contents in xylem fibers. High-resolution microarray analysis in the vascular tissues of Populus stem, combined with in silico analysis of publicly available data repositories, suggests the involvement of several previously uncharacterized transcription factors, ethylene, sphingolipids and light signaling as well as autophagy in the control of fiber cell death.

Keyword
autophagy, DNA degradation, in silico transcriptional analysis, Populus, programmed cell death, xylem fiber development
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-22104 (URN)10.1111/j.1365-313X.2008.03777.x (DOI)000265040700007 ()19175765 (PubMedID)
Note

Även med namnet Moreau-Courtois, Charleen L. samt Moreau, Charleen.

Available from: 2009-04-23 Created: 2009-04-23 Last updated: 2015-04-29Bibliographically approved
2. Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem
Open this publication in new window or tab >>Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem
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2013 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 75, no 4, 685-698 p.Article in journal (Refereed) Published
Abstract [en]

Polyamines are small polycationic amines that are widespread in living organisms. Thermospermine, synthesized by thermospermine synthase ACAULIS5 (ACL5), was recently shown to be an endogenous plant polyamine. Thermospermine is critical for proper vascular development and xylem cell specification, but it is not known how thermospermine homeostasis is controlled in the xylem. We present data in the Populus model system supporting the existence of a negative feedback control of thermospermine levels in stem xylem tissues, the main site of thermospermine biosynthesis. While over-expression of the ACL5 homologue in Populus, POPACAULIS5, resulted in strong up-regulation of ACL5 expression and thermospermine accumulation in leaves, the corresponding levels in the secondary xylem tissues of the stem were similar or lower than those in the wild-type. POPACAULIS5 over-expression had a negative effect on accumulation of indole-3-acetic acid, while exogenous auxin had a positive effect on POPACAULIS5 expression, thus promoting thermospermine accumulation. Further, over-expression of POPACAULIS5 negatively affected expression of the classIII homeodomain leucine zipper (HD-ZipIII) transcription factor gene PttHB8, a homologue of AtHB8, while up-regulation of PttHB8 positively affected POPACAULIS5 expression. These results indicate that excessive accumulation of thermospermine is prevented by a negative feedback control of POPACAULIS5 transcript levels through suppression of indole-3-acetic acid levels, and that PttHB8 is involved in the control of POPACAULIS5 expression. We propose that this negative feedback loop functions to maintain steady-state levels of thermospermine, which is required for proper xylem development, and that it is dependent on the presence of high concentrations of endogenous indole-3-acetic acid, such as those present in the secondary xylem tissues.

Keyword
POPACAULIS5, ACAULIS5 (ACL5), class III homeodomain leucine zipper transcription factors (HD-ZipIII), wood development, polyamine, Populus tremulax, Populus tremuloides, Populus trichocarpa
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-80064 (URN)10.1111/tpj.12231 (DOI)000322898800013 ()
Available from: 2013-09-11 Created: 2013-09-09 Last updated: 2017-12-06Bibliographically approved
3. Post mortem function of AtMC9 in xylem vessel elements
Open this publication in new window or tab >>Post mortem function of AtMC9 in xylem vessel elements
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2013 (English)In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 200, no 2, 498-510 p.Article in journal (Refereed) Published
Abstract [en]

Cell death of xylem elements is manifested by rupture of the tonoplast and subsequent autolysis of the cellular contents. Metacaspases have been implicated in various forms of plant cell death but regulation and execution of xylem cell death by metacaspases remains unknown. Analysis of the type II metacaspase gene family in Arabidopsis thaliana supported the function of METACASPASE 9 (AtMC9) in xylem cell death. Progression of xylem cell death was analysed in protoxylem vessel elements of 3-d-old atmc9 mutant roots using reporter gene analysis and electron microscopy. Protoxylem cell death was normally initiated in atmc9 mutant lines, but detailed electron microscopic analyses revealed a role for AtMC9 in clearance of the cell contents post mortem, that is after tonoplast rupture. Subcellular localization of fluorescent AtMC9 reporter fusions supported a post mortem role for AtMC9. Further, probe-based activity profiling suggested a function of AtMC9 on activities of papain-like cysteine proteases. Our data demonstrate that the function of AtMC9 in xylem cell death is to degrade vessel cell contents after vacuolar rupture. We further provide evidence on a proteolytic cascade in post mortem autolysis of xylem vessel elements and suggest that AtMC9 is part of this cascade.

Place, publisher, year, edition, pages
John Wiley & Sons, 2013
Keyword
Arabidopsis thaliana, autolysis, metacaspase, protease, vessel element, xylem cell death
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-81218 (URN)10.1111/nph.12387 (DOI)
Available from: 2013-10-04 Created: 2013-10-04 Last updated: 2017-12-06Bibliographically approved
4. Programmed cell death in overlying tissues facilitates lateral root emergence
Open this publication in new window or tab >>Programmed cell death in overlying tissues facilitates lateral root emergence
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(English)Manuscript (preprint) (Other academic)
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-81219 (URN)
Available from: 2013-10-04 Created: 2013-10-04 Last updated: 2015-04-29Bibliographically approved

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Bollhöner, Benjamin

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