Tonoplast%20and%20Vacuoles

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Tonoplast and Vacuoles

• presented by Tatiana Eremeeva

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Content

• Vacuoles. Functions and types.
• Vacuole biogenesis.
• Routes towards the vacuole.
• Tonoplast.
• Transport processes across the tonoplast.
• Autophagy and monitoring methods.

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Vacuoles
- (coined from vacuum) fluid-filled
compartments encompassed by a membrane called
tonoplast.
10 - Vacuole
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Plant vacuoles are multifunctional compartments

• Storage (proteins, amino acids and organic
  acids, ions, sugars, pigments)
• Digestion (acid hydrolases proteases,
  nucleases, glycosidases, lipases)
• pH and ionic homeostasis (serve as reservoirs of
  protons and metabolically important ions)
• Defense against microbial pathogens and
  herbivores (cell wall-degrading enzymes, phenolic
  compounds, alkaloids, etc.)
• Sequestration of toxic compounds
• Pigmentation

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Plants use vacuoles to produce large cells
cheaply

• By filling a large volume of the cell with
  inexpensive vacuolar contents plants are able
  to reduce the cost of making expanded structures
  such as leaves, which are essentially solar
  collectors.
• The water taken into vacuoles generates turgor
  pressure which expands the primary cell wall and
  creates stiff structures in conjunction with the
  walls.

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Types of vacuoles
Two types of vacuoles are depicted large
protein storage vacuoles (V1) and smaller
lytic/autophagic-type vacuoles (V2) that may be
involved in autophagy-associated programmed cell
death.
BiochemistryMolecular Biology of Plants/edited
by Buchmann B., Gruissem W., Russel L.J.
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Routes towards the vacuole

• In all eukaryotes, the best described mechanism
  of exiting the ER is via coat protein complex
  (COP)II-coated vesicles. The vacuole, together
  with the plasma membrane, is the most distal
  point of the secretory pathway, and many vacuolar
  proteins are transported from the ER through
  intermediate compartments.
• However, past results demonstrate the presence of
  alternative transport routes from the ER towards
  the tonoplast, which are independent of Golgi-
  and post-Golgi trafficking. Moreover, the
  transport mechanism of the vacuolar proton pumps
  challenges the current model of vacuole
  biogenesis, pointing to the ER for being the main
  membrane source for the biogenesis of the plant
  lytic compartment.

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Vacuole biogenesis
Corrado Vioti, ER and vacuoles never been closer
(pages 1-5), February 2014
Model for lytic vacuole biogenesis in
Arabidopsis.
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Routes towards the vacuole
Comparison of different pathways for the delivery
of storage proteins to vacuoles. A
Goldgi-mediated pathway for the delivery of
storage proteins to protein storage vacuoles
(PSVs) B - Goldgi-mediated pathway in which CCVs
bud off the TGN and transfer proteins to the
prevacuolar compartment (PVC) before transport to
the lytic vacuole C ER-derived protein bodies
filled with prolamins are autophaged by vacuoles.
BiochemistryMolecular Biology of Plants/edited
by Buchmann B., Gruissem W., Russel L.J.
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Tonoplast

• - the membrane delimiting plant vacuoles,
  regulates ion, water and nutrient movement
  between the cytosol and the vacuolar lumen
  through the activity of its membrane proteins.
• The relative abundance of these proteins and
  their respective activities/regulation determine
  the specific function of plant vacuoles.

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Transport processes across the tonoplast

• The regulation of solute passage across the
  tonoplast can be achieved by modified expression
  of genes encoding tonoplast proteins. However,
  also post-translational modifications of
  tonoplast proteins represent a fundamental
  principle in vacular transport regulation and
  adaptation.
• Direct post-translational modifications of the
  protein allow faster adaptation of protein.
  Reversible protein phosphorylation by specific
  protein kinases/phosphatases is a very common
  post-translational modification.

H. Ekkehard, O. Trentmann, Regulation of
transport processes across the tonoplast,
September 2014
Schematic drawing illustrating the current
knowledge on how tonoplast monosaccharide
transporters (TMTs) are regulated at the
post-translational level.
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Autophagy

• is a degradation pathway that recycles cell
  materials upon stress conditions or during
  specific developmental processes (in the lysosome
  for mammals or in the vacuole for yeast and
  plants).

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Assessment of Autophagy MonitoringMethods

• Model for monitoring autophagy in planta -
  Arabidopsis thaliana roots.
• Conditions - carbon- and nitrogen-starvation.
• Goal evaluate monitoring methods
• Green fluorescent protein (GFP)ATG8 fusion
  protein.
• ATG8 (autophagy-related) protein which is
• anchored to the autophagosomal membrane, a good
  marker for the observation of autophagosome
• movements.
• Monodansylcadaverine (MDC) - acidotropic
  fluorescent dye
• 3. LysoTracker Red (LTR) - acidotropic
  fluorescent dye

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Kinetics of autophagic activity can be
monitoredin planta using GFPATG8 transgenic
Arabidopsis
A.Merkulova, A. Guiboileau, Assessment and
Optimization of Autophagy Monitoring Methods in
Arabidopsis Roots Indicate Direct Fusion of
Autophagosomes with Vacuoles (pages 715-725),
February 2014
Monitoring of the induction of autophagy by
GFPATG8 fusion protein in Arabidopsis roots.
Behavior of autophagosomal structures and
pre-autophagosomal structures (PAS) revealed by
GFPATG8 during 24 h of carbon and nitrogen
starvation in Arabidopsis.
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Kinetics of autophagic activity can be
monitoredin planta using GFPATG8 transgenic
Arabidopsis

• Autophagy is inhibited by wortmannin treatment in
  Arabidopsis roots. White arrowheads in (B)
  indicate small dot structures, which are putative
  pre-autophagosomal structures.
• The high level of fluorescence observed at the
  interstices of root cells in (B) indicates the
  area of cytoplasm, which is enlarged, probably
  because the size of the central vacuoles is
  reduced after the action of wortmannin.

A.Merkulova, A. Guiboileau, Assessment and
Optimization of Autophagy Monitoring Methods in
Arabidopsis Roots Indicate Direct Fusion of
Autophagosomes with Vacuoles (pages 715-725),
February 2014
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Result of Assessment of Autophagy Monitoring
Methods

• The GFPATG8 transgenic line constitutes an
  excellent method for monitoring autophagy.
• These data were compared with plants stained with
  MDC and LTR. There was no appreciable MDC/LTR
  staining of small organelles in the root under
  the induction of autophagy. Extreme caution
  should therefore be used when monitoring
  autophagy with the aid of MDC/LTR.

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Thank you for attention!