Transcript Tonoplast and Vacuoles

Tonoplast and Vacuoles
presented by Tatiana Eremeeva
Content
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Vacuoles. Functions and types.
Vacuole biogenesis.
Routes towards the vacuole.
Tonoplast.
Transport processes across the tonoplast.
Autophagy and monitoring methods.
Vacuoles
- (coined from vacuum») – fluid-filled compartments
encompassed by a membrane called tonoplast.
10 - Vacuole
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
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.
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.
Biochemistry&Molecular Biology of Plants/edited by Buchmann B., Gruissem W.,
Russel L.J.
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.
Vacuole biogenesis
Corrado Vioti, ER and vacuoles: never been closer (pages 1-5), February 2014
Model for lytic vacuole biogenesis in Arabidopsis.
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.
Biochemistry&Molecular Biology of Plants/edited by Buchmann B., Gruissem W., Russel L.J.
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.
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 posttranslational 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.
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).
Assessment of Autophagy Monitoring
Methods
Model for monitoring autophagy in planta Arabidopsis thaliana roots.
Conditions - carbon- and nitrogen-starvation.
Goal – evaluate monitoring methods:
1. 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.
2. Monodansylcadaverine (MDC) - acidotropic
fluorescent dye
3. LysoTracker Red (LTR) - acidotropic fluorescent dye
Kinetics of autophagic activity can be monitored
in planta using GFP–ATG8 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 GFP–ATG8 fusion protein in
Arabidopsis roots. Behavior of autophagosomal structures and
pre-autophagosomal structures (PAS) revealed by GFP–ATG8
during 24 h of carbon and nitrogen starvation in Arabidopsis.
Kinetics of autophagic activity can be monitored
in planta using GFP–ATG8 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
Result of Assessment of Autophagy
Monitoring Methods
• The GFP–ATG8 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.
Thank you for attention!