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Complex I-complex II ratio strongly differs in various organs of

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Katrin Peters at Leibniz Universität Hannover
Katrin Peters
Markus Niessen at KWS Group
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Christoph Peterhänsel
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Abstract

In most studies, amounts of protein complexes of the oxidative phosphorylation (OXPHOS) system in different organs or tissues are quantified on the basis of isolated mitochondrial fractions. However, yield of mitochondrial isolations might differ with respect to tissue type due to varying efficiencies of cell disruption during organelle isolation procedures or due to tissue-specific properties of organelles. Here we report an immunological investigation on the ratio of the OXPHOS complexes in different tissues of Arabidopsis thaliana which is based on total protein fractions isolated from five Arabidopsis organs (leaves, stems, flowers, roots and seeds) and from callus. Antibodies were generated against one surface exposed subunit of each of the five OXPHOS complexes and used for systematic immunoblotting experiments. Amounts of all complexes are highest in flowers (likewise with respect to organ fresh weight or total protein content of the flower fraction). Relative amounts of protein complexes in all other fractions were determined with respect to their amounts in flowers. Our investigation reveals high relative amounts of complex I in green organs (leaves and stems) but much lower amounts in non-green organs (roots, callus tissue). In contrast, complex II only is represented by low relative amounts in green organs but by significantly higher amounts in non-green organs, especially in seeds. In fact, the complex I-complex II ratio differs by factor 37 between callus and leaf, indicating drastic differences in electron entry into the respiratory chain in these two fractions. Variation in amounts concerning complexes III, IV and V was less pronounced in different Arabidopsis tissues (quantification of complex V in leaves was not meaningful due to a cross-reaction of the antibody with the chloroplast form of this enzyme). Analyses were complemented by in gel activity measurements for the protein complexes of the OXPHOS system and comparative 2D blue native/SDS PAGE analyses using isolated mitochondria. We suggest that complex I has an especially important role in the context of photosynthesis which might be due to its indirect involvement in photorespiration and its numerous enzymatic side activities in plants.
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Complex I - complex II ratio strongly differs in various organs of Arabidopsis thaliana.
Plant Molecular Biology
Katrin Peters1, Markus Nießen2, Christoph Peterhänsel2, Bettina Späth3, Angela Hölzle3, Stefan
Binder3, Anita Marchfelder3, Hans-Peter Braun1*
1 Institute of Plant Genetics, Faculty of Natural Sciences, Leibniz Universität Hannover, 30419
Hannover, Germany
2 Institute of Botany, Faculty of Natural Sciences, Leibniz Universität Hannover, 30419 Hannover,
Germany
3 Molekulare Botanik, Universität Ulm, 89081 Ulm, Germany
* Corresponding author: E-mail address: braun@genetik.uni-hannover.de
Complex (subunit) Peptide 1 Peptide 2
Complex I (51-kDa subunit) EMKKSGLRGRGGAGF GAMKRGDWHRTKDLV
Complex II (SDH 1-1) CANRVAEISKPGEKQK LDDIEDTFPPKARVY
Complex III (alpha-MPP) TYGERKPVDQFLKSV VLAVPSYDTISSKFR
Complex IV (COX2) YGSRVSNQLIPQTGEA VEAVPRKDYGSRVS
Complex V (beta-subunit) GVGERTREGNDLYRE EVVAKAEKIAKESAA
Online Resource 1 Peptide sequences for the generation of IgGs Surface exposed
peptides were chosen. Synthesis of peptides was performed by Eurogentec (Seraing,
Belgium). Peptides were coupled to KLH and a mix of both peptides for each complex subunit
was used for the immunization of rabbits. The immunization protocol includes three boosts
with the peptide-mix.
Online Resource 2 Crystal structures of the five OXPHOS complexes and the selected subunits for
IgG production The upper part of the figure shows the crystal structures of the 5 OXPHOS complexes from
different species (data taken from the crystal structure database at
http://www.ncbi.nlm.nih.gov/sites/entrez?db=structure. Accessions: NuoF/Nqo1: 2FUG; sdhA: 1NEK;
peptidase M16 Seq B: 1PP9; COX2: 2OCC; beta subunit ATP-synthase: 1BMF). In the structures below the
selected subunits for IgG production are colored. These subunits were over- expressed in E. coli. Peptides
for the production of peptide specific antibodies were chosen with regard to their surface exposure to
increase prospects of antigen-IgG interactions under native conditions. The names of the corresponding
subunits in Arabidopsis thaliana are given in red at the bottom of the figure.
I+III2
I
III2
V
F1
IV
I
II
II III IV V
Online Resource 3 Specificity of the
generated OXPHOS IgGs under native
conditions Total membrane protein
(330 µg) of isolated mitochondria from
Arabidopsis thaliana Col-0 suspension
cell cultures was separated by one-
dimensional blue native PAGE. Western
blots were incubated with IgGs (dil.
1:1000) directed against one subunit of
each OXPHOS complex (for identities of
subunits see supp. Fig. 1). Detection of
immune signals was carried out by
immuno-histochemical staining using the
Vectastain ABC kit (Vector Laboratories,
Burlingame, CA, USA). Identities of the
OXPHOS complexes are given on the
left on the Coomassie stained reference
gel (for nomenclature see figure 5). The
target OXPHOS complexes of the five
immune reactions are given below the
blots, respectively. Furthermore, immune
signals are indicated by arrows.
mt cp
76
12
17
24
38
52
225
31
mt cp
Online Resource 4 The IgG directed against
the beta subunit of the mitochondrial ATP-
synthase (complex V) cross-reacts with
ATP-synthase from chloroplasts Total
protein of isolated mitochondria (‘mt’, 5 mg)
from Arabidopsis thaliana Col-0 suspension cell
cultures and isolated chloroplasts (‘cp’, 50 mg)
from Arabidopsis thaliana Col-0 plants were
separated by one-dimensional SDS-PAGE.
The reference gel on the left was stained with
Coomassie colloidal. The Western blot on the
right was incubated with the IgG directed
against the beta subunit of mitochondrial ATP-
synthase (dil. 1:1000). Detection of immune
signals was carried out by immuno-
histochemical staining using the Vectastain
ABC kit (Vector Laboratories, Burlingame, CA,
USA). The molecular masses (in kDa) of
standard proteins (High-range Molecula r
Weight Rainbow Marker, GE Healthcare,
Munich, Germany) are given on the left.
Online Resource 5 Western blot analysis for quantification of complex IV. The antibody directed against the COX2
subunit crossreacts with storage proteins present in the seed fraction. Nevertheless, separate quantification of the COX2
signal (red arrow) was possible as illustrated by two differnet immunoblots (a, b). Experimental details (see legend of
Figure 3): Total protein from six different types of tissues of Arabidopsis thaliana Col-0 (extracted from 0,6 mg FW,
respectively) was separated by 1D SDS-PAGE and subsequently transferred onto nitrocellulose membrane. Blots were
incubated with specific IgG directed against COX2 subunit of complex IV. The following proportions of the extracted
protein fractions as indicated in the figure were loaded onto the gel: leaf: 1/1, 1/2, 1/4, 1/8; flower: 1/2, 1/4, 1/8, 1/16,
1/32; root: 1/1, 1/2, 1/4, 1/8, 1/16; stem: 1/1, 1/2, 1/4, 1/8; callus: 1/2, 1/4, 1/8, 1/16, 1/32; seed: 1/2, 1/4, 1/8, 1/16, 1/32.
Leaf Flower Root
Callus
Stem
Seed
Leaf Flower Root
Callus
Stem
Seed
a b
0
25
50
75
100
Complex I Complex II Complex III Complex IV Complex V 0
25
50
75
100
flower leaf stem root callus seed
Complex I Complex II Complex III
Complex IV Complex V
flower
leaf
stem
seed
root
callus
flower
leaf
stem
seed
root
callus
flower
leaf
stem
seed
root
callus
flower
leaf
stem
seed
root
callus
flower
leaf
stem
seed
root
callus
ccomplex I complex II complex III complex IV complex V Complex I Complex II Complex III
Complex IV Complex V
d
Online Resource 6 Quantification of OXPHOS complexes in different tissues of Arabidopsis thaliana. This
figure represents an extension of Figure 4 of our publication. However, in contrast to the results shown in our
publication (part a and b of the Figure 4), quantification of the complexes here is related to total protein amount of the
investigated fractions (in part a and b of the Figure quantification is related to fresh weight of the tissues).
Experimental details (taken from the legend of Figure 4 of the publication): Data are based on immune signals
obtained by Western blotting (Fig. 3) with subsequent quantification of signals. Results refer to three replicates for
each tissue and each complex. Since all five OXPHOS complexes were most abundant in flowers, this tissue was set
as a standard (100%). c: relative amounts of OXPHOS complex per total protein amount of the fractions (y-axis),
analyzed tissue (x-axis). Identities of the complexes are given above the graph and by colors (complex I: dark-blue,
complex II: middle-blue, complex III: light-blue, complex IV: very-light-blue, complex V: turquoise). d: Same as c, but
data sorted according to tissues. The color code for the five complexes is the same as in part a of the figure.
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To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H2O2 and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.
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Two-dimensional electrophoretic comparison of mitochondrial polypeptides from different wheat (Triticum aestivum L.) tissues
Article
  • Dec 1991
  • PLANT SCI
The protein compositions of mitochondria isolated from etiolated leaves, green leaves (photosynthetic or non-photosynthetic), roots and calli from Triticum aestivum L. were compared by two-dimensional (2-D) gel electrophoresis. Qualitative and quantitative protein changes were observed between mitochondria from these tissues. When compared to etiolated leaves developed in the dark, mitochondria from leaves greened under natural light showed particularly important polypeptide changes i.e. the increase of four polypeptides belonging to the glycine decarboxylase complex (which is involved in the photorespiration phenomenon). Using non-photosynthetic green leaves developed under intermittent light (white light flashes separated by 15-min dark periods), it was shown that the quantitative increase of these four polypeptides was not correlated to efficient photosynthesis. Moreover, it was specified using a different condition of intermittent light (set of flashes separated by 24-h dark periods) leading to etiolated and of course non-photosynthetic leaves, that these polypeptide changes were not dependent on net chlorophyll synthesis. On the other hand, the analysis of mitochondrial protein composition from tissue culture has revealed some specific changes characteristic of callus. These changes were found whatever the tissue used to initiate the in vitro culture and were observed for some time in leaf mitochondria isolated from regenerated plants maintained in vitro.
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Analysis by two-dimensional gel electrophoresis of the polypeptide composition of pea mitochondria isolated from different tissues
Article
  • Jan 1987
  • ELECTROPHORESIS
The polypeptide composition of purified mitochondria isolated from pea epicotyls was studied by one- and two-dimensional electrophoresis. It was shown that two-dimensional electrophoresis was necessary to clearly resolve polypeptides of molecuar weights ranging from 50 000 to 60 000. Mitochondria were fractionated into a soluble fraction corresponding to the matrix and an insoluble fraction containing membrane components. Spots characteristic of one or the other fraction were thus localized on the two-dimensional gel map of whole mitochondria. In addition, a comparison was made of polypeptide composition in mitochondria isolated from etiolated and green leaves. It was shown that mitochondria from photosynthetic tissue differ from those on non-photosynthetic tissue by quantitative and qualitative changes of their polypeptide composition. The most characteristic changes in green leaves are the loss of one polypeptide (Mr 35 000 and pI 6.3) and the quantitative increase of four other spots. These spots belong to the matrix compartment. Their apparent molecular weights are 97 000, 51 000, 43 000 and 15 000. Conversion of glycine to serine during the photorespiration process being characteristic of mitochondria from photosynthesizing tissue, we suggest that these spots could correspond to subunits of the glycine decarboxylase multienzyme complex.
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Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis
Article
  • Jan 1985
  • ELECTROPHORESIS
A systematic analysis of protein staining in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R-250 and G-250 using a high resolution densitometer allowing for quantitative measurements during staining and destaining has revealed that none of the published procedures allows quantitative measurements. Protein staining with CBB R-250 in methanol/water/acetic acid is poor, as is staining with CBB G-250 in trichloroacetic acid or perchloric acid, the latter two, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G-250 than for R-250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albumin/mm2 gel. In addition, sensitive protein staining on a clear background is possible. Recipes are described (Section 3.11) for intensified protein staining with CBB G-250 using trichloroacetic acid or perchloric acid on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G-250 in the presence of ammonium sulfate since under these conditions the colloidal state of the dye is optimized. Furthermore, conditions are described which allow the stable fixation of the protein-dye complex. Combining the optimized staining conditions with the stable fixation in 20% ammonium sulfate allows for stepwise staining for e. g. detection of weak spots in addition to intense protein spots. The dependence of different staining procedures on gel thickness, gel concentration and compounds routinely used in polyacrylamide gel electrophoresis is also analysed. Calibration curves and application of the new procedure to biological material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining.
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Preparation of leaf mitochondria from Arabidopsis thaliana
Article
  • Jul 2005
  • PHYSIOL PLANTARUM
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.
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