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Phosphate deprivation induces transfer of DGDG galactolipid from chloroplast to mitochondria.

Jouhet J, Maréchal E, Baldan B, Bligny R, Joyard J, Block MA - J. Cell Biol. (2004)

Bottom Line: Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope.This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria.Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Physiologie Cellulaire Végétale, UMR 5168 (CNRS/CEA/Université Jseph Fourier/INRA), DRDC-PCV, CEA-Grenoble, Grenoble, France.

ABSTRACT
In many soils plants have to grow in a shortage of phosphate, leading to development of phosphate-saving mechanisms. At the cellular level, these mechanisms include conversion of phospholipids into glycolipids, mainly digalactosyldiacylglycerol (DGDG). The lipid changes are not restricted to plastid membranes where DGDG is synthesized and resides under normal conditions. In plant cells deprived of phosphate, mitochondria contain a high concentration of DGDG, whereas mitochondria have no glycolipids in control cells. Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope. The transfer of DGDG between plastid and mitochondria is investigated and detected between mitochondria-closely associated envelope vesicles and mitochondria. This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria. Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

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Characterization of mitochondria fractions isolated from either control or 3 d Pi-deprived A. thaliana cells. (A) To check purity and intactness of isolated mitochondria, succinate oxidation was followed by measuring O2 consumption. On average, each purified fraction consumed ∼280 nmol O2.min−1.mg−1 protein in the presence of succinate and ADP, and O2 consumption was stimulated 2.4 times by addition of ADP. Therefore, fractions were considered to be highly enriched in functionally intact mitochondria. Cyanide resistant pathway was slightly enhanced in Pi-deprived conditions as expected according to Rébeillé et al. (1984). (B) Comparative Western blot analysis of mitochondrial (M) and total cell extract (Ce) using antibodies specific for mitochondrial proteins, HPPK, a matrix protein, NAD9, an inner membrane protein, and TOM20 and TOM40 outer membrane proteins. (C) Western blot analysis of mitochondrial (M), chloroplast (Chl), and total cell extract (Ce) of Pi-deprived cells and of chloroplast envelope (Env) prepared from Arabidopsis plants as in Awai et al. (2001) using antibodies specific for chloroplast membrane proteins, LHCII for thylakoid, E37 for inner envelope membrane, and OEP21 for outer envelope membrane.
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fig2: Characterization of mitochondria fractions isolated from either control or 3 d Pi-deprived A. thaliana cells. (A) To check purity and intactness of isolated mitochondria, succinate oxidation was followed by measuring O2 consumption. On average, each purified fraction consumed ∼280 nmol O2.min−1.mg−1 protein in the presence of succinate and ADP, and O2 consumption was stimulated 2.4 times by addition of ADP. Therefore, fractions were considered to be highly enriched in functionally intact mitochondria. Cyanide resistant pathway was slightly enhanced in Pi-deprived conditions as expected according to Rébeillé et al. (1984). (B) Comparative Western blot analysis of mitochondrial (M) and total cell extract (Ce) using antibodies specific for mitochondrial proteins, HPPK, a matrix protein, NAD9, an inner membrane protein, and TOM20 and TOM40 outer membrane proteins. (C) Western blot analysis of mitochondrial (M), chloroplast (Chl), and total cell extract (Ce) of Pi-deprived cells and of chloroplast envelope (Env) prepared from Arabidopsis plants as in Awai et al. (2001) using antibodies specific for chloroplast membrane proteins, LHCII for thylakoid, E37 for inner envelope membrane, and OEP21 for outer envelope membrane.

Mentions: To further investigate their lipid composition, mitochondria were isolated and purified from Arabidopsis cells either deprived of Pi for 3 d or sufficiently provided with Pi (control cells). O2 consumption of isolated mitochondria was analyzed on each purified fraction (Fig. 2 A) and according to Neuburger et al. (1982), data indicated that the preparations were highly enriched in functionally intact mitochondria. To obtain sufficient amounts of lipids for analysis, three mitochondrial preparations obtained in each condition were pooled. Mitochondria purity was further controlled by Western blot on this mix. We detected approximately a fivefold enrichment of the mitochondrial inner membrane protein NAD9 (Lamattina et al., 1993), the outer membrane proteins TOM20 and TOM40 (Werhahn et al., 2001), and of the HPPK matrix protein (Mouillon et al., 2002) in the mitochondrial fractions, as compared with the whole-cell fractions (Fig. 2 B). Taking into consideration that mitochondria may represent 15–20% of total cell protein, the enrichment in mitochondria markers indicated that the isolated mitochondria were rather pure. Nevertheless, in order to ascertain galactolipid content of mitochondria, we measured the cross-contamination of the isolated organelles by plastid membranes classically reported to be enriched in galactolipids. Contamination by chloroplast membranes was measured by following the thylakoid marker LHCII (Vallon et al., 1986), the inner envelope marker E37 (Teyssier et al., 1996), and the outer envelope marker OEP21 (Bolter et al., 1999). Contamination by thylakoids was negligible because mitochondrial fractions contained 40 times less LHCII than chloroplasts (Fig. 2 C). The envelope markers OEP21 and E37 were both five times less abundant in mitochondria than in chloroplasts, and taking into account that envelope proteins likely represent ∼4% of chloroplast total proteins, this indicated that mitochondria (1 mg protein) contained <0.6% envelope proteins (6 μg protein) (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200407022/DC1).


Phosphate deprivation induces transfer of DGDG galactolipid from chloroplast to mitochondria.

Jouhet J, Maréchal E, Baldan B, Bligny R, Joyard J, Block MA - J. Cell Biol. (2004)

Characterization of mitochondria fractions isolated from either control or 3 d Pi-deprived A. thaliana cells. (A) To check purity and intactness of isolated mitochondria, succinate oxidation was followed by measuring O2 consumption. On average, each purified fraction consumed ∼280 nmol O2.min−1.mg−1 protein in the presence of succinate and ADP, and O2 consumption was stimulated 2.4 times by addition of ADP. Therefore, fractions were considered to be highly enriched in functionally intact mitochondria. Cyanide resistant pathway was slightly enhanced in Pi-deprived conditions as expected according to Rébeillé et al. (1984). (B) Comparative Western blot analysis of mitochondrial (M) and total cell extract (Ce) using antibodies specific for mitochondrial proteins, HPPK, a matrix protein, NAD9, an inner membrane protein, and TOM20 and TOM40 outer membrane proteins. (C) Western blot analysis of mitochondrial (M), chloroplast (Chl), and total cell extract (Ce) of Pi-deprived cells and of chloroplast envelope (Env) prepared from Arabidopsis plants as in Awai et al. (2001) using antibodies specific for chloroplast membrane proteins, LHCII for thylakoid, E37 for inner envelope membrane, and OEP21 for outer envelope membrane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172463&req=5

fig2: Characterization of mitochondria fractions isolated from either control or 3 d Pi-deprived A. thaliana cells. (A) To check purity and intactness of isolated mitochondria, succinate oxidation was followed by measuring O2 consumption. On average, each purified fraction consumed ∼280 nmol O2.min−1.mg−1 protein in the presence of succinate and ADP, and O2 consumption was stimulated 2.4 times by addition of ADP. Therefore, fractions were considered to be highly enriched in functionally intact mitochondria. Cyanide resistant pathway was slightly enhanced in Pi-deprived conditions as expected according to Rébeillé et al. (1984). (B) Comparative Western blot analysis of mitochondrial (M) and total cell extract (Ce) using antibodies specific for mitochondrial proteins, HPPK, a matrix protein, NAD9, an inner membrane protein, and TOM20 and TOM40 outer membrane proteins. (C) Western blot analysis of mitochondrial (M), chloroplast (Chl), and total cell extract (Ce) of Pi-deprived cells and of chloroplast envelope (Env) prepared from Arabidopsis plants as in Awai et al. (2001) using antibodies specific for chloroplast membrane proteins, LHCII for thylakoid, E37 for inner envelope membrane, and OEP21 for outer envelope membrane.
Mentions: To further investigate their lipid composition, mitochondria were isolated and purified from Arabidopsis cells either deprived of Pi for 3 d or sufficiently provided with Pi (control cells). O2 consumption of isolated mitochondria was analyzed on each purified fraction (Fig. 2 A) and according to Neuburger et al. (1982), data indicated that the preparations were highly enriched in functionally intact mitochondria. To obtain sufficient amounts of lipids for analysis, three mitochondrial preparations obtained in each condition were pooled. Mitochondria purity was further controlled by Western blot on this mix. We detected approximately a fivefold enrichment of the mitochondrial inner membrane protein NAD9 (Lamattina et al., 1993), the outer membrane proteins TOM20 and TOM40 (Werhahn et al., 2001), and of the HPPK matrix protein (Mouillon et al., 2002) in the mitochondrial fractions, as compared with the whole-cell fractions (Fig. 2 B). Taking into consideration that mitochondria may represent 15–20% of total cell protein, the enrichment in mitochondria markers indicated that the isolated mitochondria were rather pure. Nevertheless, in order to ascertain galactolipid content of mitochondria, we measured the cross-contamination of the isolated organelles by plastid membranes classically reported to be enriched in galactolipids. Contamination by chloroplast membranes was measured by following the thylakoid marker LHCII (Vallon et al., 1986), the inner envelope marker E37 (Teyssier et al., 1996), and the outer envelope marker OEP21 (Bolter et al., 1999). Contamination by thylakoids was negligible because mitochondrial fractions contained 40 times less LHCII than chloroplasts (Fig. 2 C). The envelope markers OEP21 and E37 were both five times less abundant in mitochondria than in chloroplasts, and taking into account that envelope proteins likely represent ∼4% of chloroplast total proteins, this indicated that mitochondria (1 mg protein) contained <0.6% envelope proteins (6 μg protein) (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200407022/DC1).

Bottom Line: Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope.This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria.Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Physiologie Cellulaire Végétale, UMR 5168 (CNRS/CEA/Université Jseph Fourier/INRA), DRDC-PCV, CEA-Grenoble, Grenoble, France.

ABSTRACT
In many soils plants have to grow in a shortage of phosphate, leading to development of phosphate-saving mechanisms. At the cellular level, these mechanisms include conversion of phospholipids into glycolipids, mainly digalactosyldiacylglycerol (DGDG). The lipid changes are not restricted to plastid membranes where DGDG is synthesized and resides under normal conditions. In plant cells deprived of phosphate, mitochondria contain a high concentration of DGDG, whereas mitochondria have no glycolipids in control cells. Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope. The transfer of DGDG between plastid and mitochondria is investigated and detected between mitochondria-closely associated envelope vesicles and mitochondria. This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria. Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

Show MeSH
Related in: MedlinePlus