Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

PUB11-Dependent Ubiquitination of the Phospholipid Flippase ALA10 Modifies ALA10 Localization and Affects the Pool of Linolenic Phosphatidylcholine.

Biogenesis of photosynthetic membranes depends on galactolipid synthesis, which relies on several cell compartments, notably the endoplasmic reticulum (ER) and the chloroplast envelope. Galactolipid synthesis involves lipid trafficking between both membrane compartments. In Arabidopsis, ALA10, a phospholipid flippase of the P4 type-ATPase family, counteracts the limitation of monogalactosyldiacylglycerol (MGDG) production and has a positive effect on leaf development. ALA10 locates in distinct domains of the ER depending on the ALIS (ALA interacting subunit) subunit it interacts with: close to the plasma membrane with ALIS1, or next to chloroplasts with ALIS5. It interacts with FAD2 (Fatty acid desaturase 2) and prevents accumulation of linolenic (18:3) containing phosphatidylcholine (PC) stimulating an increase of MGDG synthesis. Here we report that ALA10 interacts with PUB11 (plant U-box type 11), an E3 protein ubiquitin ligase, in vitro and in vivo. ALA10 is however ubiquitinated and degraded by the 26S proteasome in a PUB11-independent process. In pub11 null mutant, the proteasome-dependent degradation of ALA10 is retained and ALA10 is still subject to ubiquitination although its ubiquitination profile appears different. In the absence of PUB11, ALA10 is constrained to the ER close to chloroplasts, which is the usual location when ALA10 is overexpressed. Additionally, in this condition, the decrease of 18:3 containing PC is no longer observed. Taken together these results suggest, that ALA10 contributes in chloroplast-distal ER interacting domains, to reduce the 18:3 desaturation of PC and that PUB11 is involved in reconditioning of ALA10 from chloroplast-proximal to chloroplast-distal ER interacting domains.

Purification of Chloroplasts and Chloroplast Subfractions: Envelope, Thylakoids, and Stroma-From Spinach, Pea, and Arabidopsis thaliana.

Chloroplasts are specific organelles of plant cells dedicated to photosynthesis and delimited by a two-membrane chloroplast envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are clearly different from the chloroplast envelope and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three main compartments of the chloroplast: the stroma, the thylakoids, and the chloroplast envelope. Three different protocols are provided, adapted for starting leaves of spinach, Arabidopsis, and pea.

ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana.

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature.

Levels of polyunsaturated fatty acids correlate with growth rate in plant cell cultures.

In higher plants, fatty acids (FAs) with 18 carbons (18C) represent about 70% of total FAs, the most abundant species being 18:2 and 18:3. These two polyunsaturated FAs (PUFAs) represent about 55% of total FAs in Arabidopsis cell suspension cultures, whereas 18:1 represents about 10%. The level of PUFAs may vary, depending on ill-defined factors. Here, we compared various sets of plant cell cultures and noticed a correlation between the growth rate of a cell population and the level of unsaturation of 18C FAs. These observations suggest that the final level of PUFAs might depend in part on the rate of cell division, and that FAD2 and FAD3 desaturases, which are respectively responsible for the formation of 18:2 and 18:3 on phospholipids, have limiting activities in fast-growing cultures. In plant cell culture, phosphate (Pi) deprivation is known to impair cell division and to trigger lipid remodeling. We observed that Pi starvation had no effect on the expression of FAD genes, and that the level of PUFAs in this situation was also correlated with the growth rate. Thus, the level of PUFAs appears as a hallmark in determining cell maturity and aging.

The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes.

The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM-OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.

C1 metabolism and chlorophyll synthesis: the Mg-protoporphyrin IX methyltransferase activity is dependent on the folate status.

* Tetrahydrofolate derivatives are central cofactors of C1 metabolism. Using methotrexate as a specific inhibitor of folate biosynthesis, we altered the folate status in 10-d-old etiolated pea (Pisum sativum) leaves and followed the rate of chlorophyll synthesis upon illumination. * In our conditions, the folate concentration decreased only from 5.7 to 4.2 nmol g(-1) FW, but the amount of chlorophyll after 24 h of illumination was reduced 2.5 times. Folate status and rate of chlorophyll synthesis were apparently correlated through the methyl cycle. * Indeed, we observed that methyl-tetrahydrofolate was the folate derivative most affected by the treatment; the decrease of methyl-tetrahydrofolate was associated with a sharp rise in homocysteine and S-adenosylhomocysteine concentrations, which are normally maintained at very low values, shifting the methylation index (S-adenosylmethionine/S-adenosylhomocysteine ratio) from 7 to 1; the decrease of the methylation index reduced by a factor of 3 the activity of the Mg-protoporphyrin IX methyltransferase (CHLM), an essential enzyme for chlorophyll synthesis. CHLM gene expression and protein concentration remained unchanged, suggesting that this inhibition relied essentially on metabolic regulation. * These results point out that an even moderate change in the folate status may affect plant development and adaptation.

Phosphate availability affects the tonoplast localization of PLDzeta2, an Arabidopsis thaliana phospholipase D.

Under phosphate deprivation, higher plants change their lipid composition and recycle phosphate from phospholipids. A phospholipase D, PLDzeta2, is involved in this recycling and in other cellular functions related to plant development. We investigated the localization of Arabidopsis PLDzeta2 by cell fractionation and in vivo GFP confocal imaging. AtPLDzeta2 localizes to the tonoplast and the Nter regulatory domain is sufficient for its sorting. Under phosphate deprivation, AtPLDzeta2 remains located in the tonoplast but its distribution is uneven. We observed PLDzeta2-enriched tonoplast domains preferentially positioned close to mitochondria and beside chloroplasts. In absence of PLDzeta2, membrane developments were visualized inside vacuoles.

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling.

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step.

Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide.

CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. delta-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1/CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1/CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes.

The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes.

Chlorophyll biosynthesis requires a metabolic dialog between the chloroplast envelope and thylakoids where biosynthetic activities are localized. Here, we report the first plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase (MgP(IX)MT) sequence identified in the Arabidopsis genome owing to its similarity with the Synechocystis sp. MgP(IX)MT gene. After expression in Escherichia coli, the recombinant Arabidopsis thaliana cDNA was shown to encode a protein having MgP(IX)MT activity. The full-length polypeptide exhibits a chloroplast transit peptide that is processed during import into the chloroplast. The mature protein contains two functional regions. The C-terminal part aligns with the Synechocystis full-length protein. The corresponding truncated region binds to Ado-met, as assayed by UV crosslinking, and is shown to harbor the MgP(IX)MT activity. Downstream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane. In A. thaliana and spinach, the MgP(IX)MT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids. The protein is active in each membrane and has the same apparent size corresponding to the processed mature protein. The protein is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane. The rationale for a dual localization of the protein in the chloroplast is discussed.