Maryse A. Block

Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana

In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.

Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol.

The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B12-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.

Phosphate deprivation induces transfer of DGDG galactolipid from chloroplast to mitochondria

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.

Preparation and Characterization of Membrane Fractions Enriched in Outer and Inner Envelope Membranes from Spinach Chloroplasts

A gentle osmotic shock of intact and purified chloroplasts allows the preparation of envelope membranes in a reasonably pure state. Unfortunately, irreversible changes take place in the membranes (fusions?) during the osmotic shock thus making the separation of the outer membrane from the inner impossible (Douce, Joyard, 1979). Such a separation can only be achieved by strikingly different procedures. Cline et al. (1981) have described a procedure which includes freeze-thaw lysis of intact pea chloroplasts. We have developed a different method to provide the separation of two membrane fractions deriving from the chloroplast envelope membranes. Since we have characterized two polypeptides as outer envelope polypeptides (Joyard et al., 1983), we were able to characterize the membrane fractions obtained (Block et al., in press).

The Response of Nannochloropsis gaditana to Nitrogen Starvation Includes De Novo Biosynthesis of Triacylglycerols, a Decrease of Chloroplast Galactolipids, and Reorganization of the Photosynthetic Apparatus

ABSTRACT Microalgae of the genus Nannochloropsis are capable of accumulating triacylglycerols (TAGs) when exposed to nutrient limitation (in particular, nitrogen [N]) and are therefore considered promising organisms for biodiesel production. Here, after nitrogen removal from the medium, Nannochloropsis gaditana cells showed extensive triacylglycerol accumulation (38% TAG on a dry weight basis). Triacylglycerols accumulated during N deprivation harbored signatures, indicating that they mainly stemmed from freshly synthesized fatty acids, with a small proportion originating from a recycling of membrane glycerolipids. The amount of chloroplast galactoglycerolipids, which are essential for the integrity of thylakoids, decreased, while their fatty acid composition appeared to be unaltered. In starved cells, galactolipids were kept at a level sufficient to maintain chloroplast integrity, as confirmed by electron microscopy. Consistently, N-starved Nannochloropsis cells contained less photosynthetic membranes but were still efficiently performing photosynthesis. N starvation led to a modification of the photosynthetic apparatus with a change in pigment composition and a decrease in the content of all the major electron flow complexes, including photosystem II, photosystem I, and the cytochrome b6f complex. The photosystem II content was particularly affected, leading to the inhibition of linear electron flow from water to CO2. Such a reduction, however, was partially compensated for by activation of alternative electron pathways, such as cyclic electron transport. Overall, these changes allowed cells to modify their energetic metabolism in order to maintain photosynthetic growth.

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. δ-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.

Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts

The prenylquinone content and biosynthetic capabilities of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts were analyzed. Both envelope membranes contain prenylquinones, and in almost similar amounts (on a protein basis). However, the outer envelope membrane contains more alpha-tocopherol than the inner one although this prenylquinone is the major one in both fractions. On the contrary, plastoquinone-9 is present in higher amounts in the inner envelope membrane than in the outer one. In addition, it has been demonstrated that all the enzymes involved in the last steps of alpha-tocopherol and plastoquinone-9 biosynthesis, i.e., homogentisate decarboxylase polyprenyltransferase, S-adenosyl-methionine:methyl-6-phytylquinol methyltransferase, S-adenosyl-methionine: alpha-tocopherol methyltransferase, homogentisate decarboxylase solanesyltransferase, S-adenosyl-methionine:methyl-6-solanesylquinol methyltransferase, and possibly 2,3-dimethylphytylquinol cyclase, are localized on the inner envelope membrane. These results demonstrate that the inner membrane of the chloroplast envelope plays a key role in chloroplast biogenesis, and especially for the synthesis of the two major plastid prenylquinones.

Glycerolipids in photosynthesis: Composition, synthesis and trafficking☆

Glycerolipids constituting the matrix of photosynthetic membranes, from cyanobacteria to chloroplasts of eukaryotic cells, comprise monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. This review covers our current knowledge on the structural and functional features of these lipids in various cellular models, from prokaryotes to eukaryotes. Their relative proportions in thylakoid membranes result from highly regulated and compartmentalized metabolic pathways, with a cooperation, in the case of eukaryotes, of non-plastidic compartments. This review also focuses on the role of each of these thylakoid glycerolipids in stabilizing protein complexes of the photosynthetic machinery, which might be one of the reasons for their fascinating conservation in the course of evolution. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.

The galactolipid:galactolipid galactosyltransferase is located on the outer surface of the outer membrane of the chloroplast envelope

1. INTRODUCTION It is well known that the plastid envelope is in- volved in the synthesis of galactolipids (for a re- view see [l]). For instance it has been demon- strated that at least two distinct enzymes res- ponsible for the synthesis of monogalactosyldiacyl- glycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are associated with chloroplast envelope membranes. The first enzyme, UDP-galactose:di- acylglycerol galactosyltransferase, catalyses the in- corporation of galactose from UDP-galactose into MGDG [3-51:

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.