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Significance Cyanobacteria, as well as chloroplasts of plants and algae, are the sites of photosynthesis that produces oxygen. Photosynthetic membranes, also known as thylakoid membranes, in these organisms contain galactolipids, without exception, as the major components. Galactolipids are thus believed to be important for photosynthesis or at least for the formation of the flattened shape of thylakoid membranes. The biosynthetic pathway of galactolipids is definitely different in plants and cyanobacteria. Here we identified the final piece of the long-sought gene for the galactolipid synthesis in cyanobacteria, namely monoglucosyldiacylglycerol epimerase, which converts glucolipid to galactolipid. Cyanobacterial mutants in which this gene is disrupted still formed authentic thylakoid membranes without galactolipids and kept the ability to grow photosynthetically, indicating that galactolipids are not essential for either oxygenic photosynthesis or the development of the thylakoid membrane structure.

Plants show complex adaptations to freezing that prevent cell damage caused by cellular dehydration. Lipid remodeling of cell membranes during dehydration is one critical mechanism countering loss of membrane integrity and cell death. SENSITIVE TO FREEZING 2 (SFR2), a gene essential for freezing tolerance in Arabidopsis, encodes a galactolipid remodeling enzyme of the outer chloroplast envelope membrane. SFR2 processively transfers galactosyl residues from the abundant monogalactolipid to different galactolipid acceptors, forming oligogalactolipids and diacylglycerol, which is further converted to triacylglycerol. The combined activity of SFR2 and triacylglycerol-biosynthetic enzymes leads to the removal of monogalactolipids from the envelope membrane, changing the ratio of bilayer- to non-bilayer-forming membrane lipids. This SFR2-based mechanism compensates for changes in organelle volume and stabilizes membranes during freezing.

Head group-acylated chloroplast lipids were discovered in the 1960s, but interest was renewed about 15 years ago with the discovery of Arabidopsides E and G, acylated monogalactosyldiacylglycerols with oxidized fatty acyl chains originally identified in Arabidopsis thaliana. Since then, plant biologists have applied the power of mass spectrometry to identify additional oxidized and non-oxidized chloroplast lipids and quantify their levels in response to biotic and abiotic stresses. The enzyme responsible for the head-group acylation of chloroplast lipids was identified as a cytosolic protein closely associated with the chloroplast outer membrane and christened acylated galactolipid-associated phospholipase 1 (AGAP1). Despite many advances, critical questions remain about the biological functions of AGAP1 and its head group-acylated products.

Cotyledon cells of dark-germinated angiosperms develop etioplasts that are plastids containing unique internal membranes called prolamellar bodies (PLBs). Protochlorophyllide (Pchlide), a precursor of chlorophyll, accumulates in PLBs and forms a ternary complex with NADPH and light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), which allows for the rapid formation of chlorophyll after illumination while avoiding photodamage. PLBs are 3D lattice structures formed by the lipid bilayer rich in monogalactosyldiacylglycerol (MGDG). Although MGDG was found to be required for the formation and function of the thylakoid membrane in chloroplasts in various plants, the roles of MGDG in PLB formation and etioplast development are largely unknown. To analyze the roles of MGDG in etioplast development, we suppressed MGD1 encoding the major isoform of MGDG synthase by using a dexamethasone-inducible artificial microRNA in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. Strong MGD1 suppression caused a 36% loss of MGDG in etiolated seedlings, together with a 41% decrease in total Pchlide content. The loss of MGDG perturbed etioplast membrane structures and impaired the formation of the photoactive Pchlide-LPOR-NADPH complex and its oligomerization, without affecting LPOR accumulation. The MGD1 suppression also impaired the formation of Pchlide from protoporphyrin IX via multiple enzymatic reactions in etioplast membranes, which suggests that MGDG is required for the membrane-associated processes in the Pchlide biosynthesis pathway. Suppressing MGD1 at several germination stages revealed that MGDG biosynthesis at an early germination stage is particularly important for Pchlide accumulation. MGDG biosynthesis may provide a lipid matrix for Pchlide biosynthesis and the formation of Pchlide-LPOR complexes as an initial step of etioplast development.

A monogalactosyl diacylglyceride (MGDG) was isolated as an antiviral component from Coccomyxa sp. KJ (IPOD FERM BP-22254) via bioassay-guided fractionation. α-Linolenic acid (C18:3) and 7,10,13-hexadecatrienoic acid (C16:3) accounted for approximately 72% and 23%, respectively, of the MGDG total fatty acids of the MGDG. The MGDG showed virucidal activity against herpes simplex virus type 2 (HSV-2), a pathogen that causes genital herpes. Physical changes in HSV-2 shape were observed after treatment with MGDG, including a decrease in particle size, and possible damage to the viral envelope, as assessed using electron microscopy. In accordance with the morphological findings, virus particles lost their ability to bind to host cells. HSV-2 treated with high concentrations of MGDG resulted in no pathogenicity in an animal model, indicating that MGDG exhibits irreversible virucidal activity against HSV-2 particles. In the animal model of HSV-2-induced genital herpes, intravaginally administered MGDG exerted a prophylactic effect by suppressing viral yields in the genital cavity and formation of herpetic lesions, resulting in a higher survival rate in treated mice than control mice administered solvent. Thus, MGDG offers a novel prophylactic option against HSV infections.

Proteaceae species in south-western Australia occur on severely phosphorus (P)-impoverished soils. They have very low leaf P concentrations, but relatively fast rates of photosynthesis, thus exhibiting extremely high photosynthetic phosphorus-use-efficiency (PPUE). Although the mechanisms underpinning their high PPUE remain unknown, one possibility is that these species may be able to replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. For six Proteaceae species, we measured soil and leaf P concentrations and rates of photosynthesis of both young expanding and mature leaves. We also assessed the investment in galactolipids, sulfolipids and phospholipids in young and mature leaves, and compared these results with those on Arabidopsis thaliana, grown under both P-sufficient and P-deficient conditions. In all Proteaceae species, phospholipid levels strongly decreased during leaf development, whereas those of galactolipids and sulfolipids strongly increased. Photosynthetic rates increased from young to mature leaves. This shows that these species extensively replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. A considerably less pronounced shift was observed in A. thaliana. Our results clearly show that a low investment in phospholipids, relative to nonphospholipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils.

Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of thylakoid membranes and is essential for chloroplast biogenesis in plants. In Arabidopsis (Arabidopsis thaliana), MGDG is predominantly synthesized by inner envelope-localized MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (MGD1); its knockout causes albino seedlings. Because of the lethal phenotype of the null MGD1 mutant, functional details of MGDG synthesis at seedling development have remained elusive. In this study, we used an inducible gene-suppression system to investigate the impact of MGDG synthesis on cotyledon development. We created transgenic Arabidopsis lines that express an artificial microRNA targeting MGD1 (amiR-MGD1) under the control of a dexamethasone-inducible promoter. The induction of amiR-MGDl resulted in up to 75% suppression of MGD1 expression, although the resulting phenotypes related to chloroplast development were diverse, even within a line. The strong MGD1 suppression by continuous dexamethasone treatment caused substantial decreases in galactolipid content in cotyledons, leading to severe defects in the formation of thylakoid membranes and impaired photosynthetic electron transport. Time-course analyses of the MGD1 suppression during seedling germination revealed that MGDG synthesis at the very early germination stage is particularly important for chloroplast biogenesis. The MGD1 suppression down-regulated genes associated with the photorespiratory pathway in peroxisomes and mitochondria as well as those responsible for photosynthesis in chloroplasts and caused high expression of genes for the glyoxylate cycle. MGD1 function may link galactolipid synthesis with the coordinated transcriptional regulation of chloroplasts and other organelles during cotyledon greening.

Background The ripening of fleshy fruits is a complex developmental program characterized by extensive transcriptomic and metabolic remodeling in the pericarp tissues (pulp and skin) making unripe green fruits soft, tasteful and colored. The onset of ripening is regulated by a plethora of endogenous signals tuned to external stimuli. In grapevine and tomato, which are classified as non-climacteric and climacteric species respectively, the accumulation of hydrogen peroxide (H 2 O 2 ) and extensive modulation of reactive oxygen species (ROS) scavenging enzymes at the onset of ripening has been reported, suggesting that ROS could participate to the regulatory network of fruit development. In order to investigate this hypothesis, a comprehensive biochemical study of the oxidative events occurring at the beginning of ripening in Vitis vinifera cv. Pinot Noir has been undertaken. Results ROS-specific staining allowed to visualize not only H 2 O 2 but also singlet oxygen ( 1 O 2 ) in berry skin cells just before color change in distinct subcellular locations, i.e. cytosol and plastids. H 2 O 2 peak in sample skins at véraison was confirmed by in vitro quantification and was supported by the concomitant increase of catalase activity. Membrane peroxidation was also observed by HPLC-MS on galactolipid species at véraison. Mono- and digalactosyl diacylglycerols were found peroxidized on one or both α-linolenic fatty acid chains, with a 13(S) absolute configuration implying the action of a specific enzyme. A lipoxygenase (PnLOXA), expressed at véraison and localizing inside the chloroplasts, was indeed able to catalyze membrane galactolipid peroxidation when overexpressed in tobacco leaves. Conclusions The present work demonstrates the controlled, harmless accumulation of specific ROS in distinct cellular compartments, i.e. cytosol and chloroplasts, at a definite developmental stage, the onset of grape berry ripening. These features strongly candidate ROS as cellular signals in fruit ripening and encourage further studies to identify downstream elements of this cascade. This paper also reports the transient galactolipid peroxidation carried out by a véraison-specific chloroplastic lipoxygenase. The function of peroxidized membranes, likely distinct from that of free fatty acids due to their structural role and tight interaction with photosynthesis protein complexes, has to be ascertained.

• Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG-synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown. • We introduced an inducible microRNA (ami-MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA-biosynthesis and JA-responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated. • Expression of JA-biosynthesis and JA-responsive genes were reduced in amiR-MGD1-transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued. • Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG.

Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography-mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis.