An ABCB transporter regulates anisotropic cell expansion via cuticle deposition in the moss Physcomitrium patens.

Anisotropic cell expansion is crucial for the morphogenesis of land plants, as cell migration is restricted by the rigid cell wall. The anisotropy of cell expansion is regulated by mechanisms acting on the deposition or modification of cell wall polysaccharides. Besides the polysaccharide components in the cell wall, a layer of hydrophobic cuticle covers the outer cell wall and is subjected to tensile stress that mechanically restricts cell expansion. However, the molecular machinery that deposits cuticle materials in the appropriate spatiotemporal manner to accommodate cell and tissue expansion remains elusive. Here, we report that PpABCB14, an ATP-binding cassette transporter in the moss Physcomitrium patens, regulates the anisotropy of cell expansion. PpABCB14 localized to expanding regions of leaf cells. Deletion of PpABCB14 resulted in impaired anisotropic cell expansion. Unexpectedly, the cuticle proper was reduced in the mutants, and the cuticular lipid components decreased. Moreover, induced PpABCB14 expression resulted in deformed leaf cells with increased cuticle lipid accumulation on the cell surface. Taken together, PpABCB14 regulates the anisotropy of cell expansion via cuticle deposition, revealing a regulatory mechanism for cell expansion in addition to the mechanisms acting on cell wall polysaccharides.

Phosphatidic acid phosphohydrolase modulates glycerolipid synthesis in Marchantia polymorpha and is crucial for growth under both nutrient-replete and -deficient conditions.

MAIN CONCLUSION: The phosphatidic acid phosphohydrolase of Marchantia polymorpha modulates plastid glycolipid synthesis through the ER pathway and is essential for normal plant development regardless of nutrient availability. Membrane lipid remodeling is one of the strategies plant cells use to secure inorganic phosphate (Pi) for plant growth, but many aspects of the molecular mechanism and its regulation remain unclear. Here we analyzed membrane lipid remodeling using a non-vascular plant, Marchantia polymorpha. The lipid composition and fatty acid profile during Pi starvation in M. polymorpha revealed a decrease in phospholipids and an increase in both galactolipids and betaine lipids. In Arabidopsis thaliana, phosphatidic acid phosphohydrolase (PAH) is involved in phospholipid degradation and is crucial for tolerance to both Pi and nitrogen starvation. We produced two M. polymorpha PAH (MpPAH) knockout mutants (Mppah-1 and Mppah-2) and found that, unlike Arabidopsis mutants, Mppah impaired plant growth with shorter rhizoids compared with wild-type plants even under nutrient-replete conditions. Mutation of MpPAH did not significantly affect the mole percent of each glycerolipid among total membrane glycerolipids from whole plants under both Pi-replete and Pi-deficient conditions. However, the fatty acid composition of monogalactosyldiacylglycerol indicated that the amount of plastid glycolipids produced through the endoplasmic reticulum pathway was suppressed in Mppah mutants. Phospholipids accumulated in the mutants under N starvation. These results reveal that MpPAH modulates plastid glycolipid synthesis through the endoplasmic reticulum pathway more so than what has been observed for Arabidopsis PAH; moreover, unlike Arabidopsis, MpPAH is crucial for M. polymorpha growth regardless of nutrient availability.

Cis-regulatory elements and transcription factors related to auxin signaling in the streptophyte algae Klebsormidium nitens.

The phytohormone auxin affects numerous processes in land plants. The central auxin signaling machinery, called the nuclear auxin pathway, is mediated by its pivotal receptor named TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB). The nuclear auxin pathway is widely conserved in land plants, but auxin also accumulates in various algae. Although auxin affects the growth of several algae, the components that mediate auxin signaling have not been identified. We previously reported that exogenous auxin suppresses cell proliferation in the Klebsormidium nitens that is a member of streptophyte algae, a paraphyletic group sharing the common ancestor with land plants. Although K. nitens lacks TIR1/AFB, auxin affects the expression of numerous genes. Thus, elucidation of the mechanism of auxin-inducible gene expression in K. nitens would provide important insights into the evolution of auxin signaling. Here, we show that some motifs are enriched in the promoter sequences of auxin-inducible genes in K. nitens. We also found that the transcription factor KnRAV activates several auxin-inducible genes and directly binds the promoter of KnLBD1, a representative auxin-inducible gene. We propose that KnRAV has the potential to regulate auxin-responsive gene expression in K. nitens.

Developing a platform for production of the oxylipin KODA in plants.

KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low, except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were targeted to plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in crude leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific sub-cellular localization of these two enzymes and incubation of crude leaf extracts, which liberates α-linolenic acid via breakdown of endogenous lipids.

Recycling of the major thylakoid lipid MGDG and its role in lipid homeostasis in Chlamydomonas reinhardtii.

Monogalactosyldiacylglycerol (MGDG), the most abundant lipid in thylakoid membranes, is involved in photosynthesis and chloroplast development. MGDG lipase has an important role in lipid remodeling in Chlamydomonas reinhardtii. However, the process related to turnover of the lysogalactolipid that results from MGDG degradation, monogalactosylmonoacylglycerol (MGMG), remains to be clarified. Here we identified a homolog of Arabidopsis thaliana lysophosphatidylcholine acyltransferase (LPCAT) and characterized two independent knockdown (KD) alleles in C. reinhardtii. The enzyme designated as C. reinhardtiiLysolipid Acyltransferase 1 (CrLAT1) has a conserved membrane-bound O-acyl transferase domain. LPCAT from Arabidopsis has a key role in deacylation of phosphatidylcholine (PC). Chlamydomonas reinhardtii, however, lacks PC, and thus we hypothesized that CrLAT1 has some other important function in major lipid flow in this organism. In the CrLAT1 KD mutants, the amount of MGMG was increased, but triacylglycerols (TAGs) were decreased. The proportion of more saturated 18:1 (9) MGDG was lower in the KD mutants than in their parental strain, CC-4533. In contrast, the proportion of MGMG has decreased in the CrLAT1 overexpression (OE) mutants, and the proportion of 18:1 (9) MGDG was higher in the OE mutants than in the empty vector control cells. Thus, CrLAT1 is involved in the recycling of MGDG in the chloroplast and maintains lipid homeostasis in C. reinhardtii.

RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants.

Inorganic phosphate (Pi) and nitrogen (N) are essential nutrients for plant growth. We found that a five-fold oversupply of nitrate rescues Arabidopsis (Arabidopsis thaliana) plants from Pi-starvation stress. Analyses of transgenic plants that overexpressed GFP-AUTOPHAGY8 showed that an oversupply of nitrate induced autophagy flux under Pi-depleted conditions. Expression of DIN6 and DIN10, the carbon (C) starvation-responsive genes, was upregulated when nitrate was oversupplied under Pi starvation, which suggested that the plants recognized the oversupply of nitrate as C starvation stress because of the reduction in the C/N ratio. Indeed, formation of Rubisco-containing bodies (RCBs), which contain chloroplast stroma and are induced by C starvation, was enhanced when nitrate was oversupplied under Pi starvation. Moreover, autophagy-deficient mutants did not release Pi (unlike wild-type plants), exhibited no RCB accumulation inside vacuoles, and were hypersensitive to Pi starvation, indicating that RCB-mediated chlorophagy is involved in Pi starvation tolerance. Thus, our results showed that the Arabidopsis response to Pi starvation is closely linked with N and C availability and that autophagy is a key factor that controls plant growth under Pi starvation.

MYB-like transcription factor NoPSR1 is crucial for membrane lipid remodeling under phosphate starvation in the oleaginous microalga Nannochloropsis oceanica.

Membrane lipid remodeling under phosphate (Pi) limitation, a process that replaces structural membrane phospholipids with nonphosphorus lipids, is a widely observed adaptive response in plants and algae. Here, we identified the transcription factor phosphorus starvation response 1 (NoPSR1) as an indispensable player for regulating membrane lipid conversion during Pi starvation in the microalga Nannochloropsis oceanica. Knocking out NoPSR1 scarcely perturbed membrane lipid composition under Pi-sufficient conditions but significantly impaired dynamic alteration in membrane lipids during Pi starvation. In contrast, the absence of NoPSR1 led to no obvious change in cell proliferation or storage lipid accumulation under either nutrient-sufficient or Pi-deficient conditions. Our results demonstrate a key factor controlling the membrane lipid profile during the Pi starvation response in N. oceanica.

Complete Replacement of the Galactolipid Biosynthesis Pathway with a Plant-Type Pathway in the Cyanobacterium Synechococcus elongatus PCC 7942.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major components of thylakoid membranes and well-conserved from cyanobacteria to chloroplasts. However, cyanobacteria and chloroplasts synthesize these galactolipids using different pathways and enzymes, but they are believed to share a common ancestor. This fact implies that there was a replacement of the cyanobacterial galactolipid biosynthesis pathway during the evolution of a chloroplast. In this study, we first replaced the cyanobacterial MGDG biosynthesis pathway in a model cyanobacterium, Synechococcus elongatus PCC 7942, with the corresponding plant-type pathway. No obvious phenotype was observed under the optimum growth condition, and the content of membrane lipids was not largely altered in the transformants. We next replaced the cyanobacterial DGDG biosynthesis pathway with the corresponding plant-type pathway using the strain described above and isolated the strain harboring the replaced plant-type pathway instead of the whole galactolipid biosynthesis pathway. This transformant, SeGPT, can grow photoautotrophically, indicating that cyanobacterial galactolipid biosynthesis pathways can be functionally complemented by the corresponding plant-type pathways and that the lipid products MGDG and DGDG, and not biosynthesis pathways, are important. While SeGPT does not show strong growth retardation, the strain has low cellular chlorophyll content but it retained a similar oxygen evolution rate per chlorophyll content compared with the wild type. An increase in total membrane lipid content was observed in SeGPT, which was caused by a significant increase in DGDG content. SeGPT accumulated carotenoids from the xanthophyll groups. These results suggest that cyanobacteria have the capacity to accept other pathways to synthesize essential components of thylakoid membranes.

Relationship Between Glycerolipids and Photosynthetic Components During Recovery of Thylakoid Membranes From Nitrogen Starvation-Induced Attenuation in Synechocystis sp. PCC 6803.

Thylakoid membranes, the site of photochemical and electron transport reactions of oxygenic photosynthesis, are composed of a myriad of proteins, cofactors including pigments, and glycerolipids. In the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803, the size and function of thylakoid membranes are reduced under nitrogen (N) starvation but are quickly recovered after N addition to the starved cells. To understand how the functionality of thylakoid membranes is adjusted in response to N status in Synechocystis sp. PCC 6803, we examined changes in thylakoid components and the photosynthetic activity during the N starvation and recovery processes. In N-starved cells, phycobilisome content, photosystem II protein levels and the photosynthetic activity substantially decreased as compared with those in N-sufficient cells. Although the content of chlorophyll (Chl) a, total protein and total glycerolipid also decreased under the N-starved condition based on OD730 reflecting cell density, when based on culture volume, the Chl a and total protein content remained almost constant and total glycerolipid content even increased during N starvation, suggesting that cellular levels of these components decrease under the N-starved condition mainly through dilution due to cell growth. With N addition, the photosynthetic activity quickly recovered, followed by full restoration of photosynthetic pigment and protein levels. The content of phosphatidylglycerol (PG), an essential lipid constituent of both photosystems, increased faster than that of Chl a, whereas the content of glycolipids, the main constituents of the thylakoid lipid bilayer, gradually recovered after N addition. The data indicate differential regulation of PG and glycolipids during the construction of the photosynthetic machinery and regeneration of thylakoid membranes. Of note, addition of PG to the growth medium slightly accelerated the Chl a accumulation in wild-type cells during the recovery process. Because PG is required for the biosynthesis of Chl a and the formation of functional photosystem complexes, rapid PG biosynthesis in response to N acquisition may be required for the rapid formation of the photosynthetic machinery during thylakoid regeneration.

Lipid remodeling regulator 1 (LRL1) is differently involved in the phosphorus-depletion response from PSR1 in Chlamydomonas reinhardtii.

The elucidation of lipid metabolism in microalgae has attracted broad interest, as their storage lipid, triacylglycerol (TAG), can be readily converted into biofuel via transesterification. TAG accumulates in the form of oil droplets, especially when cells undergo nutrient deprivation, such as for nitrogen (N), phosphorus (P), or sulfur (S). TAG biosynthesis under N-deprivation has been comprehensively studied in the model microalga Chlamydomonas reinhardtii, during which TAG accumulates dramatically. However, the resulting rapid breakdown of chlorophyll restricts overall oil yield productivity and causes cessation of cell growth. In contrast, P-deprivation results in oil accumulation without disrupting chloroplast integrity. We used a reverse genetics approach based on co-expression analysis to identify a transcription factor (TF) that is upregulated under P-depleted conditions. Transcriptomic analysis revealed that the mutants showed repression of genes typically associated with lipid remodeling under P-depleted conditions, such as sulfoquinovosyl diacylglycerol 2 (SQD2), diacylglycerol acyltransferase (DGTT1), and major lipid droplet protein (MLDP). As accumulation of sulfoquinovosyl diacylglycerol and TAG were suppressed in P-depleted mutants, we designated the protein as lipid remodeling regulator 1 (LRL1). LRL1 mutants showed slower growth under P-depletion. Moreover, cell size in the mutant was significantly reduced, and TAG and starch accumulation per cell were decreased. Transcriptomic analysis also suggested the repression of several genes typically upregulated in adaptation to P-depletion that are associated with the cell cycle and P and lipid metabolism. Thus, our analysis of LRL1 provides insights into P-allocation and lipid remodeling under P-depleted conditions in C. reinhardtii. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The sequencing data were made publicly available under the BioProject Accession number PRJDB6733 and an accession number LC488724 at the DNA Data Bank of Japan (DDBJ). The data is available at https://trace.ddbj.nig.ac.jp/BPSearch/bioproject?acc=PRJDB6733; http://getentry.ddbj.nig.ac.jp/getentry/na/LC488724. The metabolome data were made publicly available and can be accessed at http://metabolonote.kazusa.or.jp/SE195:/; http://webs2.kazusa.or.jp/data/nur/.

Autophagy-Mediated Regulation of Lipid Metabolism and Its Impact on the Growth in Algae and Seed Plants.

Under nutrient starvation conditions, algae and seed-plant cells accumulate carbon metabolites such as storage lipids, triacylglycerols (TAGs), and starches. Recent research has suggested the involvement of autophagy in the regulation of carbon metabolites under nutrient starvation. When algae are grown under carbon starvation conditions, such as growth in darkness or in the presence of a photosynthesis inhibitor, lipid droplets are surrounded by phagophores. Indeed, the amount of TAGs in an autophagy-deficient mutant has been found to be greater than that in wild type under nitrogen starvation, and cerulenin, which is one of the inhibitors of fatty acid synthesis, induces autophagy. In land plants, TAGs accumulate predominantly in seeds and etiolated seedlings. These TAGs are degraded in peroxisomes via ß-oxidation during germination as a source of carbon for growth without photosynthesis. A global analysis of the role of autophagy in Arabidopsis seedlings under carbon starvation revealed that a lack of autophagy enhances the accumulation of TAGs and fatty acids. In Oryza sativa, autophagy-mediated degradation of TAGs and diacylglycerols has been suggested to be important for pollen development. In this review, we introduce and summarize research findings demonstrating that autophagy affects lipid metabolism and discuss the role of autophagy in membrane and storage-lipid homeostasis, each of which affects the growth and development of seed plants and algae.

Lipid remodeling under acidic conditions and its interplay with low Pi stress in Arabidopsis.

KEY MESSAGE: Here we show that accumulation of galactose-containing lipids in plastid membranes in shoots and the other membranes in roots maintains Arabidopsis growth under acidic stress and acidic phosphate deficiency. Soil acidification and phosphate deficiency are closely related to each other in natural environments. In addition to the toxicity of high proton concentrations, acid soil can lead to imbalances of ion availability and nutritional deficiencies, including inorganic phosphate (Pi). Among plants, activation of non-phosphorus-containing galactolipid, digalactosyldiacylglycerol (DGDG), synthesis concomitant with phospholipid degradation, namely membrane lipid remodeling, is crucial for coping with Pi starvation. However, regulation mechanisms of membrane lipid composition during acidic stress have not been clarified. Here, we investigated lipid metabolism in Arabidopsis thaliana grown under acidic stress with or without Pi. Under Pi-sufficient acidic conditions, DGDG was increased in shoot membranes, and some Pi starvation-responsive genes that are involved in lipid remodeling were upregulated without reducing Pi content in leaves. In contrast, under acidic Pi deficiency, membrane lipid remodeling in roots was partially repressed at a lower external pH. Nevertheless, phenotypic comparison between wild type and the double mutant of MGD2/3, which are responsible for DGDG accumulation during Pi starvation, indicated that the complete absence of lipid remodeling in roots resulted in a loss of tolerance to Pi deficiency rather specifically under acidic conditions. This result suggested important physiological roles of galactolipid-enriched membranes under acidic Pi deficiency.

Changes of aliphatic C-H bonds in cyanobacteria during experimental thermal maturation in the presence or absence of silica as evaluated by FTIR microspectroscopy.

Aliphatic C-H bonds are one of the major organic signatures detected in Proterozoic organic microfossils, and their origin is a topic of interest. To investigate the influence of the presence of silica on the thermal alteration of aliphatic C-H bonds in prokaryotic cells during diagenesis, cyanobacteria Synechocystis sp. PCC6803 were heated at temperatures of 250-450°C. Changes in the infrared (IR) signals were monitored by micro-Fourier transform infrared (FTIR) spectroscopy. Micro-FTIR shows that absorbances at 2,925 cm-1 band (aliphatic CH2 ) and 2,960 cm-1 band (aliphatic CH3 ) decrease during heating, indicating loss of the C-H bonds, which was delayed by the presence of silica. A theoretical approach using solid-state kinetics indicates that the most probable process for the aliphatic C-H decrease is three-dimensional diffusion of alteration products under both non-embedded and silica-embedded conditions. The extrapolation of the experimental results obtained at 250-450°C to lower temperatures implies that the rate constant for CH3 (kCH3 ) is similar to or lower than that for CH2 (kCH2 ; i.e., CH3 decreases at a similar rate or more slowly than CH2 ). The peak height ratio of 2,960 cm-1 band (CH3 )/2,925 cm-1 band (CH2 ; R3/2 values) either increased or remained constant during the heating. These results reveal that the presence of silica does affect the decreasing rate of the aliphatic C-H bonds in cyanobacteria during thermal maturation, but that it does not significantly decrease the R3/2 values. Meanwhile, studies of microfossils suggest that the R3/2 values of Proterozoic prokaryotic fossils from the Bitter Springs Group and Gunflint Formation have decreased during fossilization, which is inconsistent with the prediction from our experimental results that R3/2 values did not decrease after silicification. Some process other than thermal degradation, possibly preservation of specific classes of biomolecules with low R3/2 values, might have occurred during fossilization.

Betaine Lipid Is Crucial for Adapting to Low Temperature and Phosphate Deficiency in Nannochloropsis.

Diacylglyceryl-N,N,N-trimethylhomo-Ser (DGTS) is a nonphosphorous, polar glycerolipid that is regarded as analogous to the phosphatidylcholine in bacteria, fungi, algae, and basal land plants. In some species of algae, including the stramenopile microalga Nannochloropsis oceanica, DGTS contains an abundance of eicosapentaenoic acid (EPA), which is relatively scarce in phosphatidylcholine, implying that DGTS has a unique physiological role. In this study, we addressed the role of DGTS in N. oceanica We identified two DGTS biosynthetic enzymes that have distinct domain configurations compared to previously identified DGTS synthases. Mutants lacking DGTS showed growth retardation under phosphate starvation, demonstrating a pivotal role for DGTS in the adaptation to this condition. Under normal conditions, DGTS deficiency led to an increase in the relative amount of monogalactosyldiacylglycerol, a major plastid membrane lipid with high EPA content, whereas excessive production of DGTS induced by gene overexpression led to a decrease in monogalactosyldiacylglycerol. Meanwhile, lipid analysis of partial phospholipid-deficient mutants revealed a role for phosphatidylcholine and phosphatidylethanolamine in EPA biosynthesis. These results suggest that DGTS and monogalactosyldiacylglycerol may constitute the two major pools of EPA in extraplastidic and plastidic membranes, partially competing to acquire EPA or its precursors derived from phospholipids. The mutant lacking DGTS also displayed impaired growth and a lower proportion of EPA in extraplastidic compartments at low temperatures. Our results indicate that DGTS is involved in the adaptation to low temperatures through a mechanism that is distinct from the DGTS-dependent adaptation to phosphate starvation in N. oceanica.

Rice leaf hydrophobicity and gas films are conferred by a wax synthesis gene (LGF1) and contribute to flood tolerance.

Floods impede gas (O2 and CO2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas.

Physcomitrella MADS-box genes regulate water supply and sperm movement for fertilization.

MIKC classic (MIKCC)-type MADS-box genes encode transcription factors that function in various developmental processes, including angiosperm floral organ identity. Phylogenetic analyses of the MIKCC-type MADS-box family, including genes from non-flowering plants, suggest that the increased numbers of these genes in flowering plants is related to their functional divergence; however, their precise functions in non-flowering plants and their evolution throughout land plant diversification are unknown. Here, we show that MIKCC-type MADS-box genes in the moss Physcomitrella patens function in two ways to enable fertilization. Analyses of protein localization, deletion mutants and overexpression lines of all six genes indicate that three MIKCC-type MADS-box genes redundantly regulate cell division and growth in the stems for appropriate external water conduction, as well as the formation of sperm with motile flagella. The former function appears to be maintained in the flowering plant lineage, while the latter was lost in accordance with the loss of sperm.

Arabidopsis Phosphatidic Acid Phosphohydrolases Are Essential for Growth under Nitrogen-Depleted Conditions.

The Arabidopsis homologs of mammalian lipin, PAH1 and PAH2, are cytosolic phosphatidic acid phosphohydrolases that are involved in phospholipid biosynthesis and are essential for growth under phosphate starvation. Here, pah1 pah2 double-knockout mutants were found to be hypersensitive to nitrogen (N) starvation, whereas transgenic plants overexpressing PAH1 or PAH2 in the pah1 pah2 mutant background showed a similar growth phenotype as compared with wild type (WT) under N starvation. The chlorophyll content of pah1 pah2 was significantly lower than that of WT, whereas the chlorophyll content and photosynthetic activity of the transgenic plants were significantly higher than those of WT under N-depleted conditions. Membrane glycerolipid composition of the pah1 pah2 mutants showed a significant decrease in the mole percent of chloroplast lipids to other phospholipids, whereas membrane lipid composition did not differ between transgenic plants and WT plants. Pulse-chase labeling experiments using plants grown under N-depleted conditions showed that, in pah1 pah2 plants, the labeling percent of chloroplast lipids such as phosphatidylglycerol and monogalactosyldiacylglycerol in the total glycerolipids was significantly lower than in WT. Moreover, N starvation-induced degradation of chloroplast structure was enhanced in pah1 pah2 mutants, and the membrane structure was recovered by complementation with PAH1. Thus, PAH is involved in maintaining chloroplast membrane structure and is required for growth under N-depleted conditions.

Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence.

Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles.

Tangled evolutionary processes with commonality and diversity in plastidial glycolipid synthesis in photosynthetic organisms.

In photosynthetic organisms, the photosynthetic membrane constitutes a scaffold for light-harvesting complexes and photosynthetic reaction centers. Three kinds of glycolipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, constitute approximately 80-90% of photosynthetic membrane lipids and are well conserved from tiny cyanobacteria to the leaves of huge trees. These glycolipids perform a wide variety of functions beyond biological membrane formation. In particular, the capability of adaptation to harsh environments through regulation of membrane glycolipid composition is essential for healthy growth and development of photosynthetic organisms. The genome analysis and functional genetics of the model seed plant Arabidopsis thaliana have yielded many new findings concerning the biosynthesis, regulation, and functions of glycolipids. Nevertheless, it remains to be clarified how the complex biosynthetic pathways and well-organized functions of glycolipids evolved in early and primitive photosynthetic organisms, such as cyanobacteria, to yield modern photosynthetic organisms like land plants. Recently, genome data for many photosynthetic organisms have been made available as the fruit of the rapid development of sequencing technology. We also have reported the draft genome sequence of the charophyte alga Klebsormidium flaccidum, which is an intermediate organism between green algae and land plants. Here, we performed a comprehensive phylogenic analysis of glycolipid biosynthesis genes in oxygenic photosynthetic organisms including K. flaccidum. Based on the results together with membrane lipid analysis of this alga, we discuss the evolution of glycolipid synthesis in photosynthetic organisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.