Wounding Triggers Wax Biosynthesis in Arabidopsis Leaves in an Abscisic Acid and Jasmonoyl-Isoleucine Dependent Manner.

Wounding caused by insects or abiotic factors such as wind and hail can cause severe stress for plants. Intrigued by the observation that wounding induces expression of genes involved in surface wax synthesis in a jasmonoyl-isoleucine (JA-Ile)-independent manner, the role of wax biosynthesis and respective genes upon wounding was investigated. Wax, a lipid-based barrier, protects plants both from environmental threats as well as from an uncontrolled loss of water. Its biosynthesis is described to be regulated by abscisic acid (ABA), whereas the main wound-signal is the hormone JA-Ile. We show in this study, that genes coding for enzymes of surface wax synthesis are induced upon wounding in Arabidopsis thaliana leaves in a JA-Ile-independent but ABA-dependent manner. Furthermore, the ABA-dependent transcription factor MYB96 is a key regulator of wax biosynthesis upon wounding. On the metabolite level, wound-induced wax accumulation is strongly reduced in JA-Ile-deficient plants, but this induction is only slightly decreased in ABA-reduced plants. To further analyze the ABA-dependent wound response, we conducted wounding experiments in high humidity. They show that high humidity prevents the wound-induced wax accumulation in A. thaliana leaves. Together the data presented in this study show that wound-induced wax accumulation is JA-Ile-dependent on the metabolite level, but the expression of genes coding for enzymes of wax synthesis is regulated by ABA.

Overexpression of the vacuolar sugar importer BvTST1 from sugar beet in Camelina improves seed properties and leads to altered root characteristics.

Overexpression of the vacuolar sugar transporter TST1 in Arabidopsis leads to higher seed lipid levels and higher total seed yield per plant. However, effects on fruit biomass have not been observed in crop plants like melon, strawberry, cotton, apple, or tomato with increased tonoplast sugar transporter (TST) activity. Thus, it was unclear whether overexpression of TST in selected crops might lead to increased fruit yield, as observed in Arabidopsis. Here, we report that constitutive overexpression of TST1 from sugar beet in the important crop species Camelina sativa (false flax) resembles the seed characteristics observed for Arabidopsis upon increased TST activity. These effects go along with a stimulation of sugar export from source leaves and not only provoke optimised seed properties like higher lipid levels and increased overall seed yield per plant, but also modify the root architecture of BvTST1 overexpressing Camelina lines. Such mutants grew longer primary roots and showed an increased number of lateral roots, especially when developed under conditions of limited water supply. These changes in root properties result in a stabilisation of total seed yield under drought conditions. In summary, we demonstrate that increased vacuolar TST activity may lead to optimised yield of an oil-seed crop species with high levels of healthy ω3 fatty acids in storage lipids. Moreover, since BvTST1 overexpressing Camelina mutants, in addition, exhibit optimised yield under limited water availability, we might devise a strategy to create crops with improved tolerance against drought, representing one of the most challenging environmental cues today and in future.

The glycosyltransferase UGT76E1 significantly contributes to 12-O-glucopyranosyl-jasmonic acid formation in wounded Arabidopsis thaliana leaves.

Jasmonoyl-isoleucine (JA-Ile) is a phytohormone that orchestrates plant defenses in response to wounding, feeding insects, or necrotrophic pathogens. JA-Ile metabolism has been studied intensively, but its catabolism as a potentially important mechanism for the regulation of JA-Ile-mediated signaling is not well-understood. Especially the enzyme(s) responsible for specifically glycosylating 12-hydroxy-jasmonic acid (12-OH-JA) and thereby producing 12-O-glucopyranosyl-jasmonic acid (12-O-Glc-JA) is still elusive. Here, we used co-expression analyses of available Arabidopsis thaliana transcriptomic data, identifying four UDP-dependent glycosyltransferase (UGT) genes as wound-induced and 12-OH-JA-related, namely, UGT76E1, UGT76E2, UGT76E11, and UGT76E12 We heterologously expressed and purified the corresponding proteins to determine their individual substrate specificities and kinetic parameters. We then used an ex vivo metabolite-fingerprinting approach to investigate these proteins in conditions as close as possible to their natural environment, with an emphasis on greatly extending the range of potential substrates. As expected, we found that UGT76E1 and UGT76E2 are 12-OH-JA-UGTs, with UGT76E1 contributing a major in vivo UGT activity, as deduced from Arabidopsis mutants with abolished or increased UGT gene expression. In contrast, recombinant UGT76E11 acted on an unidentified compound and also glycosylated two other oxylipins, 11-hydroxy-7,9,13-hexadecatrienoic acid (11-HHT) and 13-hydroxy-9,11,15-octadecatrienoic acid (13-HOT), which were also accepted by recombinant UGT76E1, UGT76E2, and UGT76E12 enzymes. UGT76E12 glycosylated 12-OH-JA only to a low extent, but also accepted an artificial hydroxylated fatty acid and low amounts of kaempferol. In conclusion, our findings have elucidated the missing step in the wound-induced synthesis of 12-O-glucopyranosyl-jasmonic acid in A. thaliana.

Natural variation in acyl editing is a determinant of seed storage oil composition.

Seeds exhibit wide variation in the fatty acid composition of their storage oil. However, the genetic basis of this variation is only partially understood. Here we have used a multi-parent advanced generation inter-cross (MAGIC) population to study the genetic control of fatty acid chain length in Arabidopsis thaliana seed oil. We mapped four quantitative trait loci (QTL) for the quantity of the major very long chain fatty acid species 11-eicosenoic acid (20:1), using multiple QTL modelling. Surprisingly, the main-effect QTL does not coincide with FATTY ACID ELONGASE 1 and a parallel genome wide association study suggested that LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE 2 (LPCAT2) is a candidate for this QTL. Regression analysis also suggested that LPCAT2 expression and 20:1 content in seeds of the 19 MAGIC founder accessions are related. LPCAT is a key component of the Lands cycle; an acyl editing pathway that enables acyl-exchange between the acyl-Coenzyme A and phosphatidylcholine precursor pools used for microsomal fatty acid elongation and desaturation, respectively. We Mendelianised the main-effect QTL using biparental chromosome segment substitution lines and carried out complementation tests to show that a single cis-acting polymorphism in the LPCAT2 promoter causes the variation in seed 20:1 content, by altering the LPCAT2 expression level and total LPCAT activity in developing siliques. Our work establishes that oilseed species exhibit natural variation in the enzymic capacity for acyl editing and this contributes to the genetic control of storage oil composition.

Genome Wide Analysis of Fatty Acid Desaturation and Its Response to Temperature.

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5' untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL.

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 Are Responsible for Making Omega-7 Fatty Acids in the Arabidopsis Aleurone.

Omega-7 monounsaturated fatty acids (ω-7s) are specifically enriched in the aleurone of Arabidopsis (Arabidopsis thaliana) seeds. We found significant natural variation in seed ω-7 content and used a Multiparent Advanced Generation Inter-Cross population to fine-map a major quantitative trait loci to a region containing ACYL-ACYL CARRIER PROTEIN DESATURASE1 (AAD1) and AAD3 We found that AAD3 expression is localized to the aleurone where mutants show an approximately 50% reduction in ω-7 content. By contrast, AAD1 is localized to the embryo where mutants show a small reduction in ω-9 content. Enzymatic analysis has previously shown that AAD family members possess both stearoyl- and palmitoyl-ACP Δ(9) desaturase activity, including the predominant isoform SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2. However, aad3 ssi2 aleurone contained the same amount of ω-7s as aad3 Within the AAD family, AAD3 shares the highest degree of sequence similarity with AAD2 and AAD4. Mutant analysis showed that AAD2 also contributes to ω-7 production in the aleurone, and aad3 aad2 exhibits an approximately 85% reduction in ω-7s Mutant analysis also showed that FATTY ACID ELONGASE1 is required for the production of very long chain ω-7s in the aleurone. Together, these data provide genetic evidence that the ω-7 pathway proceeds via Δ(9) desaturation of palmitoyl-ACP followed by elongation of the product. Interestingly, significant variation was also identified in the ω-7 content of Brassica napus aleurone, with the highest level detected being approximately 47% of total fatty acids.

Oil is on the agenda: Lipid turnover in higher plants.

Lipases hydrolyze ester bonds within lipids. This process is called lipolysis. They are key players in lipid turnover and involved in numerous metabolic pathways, many of which are shared between organisms like the mobilization of neutral or storage lipids or lipase-mediated membrane lipid homeostasis. Some reactions though are predominantly present in certain organisms, such as the production of signaling molecules (endocannabinoids) by diacylglycerol (DAG) and monoacylglycerol (MAG) lipases in mammals and plants or the jasmonate production in flowering plants. This review aims at giving an overview of the different functional classes of lipases and respective well-known activities, with a focus on the most recent findings in plant biology for selected classes. Here we will put an emphasis on the physiological role and contribution of lipases to the turnover of neutral lipids found in seed oil and other vegetative tissue as candidates for increasing the economical values of crop plants. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

The Escherichia coli Envelope Stress Sensor CpxA Responds to Changes in Lipid Bilayer Properties.

The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.

Multigene engineering of triacylglycerol metabolism boosts seed oil content in Arabidopsis.

Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased.

bZIP67 regulates the omega-3 fatty acid content of Arabidopsis seed oil by activating fatty acid desaturase3.

Arabidopsis thaliana seed maturation is accompanied by the deposition of storage oil, rich in the essential ω-3 polyunsaturated fatty acid α-linolenic acid (ALA). The synthesis of ALA is highly responsive to the level of fatty acid desaturase3 (FAD3) expression, which is strongly upregulated during embryogenesis. By screening mutants in leafy cotyledon1 (LEC1)-inducible transcription factors using fatty acid profiling, we identified two mutants (lec1-like and bzip67) with a seed lipid phenotype. Both mutants share a substantial reduction in seed ALA content. Using a combination of in vivo and in vitro assays, we show that bZIP67 binds G-boxes in the FAD3 promoter and enhances FAD3 expression but that activation is conditional on bZIP67 association with LEC1-like (L1L) and nuclear factor-YC2 (NF-YC2). Although FUSCA3 and abscisic acid insensitive3 are required for L1L and bZIP67 expression, neither protein is necessary for [bZIP67:L1L:NF-YC2] to activate FAD3. We conclude that a transcriptional complex containing L1L, NF-YC2, and bZIP67 is induced by LEC1 during embryogenesis and specifies high levels of ALA production for storage oil by activating FAD3 expression.

The sugar-dependent1 lipase limits triacylglycerol accumulation in vegetative tissues of Arabidopsis.

There has been considerable interest recently in the prospect of engineering crops to produce triacylglycerol (TAG) in their vegetative tissues as a means to achieve a step change in oil yield. Here, we show that disruption of TAG hydrolysis in the Arabidopsis (Arabidopsis thaliana) lipase mutant sugar-dependent1 (sdp1) leads to a substantial accumulation of TAG in roots and stems but comparatively much lower TAG accumulation in leaves. TAG content in sdp1 roots increases with the age of the plant and can reach more than 1% of dry weight at maturity, a 50-fold increase over the wild type. TAG accumulation in sdp1 roots requires both ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL ACYLTRANSFERASE1 and can also be strongly stimulated by the provision of exogenous sugar. In transgenic plants constitutively coexpressing WRINKLED1 and DGAT1, sdp1 also doubles the accumulation of TAG in roots, stems, and leaves, with levels ranging from 5% to 8% of dry weight. Finally, provision of 3% (w/v) exogenous Suc can further boost root TAG content in these transgenic plants to 17% of dry weight. This level of TAG is similar to seed tissues in many plant species and establishes the efficacy of an engineering strategy to produce oil in vegetative tissues that involves simultaneous manipulation of carbohydrate supply, fatty acid synthesis, TAG synthesis, and also TAG breakdown.

Acyl-lipid metabolism.

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Suppression of the SUGAR-DEPENDENT1 triacylglycerol lipase family during seed development enhances oil yield in oilseed rape (Brassica napus L.).

Increasing the productivity of oilseed crops is an important challenge for plant breeders and biotechnologists. To date, attempts to increase oil production in seeds via metabolic pathway engineering have focused on boosting synthetic capacity. However, in the tissues of many organisms, it is well established that oil levels are determined by both anabolism and catabolism. Indeed, the oil content of rapeseed (Brassica napus L.) has been reported to decline by approximately 10% in the final stage of development, as the seeds desiccate. Here, we show that RNAi suppression of the SUGAR-DEPENDENT1 triacylglycerol lipase gene family during seed development results in up to an 8% gain in oil yield on either a seed, plant or unit area basis in the greenhouse, with very little adverse impact on seed vigour. Suppression of lipolysis could therefore constitute a new method for enhancing oil yield in oilseed crops.

Seed storage oil mobilization is important but not essential for germination or seedling establishment in Arabidopsis.

Triacylglycerol (TAG) is a major storage reserve in many plant seeds. We previously identified a TAG lipase mutant called sugar-dependent1 (sdp1) that is impaired in TAG hydrolysis following Arabidopsis (Arabidopsis thaliana) seed germination (Eastmond, 2006). The aim of this study was to identify additional lipases that account for the residual TAG hydrolysis observed in sdp1. Mutants were isolated in three candidate genes (SDP1-LIKE [SDP1L], ADIPOSE TRIGLYCERIDE LIPASE-LIKE, and COMPARATIVE GENE IDENTIFIER-58-LIKE). Analysis of double, triple, and quadruple mutants showed that SDP1L is responsible for virtually all of the residual TAG hydrolysis present in sdp1 seedlings. Oil body membranes purified from sdp1 sdp1L seedlings were deficient in TAG lipase activity but could still hydrolyze di- and monoacylglycerol. SDP1L is expressed less strongly than SDP1 in seedlings. However, SDP1L could partially rescue TAG breakdown in sdp1 seedlings when expressed under the control of the SDP1 or 35S promoters and in vitro assays showed that both SDP1 and SDP1L can hydrolyze TAG, in preference to diacylglycerol or monoacylglycerol. Seed germination was slowed in sdp1 sdp1L and postgerminative seedling growth was severely retarded. The frequency of seedling establishment was also reduced, but sdp1 sdp1L was not seedling lethal under normal laboratory growth conditions. Our data show that together SDP1 and SDP1L account for at least 95% of the rate of TAG hydrolysis in Arabidopsis seeds, and that this hydrolysis is important but not essential for seed germination or seedling establishment.

Tryptophan residues promote membrane association for a plant lipid glycosyltransferase involved in phosphate stress.

Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

Acyl-lipid metabolism.

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function.

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.

Monogalactosyldiacylglycerol deficiency in Arabidopsis affects pigment composition in the prolamellar body and impairs thylakoid membrane energization and photoprotection in leaves.

Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of chloroplast membranes and has been proposed to act directly in several important plastidic processes, particularly during photosynthesis. In this study, the effect of MGDG deficiency, as observed in the monogalactosyldiacylglycerol synthase1-1 (mgd1-1) mutant, on chloroplast protein targeting, phototransformation of pigments, and photosynthetic light reactions was analyzed. The targeting of plastid proteins into or across the envelope, or into the thylakoid membrane, was not different from wild-type in the mgd1 mutant, suggesting that the residual amount of MGDG in mgd1 was sufficient to maintain functional targeting mechanisms. In dark-grown plants, the ratio of bound protochlorophyllide (Pchlide, F656) to free Pchlide (F631) was increased in mgd1 compared to the wild type. Increased levels of the photoconvertible pigment-protein complex (F656), which is photoprotective and suppresses photooxidative damage caused by an excess of free Pchlide, may be an adaptive response to the mgd1 mutation. Leaves of mgd1 suffered from a massively impaired capacity for thermal dissipation of excess light due to an inefficient operation of the xanthophyll cycle; the mutant contained less zeaxanthin and more violaxanthin than wild type after 60 min of high-light exposure and suffered from increased photosystem II photoinhibition. This is attributable to an increased conductivity of the thylakoid membrane at high light intensities, so that the proton motive force is reduced and the thylakoid lumen is less acidic than in wild type. Thus, the pH-dependent activation of the violaxanthin de-epoxidase and of the PsbS protein is impaired.

Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site.

The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.