Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.     

AGPAT6 is a novel microsomal glycerol-3-phosphate acyltransferase

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [(13)C(7)]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.     

Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6-/- mice

Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6(-/-)) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [(14)C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that "Agpat6(-/-) mice" are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4.     

Introduction of the cDNA for shape Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice

The chilling sensitivity of several plant species is closely correlated with the levels of unsaturation of fatty acids in the phosphatidylglycerol (PG) of chloroplast membranes. Plants with a high proportion of unsaturated fatty acids, such as Arabidopsis thaliana, are resistant to chilling, whereas species like squash with only a low proportion are rather sensitive to chilling. The glycerol-3-phosphate O-acyltransferase (GPAT) enzyme of chloroplasts plays an important role in determining the levels of PG fatty acid desaturation.A cDNA for oleate-selective GPAT of Arabidopsis under the control of a maize Ubiquitin promoter was introduced into rice (Oryza sativa L.) using the Agrobacterium-mediated gene transfer method. The levels of unsaturated fatty acids in the phosphatidylglycerol of transformed rice leaves were found to be 28% higher than that of untransformed controls. The net photosynthetic rate of leaves of transformed rice plants was 20% higher than that of the wild type at 17°C. Thus, introduction of cDNA for the Arabidopsis GPAT causes greater unsaturation of fatty acids and confers chilling tolerance of photosynthesis on rice.     

Effect of growth temperature on cellular fatty acids in sulphate-reducing bacteria

The effect of growth temperature on the cellular fatty acid composition of sulphate-reducing bacteria (SRB) was studied in 12 species belonging to eight genera including psychrophiles and mesophiles. Most of these species were of marine origin. The investigated SRB with the exception of four Desulfobacter species exhibited only a minor increase in the proportion of cis-unsaturated fatty acids (by < or = 5% per 10 degrees C) when the growth temperature was decreased; psychrophiles maintained their typically high content of cis-unsaturated fatty acids (around 75% of total fatty acids) nearly constant. The four Desulfobacter species, however, increased the proportion of cis-unsaturated among total fatty acids significantly (by > or =14% per 10 degrees C; measured in late growth phase) with decreasing growth temperature. The ratio between unsaturated and saturated fatty acids in Desulfobacter species changed not only with the growth temperature, but also with the growth state in batch cultures at constant temperature. Changes of cellular fatty acids were studied in detail with D. hydrogenophilus, the most psychrotolerant (growth range 0-35 degrees C) among the mesophilic SRB examined. Desulfobacter hydrogenophilus also formed cis-9,10-methylenehexadecanoic acid (a cyclopropane fatty acid) and 10-methylhexadecanoic acid. At low growth temperature (12 degrees C), the relative amount of these fatty acids was at least threefold lower; this questions the usefulness of 10-methylhexadecanoic acid as a reliable biomarker of Desulfobacter in cold sediments.     

Cis-unsaturated fatty acids induce both lipogenesis and calcium binding in adipocytes

The addition of 0.4-3 mM of cis-unsaturated fatty acids such as oleic acid (18:1) or linoleic acid (18:2) to intact rat adipocytes stimulated lipogenesis at 37 degrees C. Saturated or trans-unsaturated fatty acids were ineffective. Fluorescence photobleaching recovery studies performed under similar conditions indicated that the cis-unsaturated fatty acids do not alter lateral mobility of either a lipid probe or a general protein marker in the plasma membrane. A high concentration (7 mM) of Ca2+, which by itself has some stimulatory effect on lipogenesis, significantly potentiated the effect of oleic acid on this insulin-like activity. Measurement of 45Ca2+ binding by fat cells has indicated that cis-unsaturated (but not saturated) fatty acids increased 12- to 20-fold the amount of Ca2+ associated with the cells. The dependence of this effect on the fatty acid concentration correlates well with the effect of the fatty acid on the induction of lipogenesis. Our results suggest that cis-unsaturated fatty acids affect membrane organization in a manner which induces a significant increase in membrane associated or intracellular Ca2+. This increase may be responsible for inducing exocytotic-like processes which facilitate translocation of glucose transport activity from storage sites to the plasma membrane and thus produce an insulin-like effect.     

In vivo substrates and the contribution of the common phospholipase D, PLDalpha, to wound-induced metabolism of lipids in Arabidopsis

The common plant phospholipase D (PLD), PLDalpha, has been proposed to be involved in wound-induced production of jasmonic acid. To better understand the role(s) of PLDalpha in the wound response, detailed lipid analysis was carried out to determine the in vivo substrates and the contribution of PLDalpha in wound-induced lipid metabolism in Arabidopsis thaliana. Mechanical wounding of Arabidopsis leaves resulted in significantly less hydrolysis of phosphatidylcholine (PC) in PLDalpha-deficient than in wild-type plants. Hydrolysis of phosphatidylethanolamine, phosphatidylglycerol (PG), and phosphatidylinositol within 30 min of wounding was not significantly different in PLDalpha-deficient and wild-type leaves. Phosphatidic acid (PA) levels increased rapidly in wild-type and, to a lesser extent, in PLDalpha-deficient plants. The acyl composition of the PA generated by wounding suggests that the major in vivo substrate of PLD in wild-type leaves was PC, and that PG hydrolysis accounted for 10-15% of the wound-induced PA in wild-type leaves. Comparison of the acyl compositions of the wound-induced PA of wild-type and PLDalpha-deficient leaves indicated that PLDalpha hydrolyzed PG more readily than other PLD isoforms did. Wounding produced substantial increases in free linoleic and linolenic acids in wild-type plants, whereas PLDalpha-deficient plants showed only a slight increase in linoleic acid and no significant increase in linolenic acid. These results demonstrate that PLDalpha and at least one other PLD isoform, as well as other hydrolytic enzymes, are active in mechanically wounded Arabidopsis leaves, and PLDalpha is involved in wound-induced metabolism of polyunsaturated fatty acids.     

Interactions of Pseudomonas syringae pv. glycinea with host and nonhost plants in relation to temperature and phytotoxin synthesis

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C. CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.     

Enzymatic activities of the human AGPAT isoform 3 and isoform 5: localization of AGPAT5 to mitochondria

The enzyme 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) converts lysophosphatidic acid (LPA) to phosphatidic acid (PA). In this study, we show enzymatic properties, tissue distribution, and subcellular localization of human AGPAT3 and AGPAT5. In cells overexpressing these isoforms, the proteins were detected in the nuclear envelope and the endoplasmic reticulum. AGPAT5-GFP fusion protein was localized in the mitochondria of both Chinese hamster ovary and human epithelial cervical cancer cells. Using lysates of AD293 cells infected with AGPAT3 and AGPAT5 recombinant adenovirus, we show that AGPAT3 and AGPAT5 proteins have AGPAT activity. Both the isoforms have similar apparent V(max) of 6.35 and 2.42 nmol/min/mg protein, respectively, for similar LPA. The difference between the two isoforms is in their use of additional lysophospholipids. AGPAT3 shows significant esterification of lysophosphatidylinositol (LPI) in the presence of C20:4 fatty acid, whereas AGPAT5 demonstrates significant acyltransferase activity toward lysophosphatidylethanolamine (LPE) in the presence of C18:1 fatty acid. The AGPAT3 mRNA is ubiquitously expressed in human tissues with several-fold differences in the expression pattern compared with the closely related AGPAT4. In summary, we show that in the presence of different fatty acids, AGPAT3 and AGPAT5 prefer different lysophospholipids as acyl acceptors. More importantly, localization of overexpressed AGPAT5 (this study) as well as GPAT1 and 2 (previous studies) in mitochondria supports the idea that the mitochondria might be capable of synthesizing some of their own glycerophospholipids.     

Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic Acid into triacylglycerols

Background

De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid.

Methods and Results

Incubation of GPAT2-transfected CHO-K1 cells with [1-¹⁴C]arachidonate for 3 h increased incorporation of [¹⁴C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2''s role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein.

Conclusions

These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.     

Biosynthesis of the unique trans-delta 3-hexadecenoic acid component of chloroplast phosphatidylglycerol: evidence concerning its site and mechanism of formation

As in most higher plants, chloroplast membranes of the green alga Dunaliella salina contain phosphatidylglycerol (PG) that is rich in trans-delta 3-hexadecenoic acid (16:1t), a fatty acid found nowhere else in the cell. After labeling D. salina with exogenous [3H]myristic acid [( 3H]14:0), the cis-unsaturated fatty acids of monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol as well as PG had higher specific radioactivities in chloroplast envelopes than in thylakoids. In contrast, 16:1t was very slow to become radioactive, and its specific radioactivity was several times higher in isolated thylakoids than in envelopes after brief (3-20 min) labeling with [3H]14:0. Analysis of individual PG molecular species revealed that the fatty acid paired with 16:1t was also labeled slowly. Thus linoleate (18:2) released from a 16:1t-containing PG had a 350-fold (at 3 min) to 20-fold (at 60 min) lower specific radioactivity than did 18:2 from a palmitate (16:0)-containing PG. The findings suggest that the substrates for trans-desaturation are 16:0-containing PG molecular species which are readily labeled from [3H]14:0 in the envelope but are diluted by the large pool of thylakoid PG before penetrating to the desaturation site. By examining the labeling patterns of individual PG molecular species classes, it was concluded that D. salina 16:1t is formed from 16:0 linked to 18:2/16:0 PG and 18:3/16:0 PG by a trans-desaturase located within the inner recesses of the thylakoid compartment.     

A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed.     

Functional characterization of human 1-acylglycerol-3-phosphate acyltransferase isoform 8: cloning, tissue distribution, gene structure, and enzymatic activity

Glycerophospholipids and triglycerides are synthesized de novo by cells through an evolutionary conserved process involving serial acylations of phosphorylated glycerol. Various isoforms of the enzyme, 1-acylglycerol-3-phosphate acyltransferase (AGPAT), acylate lysophosphatidic acid at the sn-2 position to produce phosphatidic acid. We cloned a cDNA predicted to be AGPAT isoform and designated it AGPAT8. Human and mouse AGPAT8 proteins are 89% homologous, and their gene structure is also highly conserved. AGPAT8 is most closely related to AGPAT5, and its cDNA is expressed most in the heart, while AGPAT5 is expressed more in the prostate and testis. In cell lysates, AGPAT8 shows moderate acyltransferase activity with [(3)H]oleoyl-CoA but lacks acyl-CoA:lysocardiolipin acyltransferase activity. In whole cells upon incubation with [(14)C]linoleic acid, most of the radioactivity was recovered in phosphatidyl ethanolamine, phosphatidyl choline and phosphatidic acid fraction. Of the two well conserved acyltransferase motifs, NHX(4)D is present in AGPAT8, whereas arginine in the EGTR motif is substituted by aspartate. However, mutation of EGTD to EGTR did not increase enzymatic activity significantly. Based on the X-ray crystallographic structure of a related acyltransferase, squash gpat, a model is proposed in which a hydrophobic pocket in AGPAT8 accommodates fatty acyl chains of both substrates in an orientation where the NHX(4)D motif participates in catalysis.     

Studies on Composition of Molecular Species of Phosphatidylglycerol in Relation to Cold-resistance of Poplar

The composition of molecular species and the positional distribution in fatty acids of phosphatidylglycerol (PG) isolated from poplar ( Populus deltoides cv. Lux 1-69/55 and Poeuramericarla cv.I- 45/51 ) leaves were analyzed by high-performance liquid chromatography (HPLC), enzym hydrolysis and gas phase chromatography (C,C), and the different cold-resistant poplars were compared with respect to the compositions of molecular species of PG isolated from their leaves. The results showed that the fatty acid compositions ( sn- 1/sn-2) of the major molecular species in PCs from poplar leaves were as follows: 18:3/18:2(18:2/18:3), 18:3/16: 1(3t); 18:3/16:0; 18:2/ 16:1 (3t); 16:0/18:2,18:2/16:0; 18: 1/16: l(3t); 16:0/16: l(3t); 18: 1/18: 1,16:0/18: 1( 18: 1/16:0); 16:0/16:0o The positional distribution of fatty acids in lPG from poplar leaves was found that 16:1(30 was exclusively occupied the sn-2 position, whereas 16:0 was present in both the sn1 position and the sn-2 position. The C18 acids were principally localized at the sn-2 position. The relative contents of the unsaturated molecular species of leaf PCs were more than 70% in both coldresistant poplar and cold-sensitive poplar. The ratio of the unsaturated/saturated molecular species of PG isolated from the cold-resistant Ⅰ -45 poplar was 3.10, which was higher than that of the PG from the cold-sensitive cottonwood, which was 2.38. The sum of the relative contents of the disaturated molecular species of the PG from poplar leaves was closely associated with the cold-resistance of plants. The ∑[ 16:0/16:0+ 16:0/16: l(3t) ] of the PG from cottonwood was higher than that of the PG from cold-resistant I -45 poplar. The differences in the compositions of molecular species and the phase transition temperatures of PCs between cold-resistant and cold-sensitive plants were discussed in terms of the pathways and the activities of selective acyhransferases involved in the PG biosynthesis in chloroplast.     

In vivo substrates and the contribution of the common phospholipase D,PLDα, to wound-induced metabolism of lipids in Arabidopsis

The common plant phospholipase D (PLD), PLDα, has been proposed to be involved in wound-induced production of jasmonic acid. To better understand the role(s) of PLDα in the wound response, detailed lipid analysis was carried out to determine the in vivo substrates and the contribution of PLDα in wound-induced lipid metabolism in Arabidopsis thaliana. Mechanical wounding of Arabidopsis leaves resulted in significantly less hydrolysis of phosphatidylcholine (PC) in PLDα-deficient than in wild-type plants. Hydrolysis of phosphatidylethanolamine, phosphatidylglycerol (PG), and phosphatidylinositol within 30 min of wounding was not significantly different in PLDα-deficient and wild-type leaves. Phosphatidic acid (PA) levels increased rapidly in wild-type and, to a lesser extent, in PLDα-deficient plants. The acyl composition of the PA generated by wounding suggests that the major in vivo substrate of PLD in wild-type leaves was PC, and that PG hydrolysis accounted for 10–15% of the wound-induced PA in wild-type leaves. Comparison of the acyl compositions of the wound-induced PA of wild-type and PLDα-deficient leaves indicated that PLDα hydrolyzed PG more readily than other PLD isoforms did. Wounding produced substantial increases in free linoleic and linolenic acids in wild-type plants, whereas PLDα-deficient plants showed only a slight increase in linoleic acid and no significant increase in linolenic acid. These results demonstrate that PLDα and at least one other PLD isoform, as well as other hydrolytic enzymes, are active in mechanically wounded Arabidopsis leaves, and PLDα is involved in wound-induced metabolism of polyunsaturated fatty acids.     

The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis

Suberin and cutin are fatty acid- and glycerol-based plant polymers that act as pathogen barriers and function in the control of water and solute transport. However, despite important physiological roles, their biosynthetic pathways, including the acyl transfer reactions, remain hypothetical. We report the characterization of two suberin mutants (gpat5-1 and gpat5-2) of Arabidopsis thaliana GPAT5, encoding a protein with acyl-CoA:glycerol-3-phosphate acyltransferase activity. RT-PCR and beta-glucuronidase-promoter fusion analyses demonstrated GPAT5 expression in seed coat, root, hypocotyl, and anther. The gpat5 plants showed a 50% decrease in aliphatic suberin in young roots and produced seed coats with a severalfold reduction in very long chain dicarboxylic acid and omega-hydroxy fatty acids typical of suberin but no change in the composition or content of membrane or storage glycerolipids or surface waxes. Consistent with their altered suberin, seed coats of gpat5 mutants had a steep increase in permeability to tetrazolium salts compared with wild-type seed coats. Furthermore, the germination rate of gpat5 seeds under high salt was reduced, and gpat5 seedlings had lower tolerance to salt stress. These results provide evidence for a critical role of GPAT5 in polyester biogenesis in seed coats and roots and for the importance of lipid polymer structures in the normal function of these organs.     

Genetic decrease in fatty acid unsaturation of phosphatidylglycerol increased photoinhibition of photosystem I at low temperature in tobacco leaves

Leaves of transgenic tobacco plants with decreased levels of fatty acid unsaturation in phosphatidylglycerol (PG) exhibited a slightly lower level of the steady state oxidation of the photosystem I (PSI) reaction center P700 (P700(+)) than wild-type plants. The PSI photochemistry of wild-type plants was only marginally affected by high light treatments. Surprisingly, all plants of transgenic lines exhibited much higher susceptibility to photoinhibition of PSI than wild-type plants. This was accompanied by a 2.5-fold faster re-reduction rate of P700(+) in the dark, indicating a higher capacity for cyclic electron flow around PSI in high light treated transgenic leaves. This was associated with a much higher intersystem electron pool size suggesting over-reduction of the PQ pool in tobacco transgenic lines with altered PG unsaturation compared to wild-type plants. The physiological role of PG unsaturation in PSI down-regulation and modulation of the capacity of PSI-dependent cyclic electron flows and distribution of excitation light energy in tobacco plants under photoinhibitory conditions at low temperatures is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Effects of low-temperature stress on the metabolism of phosphatidylglycerol molecular species in Dunaliella salina

A detailed analysis was made of individual phosphatidylglycerol (PG) molecular species isolated from microsomes and chloroplasts at various times after labeling Dunaliella salina cells with [14C]palmitic, [14C]oleic, or [14C]lauric acid. The patterns of [14C]fatty acid incorporation were in agreement with PG being formed by the "eucaryotic" type pathway in microsomes and the "procaryotic" type pathway in chloroplasts. In Dunaliella, which lacks a quantitatively significant flux of eucaryotic-type lipids from microsomes into chloroplast glycolipids, indications were found for a more subtle movement of microsomally synthesized PG into the chloroplasts. This transfer was more evident in cells stressed by exposure to 12 degrees C than it was at 30 degrees C, and may afford a mechanism for recruiting key microsomal PG molecular species toward low-temperature acclimation in chloroplasts.     

Increased lipogenesis in isolated hepatocytes from cold-acclimated ducklings

Thermogenic endurance and development of metabolic cold adaptation in birds may critically depend on their ability to synthesize and use fatty acids (FA) as fuel substrates. Hepatic lipogenesis and the capacity to oxidize FA in thermogenic tissues were measured in cold-acclimated (CA) ducklings (Cairina moschata) showing original mechanisms of metabolic cold adaptation in the absence of brown adipose tissue, the specialized thermogenic tissue of rodents. The rate of FA synthesis from [U-(14)C]glucose and from [1-(14)C]acetate, measured in incubated hepatocytes isolated from 5-wk-old thermoneutral (TN; 25 degrees C) or CA (4 degrees C) fed ducklings, was higher than in other species. Hepatic de novo lipogenesis was further increased by cold acclimation with both glucose (+194%) and acetate (+111%) as precursor. Insulin slightly increased (+11-14%) hepatic lipogenesis from both precursors in CA ducklings, whereas glucagon was clearly inhibitory (-29 to -51%). Enhanced de novo lipogenesis was associated with higher (+171%) hepatocyte activity of glucose oxidation and larger capacity (+50 to +100%) of key lipogenic enzymes. The potential for FA oxidation was higher in liver (+61%) and skeletal muscle (+29 to +81%) homogenates from CA than from TN ducklings, suggesting that the higher hepatic lipogenesis may fuel oxidation in thermogenic tissues. Present data underline the high capacity to synthesize lipids from glucose in species like muscovy ducks susceptible to hepatic steatosis. Lipogenic capacity can be further increased in the cold and may represent an important step in the metabolic adaptation to cold of growing ducklings.