Head‐group acylation of monogalactosyldiacylglycerol is a common stress response,and the acyl‐galactose acyl composition varies with the plant species and applied stress

Formation of galactose‐acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap‐freezing. Here, lipidomic analysis using mass spectrometry showed that galactose‐acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl‐galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl‐galactose components in response to the same stress. Additionally, the composition of the acyl‐galactose component of Arabidopsis acMGDG (galactose‐acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub‐lethal freezing treatment, acMGDG contained mainly non‐oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid‐containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose‐acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.     

Lipid changes after leaf wounding in Arabidopsis thaliana: expanded lipidomic data form the basis for lipid co‐occurrence analysis

A direct‐infusion electrospray ionization triple–quadrupole mass spectrometry method with multiple reaction monitoring (MRM) was employed to measure 264 lipid analytes extracted from leaves of Arabidopsis thaliana subjected to mechanical wounding. The method provided precise measurements with an average coefficient of variation of 6.1%. Lipid classes analyzed comprised galactolipids and phospholipids (including monoacyl molecular species, molecular species with oxidized acyl chains, phosphatidic acids (PAs)), tri‐ and tetra‐galactosyldiacylglycerols (TrGDGs and TeGDGs), head‐group‐acylated galactolipids, and head‐group‐acylated phosphatidylglycerol (acPG), sulfoquinovosyldiacylglycerols (SQDGs), sphingolipids, di‐ and tri‐acylglycerols (DAGs and TAGs), and sterol derivatives. Of the 264 lipid analytes, 254 changed significantly in response to wounding. In general, levels of structural lipids decreased, whereas monoacyl molecular species, galactolipids and phosphatidylglycerols (PGs) with oxidized fatty acyl chains, PAs, TrGDGs, TeGDGs, TAGs, head‐group‐acylated galactolipids, acPG, and some sterol derivatives increased, many transiently. The observed changes are consistent with activation of lipid oxidizing, hydrolyzing, glycosylating, and acylating activities in the wounding response. Correlation analysis of the levels of lipid analytes across individual control and treated plants was used to construct a lipid dendrogram and to define clusters and sub‐clusters of lipid analytes, each composed of a group of lipids which occurred in a coordinated manner. Current knowledge of metabolism supports the notion that observed sub‐clusters comprise lipids generated by a common enzyme and/or metabolically downstream of a common enzyme. This work demonstrates that co‐occurrence analysis, based on correlation of lipid levels among plants, is a powerful approach to defining lipids generated in vivo by a common enzymatic pathway.     

The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach

Dehydration leads to different physiological and biochemical responses in plants. We analysed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation‐tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant) and two desiccation‐sensitive species, Lindernia subracemosa and Arabidopsis thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolysed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x‐DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x‐DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation‐sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer‐ to non‐bilayer‐forming lipids, thus contributing to protein and membrane stabilization.     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

The seeds of many nondomesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered seeds remains low and is often hampered by their inefficient exclusion from phospholipids. Recent studies have established the feasibility of increasing triacylglycerol content in plant leaves, which provides a novel approach for increasing energy density of biomass crops. Here, we determined whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids. Eleostearic acid (ESA) is a conjugated fatty acid produced in seeds of the tung tree (Vernicia fordii) and has both industrial and nutritional end‐uses. Arabidopsis thaliana lines with elevated leaf oil were first generated by transforming wild‐type, cgi‐58 or pxa1 mutants (the latter two of which contain mutations disrupting fatty acid breakdown) with the diacylglycerol acyltransferases (DGAT1 or DGAT2) and/or oleosin genes from tung. High‐leaf‐oil plant lines were then transformed with tung FADX, which encodes the fatty acid desaturase/conjugase responsible for ESA synthesis. Analysis of lipids in leaves revealed that ESA was efficiently excluded from phospholipids, and co‐expression of tung FADX and DGAT2 promoted a synergistic increase in leaf oil content and ESA accumulation. Taken together, these results provide a new approach for increasing leaf oil content that is coupled with accumulation of unusual fatty acids. Implications for production of biofuels, bioproducts, and plant–pest interactions are discussed.     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Fatty-acid composition of polar lipids in fruit and leaf chloroplasts of “16:3”- and “18:3”-plant species

The fatty-acid composition of polar lipids from fruit and leaf chloroplasts was compared in five Solanaceous and two cucurbit species. The acylated fatty acids in monogalactosyl diglycerides (MGDG) from leaf chloroplasts of all five Solanaceous species included substantial amounts of ^7,10,13-hexadecatrienoic acid (16:3). In contrast, the MGDG from fruit chloroplasts of the Solanaceae contained very little of this plastid-specific polyunsaturate, and instead included a proportionately greater percentage of linoleic acid (18:2). In MGDG from leaf chloroplasts of two cucurbits, -linolenic acid (18:3) constituted 94–95% of the acylated fatty acids. Fruit-chloroplast galactolipids of the cucurbits had a greater abundance of 18:2, and hence a higher 18:2/18:3 ratio, than found in the corresponding leaf lipids. Among the phosphoglycerides, the unusual fatty acid 3-trans-hexadecenoate (trans-16:1) constituted from 15 to 24% of the acylated fatty acids in phosphatidyl glycerol (PG) from leaf chloroplasts (all species). In sharp contrast, trans-16:1 was virtually absent in PG from fruit chloroplasts of both Solanaceous and cucurbit species, and was replaced by a proportionate increase in the content of palmitate (16:0). The observed differences in the polar lipid fatty-acid composition of fruit and leaf chloroplasts are discussed in terms of the relative activity of several intrachloroplastic enzymes involved in lipid synthesis and fatty-acyl desaturation.Abbreviations MGDG monogalactosyldiglyceride - DGDG digalactosyl diglyceride - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PG phosphatidyl glycerol     

Temperature modulation and the presence of C20 fatty acids in mono‐ and digalactosyldiacylglycerol of Euglena gracilis and Lepocinclis acus: A modern interpretation of euglenid galactolipids using positive‐ion electrospray ionization/mass spectrometry

Mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) are important galactolipids that comprise photosynthetic membranes in almost all photosynthetic organisms. Intact forms of MGDG and DGDG of Euglena gracilis and Lepocinclis acus, two example euglenids with secondary plastids of green algal origin, were elucidated with fatty acid regiochemistry via positive‐ion electrospray ionization/mass spectrometry at two growth temperatures. At 20°C, E. gracilis and L. acus produced predominantly 18:3/16:4 (sn‐1/sn‐2) MGDG, whereas at 30°C this was supplanted by 18:2/16:2 MGDG. At both temperatures were also observed a variety of other MGDG and DGDG forms, including C₂₀ fatty acid‐containing forms not expected in a green algal‐derived plastid. In addition to providing structural details of MGDG and DGDG not available in past studies, these results suggest a previously unknown relationship between these two organisms and the red algae. This study also illustrates that temperature modulation of galactolipids occurs via modification of unsaturation of both the sn‐1 and sn‐2 fatty acids; this is fundamentally different from previously published studies from our laboratory on other algal classes.     

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

Two combinatorial patterns of telomere histone marks in plants with canonical and non‐canonical telomere repeats

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non‐canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate‐type telomere repeat TTAGGG or Allium genus‐specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non‐canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR‐dCas9‐eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C‐3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis‐like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco‐like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere‐associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.     

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.     

Oxo-phytodienoic acid (OPDA) is formed on fatty acids esterified to galactolipids after tissue disruption in Arabidopsis thaliana

Biotic and abiotic stress induces the formation of galactolipids esterified with the phytohormones 12-oxo-phytodienoic acid (OPDA) and dinor-oxo-phytodienoic acid (dnOPDA) in Arabidopsis thaliana. The biosynthetic pathways of free (dn)OPDA is well described, but it is unclear how they are incorporated into galactolipids. We herein show that (dn)OPDA containing lipids are formed rapidly after disruption of cellular integrity in leaf tissue. Five minutes after freeze-thawing, 60-70% of the trienoic acids esterified to chloroplast galactolipids are converted to (dn)OPDA. Stable isotope labeling with (18)O-water provides strong evidence for that the fatty acids remain attached to galactolipids during the enzymatic conversion to (dn)OPDA.     

Traumatin- and Dinortraumatin-containing Galactolipids in Arabidopsis: THEIR FORMATION IN TISSUE-DISRUPTED LEAVES AS COUNTERPARTS OF GREEN LEAF VOLATILES*

Green leaf volatiles (GLVs) consisting of six-carbon aldehydes, alcohols, and their esters, are biosynthesized through the action of fatty acid hydroperoxide lyase (HPL), which uses fatty acid hydroperoxides as substrates. GLVs form immediately after disruption of plant leaf tissues by herbivore attacks and mechanical wounding and play a role in defense against attackers that attempt to invade through the wounds. The fates and the physiological significance of the counterparts of the HPL reaction, the 12/10-carbon oxoacids that are formed from 18/16-carbon fatty acid 13-/11-hydroperoxides, respectively, are largely unknown. In this study, we detected monogalactosyl diacylglycerols (MGDGs) containing the 12/10-carbon HPL products in disrupted leaf tissues of Arabidopsis, cabbage, tobacco, tomato, and common bean. They were identified as an MGDG containing 12-oxo-9-hydroxy-(E)-10-dodecenoic acid and 10-oxo-7-hydroxy-(E)-8-decenoic acid and an MGDG containing two 12-oxo-9-hydroxy-(E)-10-dodecenoic acids as their acyl groups. Analyses of Arabidopsis mutants lacking HPL indicated that these MGDGs were formed enzymatically through an active HPL reaction. Thus, our results suggested that in disrupted leaf tissues, MGDG-hydroperoxides were cleaved by HPL to form volatile six-carbon aldehydes and non-volatile 12/10-carbon aldehyde-containing galactolipids. Based on these results, we propose a novel oxylipin pathway that does not require the lipase reaction to form GLVs.     

Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

Root colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H₂O₂ and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.     

Wheat leaf lipids during heat stress: I. High day and night temperatures result in major lipid alterations

Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate‐resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization‐tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat‐tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3‐containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd‐numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0‐acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0‐ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT.     

Metabolism of Unsaturated Monogalactosyldiacylglycerol Molecular Species in Arabidopsis thaliana Reveals Different Sites and Substrates for Linolenic Acid Synthesis

Synthesis of unsaturated monogalactosyldiacylglycerol (MGDG) was examined in a mutant of Arabidopsis thaliana (L.) Heynh. containing reduced levels of hexadecatrienoic (16:3) and linolenic (18:3) acids in leaf lipids. Molecular species composition and labeling kinetics following the incorporation of exogenous [¹⁴C]fatty acids suggest that at least two pathways and multiple substrates are involved in desaturation of linoleic acid (18:2) to 18:3 for production of unsaturated galactolipids. A reduction in 18:3/16:3 MGDG and an increase in 18:2/16:2 MGDG, together with labeling kinetics of these molecular species following the incorporation of exogenous [¹⁴C]12:0 fatty acids, suggests that a chloroplastic pathway for production of 18:3 at the sn-1 position of MGDG utilizes 18:2/16:2 MGDG as a substrate. This chloroplastic (prokaryotic) pathway is deficient in the mutant. When exogenous [¹⁴C]18:1 was supplied, a eukaryotic (cytoplasmic) pathway involving the desaturation of 18:2 to 18:3 on phosphatidylcholine serves as the source of 18:3 for the sn-2 position of MGDG. This eucaryotic pathway predominates in the mutant.