Type A and type B monogalactosyldiacylglycerol synthases are spatially and functionally separated in the plastids of higher plants

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. In Arabidopsis, the three MGDG synthases are classified into type A (atMGD1) and type B MGD isoforms (atMGD2 and atMGD3). atMGD1 is an inner envelope membrane-associated protein of chloroplasts and is responsible for the bulk of galactolipid biosynthesis in green tissues. MGD1 function is indispensable for thylakoid membrane biogenesis and embryogenesis. By contrast, type B atMGD2 and atMGD3 are localized in the outer envelopes and have no important role in chloroplast biogenesis or plant development under nutrient-sufficient conditions. These type B MGD genes are, however, strongly induced by phosphate (Pi) starvation and are essential for alternative galactolipid biosynthesis during Pi starvation. MGD1 gene expression is up-regulated by light and cytokinins. By contrast, Pi starvation-dependent expression of atMGD2/3 is suppressed by cytokinins but induced through auxin signaling pathways. These growth factors may control the functional sharing of the inner envelope pathway by atMGD1 and the outer envelope pathway by atMGD2/3 according to the growth environment.     

Critical regulation of galactolipid synthesis controls membrane differentiation and remodeling in distinct plant organs and following environmental changes

The plant galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the most abundant lipids in chloroplast membranes, and they constitute the majority of total membrane lipids in plants. MGDG is synthesized by two types of MGDG synthase, type-A (MGD1) and type-B (MGD2, MGD3). These MGDG synthases have distinct roles in Arabidopsis. In photosynthetic organs, Type A MGD is responsible for the bulk of MGDG synthesis, whereas Type B MGD is expressed in non-photosynthetic organs such as roots and flowers and mainly contributes to DGDG accumulation under phosphate deficiency. Similar to MGDG synthesis, DGDG is synthesized by two synthases, DGD1 and DGD2; DGD1 is responsible for the majority of DGDG synthesis, whereas DGD2 makes its main contribution under phosphate deficiency. These galactolipid synthases are regulated by light, plant hormones, redox state, phosphatidic acid levels, and various stress conditions such as drought and nutrient limitation. Maintaining the appropriate ratio of these two galactolipids in chloroplasts is important for stabilizing thylakoid membranes and maximizing the efficiency of photosynthesis. Here we review progress made in the last decade towards a better understanding of the pathways regulating plant galactolipid biosynthesis.     

Disruption of the two digalactosyldiacylglycerol synthase genes DGD1 and DGD2 in Arabidopsis reveals the existence of an additional enzyme of galactolipid synthesis

Two genes (DGD1 and DGD2) are involved in the synthesis of the chloroplast lipid digalactosyldiacylglycerol (DGDG). The role of DGD2 for galactolipid synthesis was studied by isolating Arabidopsis T-DNA insertional mutant alleles (dgd2-1 and dgd2-2) and generating the double mutant line dgd1 dgd2. Whereas the growth and lipid composition of dgd2 were not affected, only trace amounts of DGDG were found in dgd1 dgd2. The growth and photosynthesis of dgd1 dgd2 were affected more severely compared with those of dgd1, indicating that the residual amount of DGDG in dgd1 is crucial for normal plant development. DGDG synthesis was increased after phosphate deprivation in the wild type, dgd1, and dgd2 but not in dgd1 dgd2. Therefore, DGD1 and DGD2 are involved in DGDG synthesis during phosphate deprivation. DGD2 was localized to the outer side of chloroplast envelope membranes. Like DGD2, heterologously expressed DGD1 uses UDP-galactose for galactosylation. Galactolipid synthesis activity for monogalactosyldiacylglycerol (MGDG), DGDG, and the unusual oligogalactolipids tri- and tetragalactosyldiacylglycerol was detected in isolated chloroplasts of all mutant lines, including dgd1 dgd2. Because dgd1 and dgd2 carry null mutations, an additional, processive galactolipid synthesis activity independent from DGD1 and DGD2 exists in Arabidopsis. This third activity, which is related to the Arabidopsis galactolipid:galactolipid galactosyltransferase, is localized to chloroplast envelope membranes and is capable of synthesizing DGDG from MGDG in the absence of UDP-galactose in vitro, but it does not contribute to net galactolipid synthesis in planta.     

DGD2, an arabidopsis gene encoding a UDP-galactose-dependent digalactosyldiacylglycerol synthase is expressed during growth under phosphate-limiting conditions.

The galactolipid digalactosyldiacylglycerol (DGDG), one of the main chloroplast lipids in higher plants, is believed to be synthesized by the galactolipid:galactolipid galactosyltransferase, which transfers a galactose moiety from one molecule of monogalactosyldiacylglycerol (MGDG) to another. Here, we report that Arabidopsis as well as other plant species contain two genes, DGD1 and DGD2, encoding enzymes with DGDG synthase activity. Using MGDG and UDP-galactose as substrates for in vitro assays with DGD2 we could for the first time measure DGDG synthase activity of a heterologously expressed plant cDNA. UDP-galactose, but not MGDG, serves as the galactose donor for DGDG synthesis catalyzed by DGD2, providing clear evidence for the existence of a UDP-galactose-dependent DGDG synthase in higher plants. In in vitro assays, DGD2 was capable of galactosylating DGDG, resulting in the synthesis of an oligogalactolipid tentatively identified as trigalactosyldiacylglycerol. DGD2 mRNA expression in leaves was very low but was strongly induced during growth under phosphate-limiting conditions. This induction correlates with the previously described increase in DGDG during phosphate deprivation. Therefore, in contrast to DGD1, which is responsible for the synthesis of the bulk of DGDG found in chloroplasts, DGD2 apparently is involved in the synthesis of DGDG under specific growth conditions.     

Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential for chloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposed to participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDG occurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substitute DGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthase genes ( MGD1 , MGD2 and MGD3 ), the type-B MGD2 and MGD3 are upregulated in parallel with DGDG synthase genes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertional mutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drastic reduction in DGDG accumulation, particularly in the root, indicating that MGD3 is the main isoform responsible for DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-induced accumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential for membrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosynthetic performance were also observed in these mutants under Pi-starved conditions. These results demonstrate that Pi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. The widespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodeling mediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.     

Galactolipids rule in seed plants

Chloroplast membranes contain high levels of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). The isolation of the genes involved in the biosynthesis of MGDG and DGDG, and the identification of galactolipid-deficient Arabidopsis mutants has greatly facilitated the analysis of galactolipid biosynthesis and function. Galactolipids are found in X-ray structures of photosynthetic complexes, suggesting a direct role in photosynthesis. Furthermore, galactolipids can substitute for phospholipids, as suggested by increases in the galactolipid:phospholipid ratio after phosphate deprivation. The ratio of MGDG to DGDG is also crucial for the physical phase of thylakoid membranes and might be regulated.     

Interspecies compatibility of NAS1 gene promoters.

Nicotianamine and nicotianamine synthase (NAS) play key roles in iron nutrition in all higher plants. However, the mechanism underlying the regulation of NAS expression differs among plant species. Sequences homologous to iron deficiency-responsive elements (IDEs), i.e., cis-acting elements, are found on the promoters of these genes. We aimed to verify the interspecies compatibility of the Fe-deficiency response of NAS1 genes and understand the universal mechanisms that regulate their expression patterns in higher plants. Therefore, we introduced the graminaceous (Hordeum vulgare L. and Oryza sativa L.) NAS1 promoter::GUS into dicots (Nicotiana tabacum L. and Arabidopsis thaliana L.). Fe deficiency induced HvNAS1 expression in the shoots and roots when introduced into rice. HvNAS1 promoter::GUS and OsNAS1 promoter::GUS induced strong expression of GUS under Fe-deficient conditions in transformed tobacco. In contrast, these promoters only definitely functioned in Arabidopsis transformants. These results suggest that some Fe nutrition-related trans-factors are not compatible between graminaceous plants and Arabidopsis. HvNAS1 promoter::GUS induced GUS activity only in the roots of transformed tobacco under Fe-deficient conditions. On the other hand, OsNAS1 promoter::GUS induced GUS activity in both the roots and shoots of transformed tobacco under conditions of Fe deficiency. In tobacco transformants, the induction of GUS activity was induced earlier in the shoots than roots. These results suggest that the HvNAS1 and OsNAS1 promoters are compatible with Fe-acquisition-related trans-factors in the roots of tobacco and that the OsNAS1 promoter is also compatible with some shoot-specific Fe deficiency-related trans-factors in tobacco.     

The digalactosyldiacylglycerol (DGDG) synthase DGD1 is inserted into the outer envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on direct interaction with monogalactosyldiacylglycerol synthase for DGDG biosynthesis.

Galactolipids make up the bulk of chloroplast lipids. Therefore, the genes involved in the synthesis of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) play a critical role in chloroplast development. In this study, we analyzed the subcellular localization of the Arabidopsis DGDG synthase DGD1, which was recently identified by complementation of the Arabidopsis dgd1 mutant. In vitro import experiments demonstrated that DGD1 was targeted to the chloroplast outer envelope in an ATP-independent manner. DGD1 could not be extracted from the membranes by high salt or alkali, suggesting that it is an integral membrane protein. Uptake experiments with truncated versions of DGD1 indicated that the information for targeting and insertion into the outer envelope resides in the N-terminal half of DGD1, but not in the first 33 amino acids. DGD1 apparently does not contain a cleavable signal peptide. Antibodies to Arabidopsis DGD1 detected a 90-kDa protein localized to the chloroplast envelopes of both pea and Arabidopsis. Transformation of DGD1 constructs into cyanobacteria resulted in the expression of active DGDG synthase and demonstrated that DGDG synthesis depends on MGDG lipid, but does not require direct interaction with the plant MGDG synthase.     

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.     

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

Drought stress and rehydration affect the balance between MGDG and DGDG synthesis in cowpea leaves

Membranes are main targets of drought, and there is growing evidence for the involvement of membrane lipid in plant adaptation to such an environmental stress. Biosynthesis of the galactosylglycerolipids, monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG), which are the main components of chloroplast envelope and thylakoid membranes, could be important for plant tolerance to water deficit and for recovery after rehydration. In this study, galactolipid (GL) biosynthesis in cowpea ( Vigna unguiculata L. Walp) leaves was analysed during drought stress and subsequent rewatering. Comparison of two cowpea cutivars, one drought tolerant and the other drought susceptible submitted to moderate drought stress, revealed patterns associated with water-deficit tolerance: increase in DGDG leaf content, stimulation of DGDG biosynthesis in terms of ¹⁴C-acetate incorporation and messenger accumulation corresponding to four genes coding for GL synthases ( MDG1 , MGD2 , DGD1 and DGD2 ). Similar to phosphate starvation, lack of water enhanced DGDG biosynthesis and it was hypothesized that the drought-induced DGDG accumulated in extrachloroplastic membranes, and thus contributes to plant tolerance to arid environments.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate

The rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a specific expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell files in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young inflorescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These findings are discussed in relation to the role of cytokinins in plant development.     

Membrane lipid alteration during phosphate starvation is regulated by phosphate signaling and auxin/cytokinin cross-talk

During phosphate (Pi) starvation in plants, membrane phospholipid content decreases concomitantly with an increase in non-phosphorus glycolipids. Although several studies have indicated the involvement of phytohormones in various physiological changes upon Pi starvation, the regulation of Pi-starvation induced membrane lipid alteration remains unknown. Previously, we reported the response of type B monogalactosyl diacylglycerol synthase genes (atMGD2 and atMGD3) to Pi starvation, and suggested a role for these genes in galactolipid accumulation during Pi starvation. We now report our investigation of the regulatory mechanism for the response of atMGD2/3 and changes in membrane lipid composition to Pi starvation. Exogenous auxin activated atMGD2/3 expression during Pi starvation, whereas their expression was repressed by cytokinin treatment in the root. Moreover, auxin inhibitors and the axr4 aux1 double mutation in auxin signaling impaired the increase of atMGD2/3 expression during Pi starvation, showing that auxin is required for atMGD2/3 activation. The fact that hormonal effects during Pi starvation were also observed with regard to changes in membrane lipid composition demonstrates that both auxin and cytokinin are indeed involved in the dynamic changes in membrane lipids during Pi starvation. Phosphite is not metabolically available in plants; however, when we supplied phosphite to Pi-starved plants, the Pi-starvation response disappeared with respect to both atMGD2/3 expression and changes in membrane lipids. These results indicate that the observed global change in plant membranes during Pi starvation is not caused by Pi-starvation induced damage in plant cells but rather is strictly regulated by Pi signaling and auxin/cytokinin cross-talk.     

Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves

Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.     

Biosynthesis and desaturation of prokaryotic galactolipids in leaves and isolated chloroplasts from spinach

Mono- and digalactosyldiacylglycerol (MGDG and DGDG) were isolated from the leaves of sixteen 16:3 plants. In all of these plant species, the sn-2 position of MGDG was more enriched in C₁₆ fatty acids than sn-2 of DGDG. The molar ratios of prokaryotic MGDG to prokaryotic DGDG ranged from 4 to 10. This suggests that 16:3 plants synthesize more prokaryotic MGDG than prokaryotic DGDG. In the 16:3 plant Spinacia oleracea L. (spinach), the formation of prokaryotic galactolipids was studied both in vivo and in vitro. In intact spinach leaves as well as in chloroplasts isolated from these leaves, radioactivity from [1-¹⁴C]acetate accumulated 10 times faster in MGDG than in DGDG. After 2 hours of incorporation, most labeled galactolipids from leaves and all labeled galactolipids from isolated chloroplasts were in the prokaryotic configuration. Both in vivo and in vitro, the desaturation of labeled palmitate and oleate to trienoic fatty acids was higher in MGDG than in DGDG. In leaves, palmitate at the sn-2 position was desaturated in MGDG but not in DGDG. In isolated chloroplasts, palmitate at sn-2 similarly was desaturated only in MGDG, but palmitate and oleate at the sn-1 position were desaturated in MGDG as well as in DGDG. Apparently, palmitate desaturase reacts with sn-1 palmitate in either galactolipid, but does not react with the sn-2 fatty acid of DGDG. These results demonstrate that isolated spinach chloroplasts can synthesize and desaturate prokaryotic MGDG and DGDG. The finally accumulating molecular species, MGDG(18:3/16:3) and DGDG(18:3/16:0), are made by the chloroplasts in proportions similar to those found in leaves.