Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and Synthesis of Structurally Defined Fructan Molecules from Sucrose

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.     

A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber

The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers.     

Molecular dissection of variation in carbohydrate metabolism related to water-soluble carbohydrate accumulation in stems of wheat

Water-soluble carbohydrates (WSCs; composed of mainly fructans, sucrose [Suc], glucose [Glc], and fructose) deposited in wheat (Triticum aestivum) stems are important carbon sources for grain filling. Variation in stem WSC concentrations among wheat genotypes is one of the genetic factors influencing grain weight and yield under water-limited environments. Here, we describe the molecular dissection of carbohydrate metabolism in stems, at the WSC accumulation phase, of recombinant inbred Seri/Babax lines of wheat differing in stem WSC concentrations. Affymetrix GeneChip analysis of carbohydrate metabolic enzymes revealed that the mRNA levels of two fructan synthetic enzyme families (Suc:Suc 1-fructosyltransferase and Suc:fructan 6-fructosyltransferase) in the stem were positively correlated with stem WSC and fructan concentrations, whereas the mRNA levels of enzyme families involved in Suc hydrolysis (Suc synthase and soluble acid invertase) were inversely correlated with WSC concentrations. Differential regulation of the mRNA levels of these Suc hydrolytic enzymes in Seri/Babax lines resulted in genotypic differences in these enzyme activities. Down-regulation of Suc synthase and soluble acid invertase in high WSC lines was accompanied by significant decreases in the mRNA levels of enzyme families related to sugar catabolic pathways (fructokinase and mitochondrion pyruvate dehydrogenase complex) and enzyme families involved in diverting UDP-Glc to cell wall synthesis (UDP-Glc 6-dehydrogenase, UDP-glucuronate decarboxylase, and cellulose synthase), resulting in a reduction in cell wall polysaccharide contents (mainly hemicellulose) in the stem of high WSC lines. These data suggest that differential carbon partitioning in the wheat stem is one mechanism that contributes to genotypic variation in WSC accumulation.     

The role of transporters in supplying energy to plant plastids

The energy status of plant cells strongly depends on the energy metabolism in chloroplasts and mitochondria, which are capable of generating ATP either by photosynthetic or oxidative phosphorylation, respectively. Another energy-rich metabolite inside plastids is the glycolytic intermediate phosphoenolpyruvate (PEP). However, chloroplasts and most non-green plastids lack the ability to generate PEP via a complete glycolytic pathway. Hence, PEP import mediated by the plastidic PEP/phosphate translocator or PEP provided by the plastidic enolase are vital for plant growth and development. In contrast to chloroplasts, metabolism in non-green plastids (amyloplasts) of starch-storing tissues strongly depends on both the import of ATP mediated by the plastidic nucleotide transporter NTT and of carbon (glucose 6-phosphate, Glc6P) mediated by the plastidic Glc6P/phosphate translocator (GPT). Both transporters have been shown to co-limit starch biosynthesis in potato plants. In addition, non-photosynthetic plastids as well as chloroplasts during the night rely on the import of energy in the form of ATP via the NTT. During energy starvation such as prolonged darkness, chloroplasts strongly depend on the supply of ATP which can be provided by lipid respiration, a process involving chloroplasts, peroxisomes, and mitochondria and the transport of intermediates, i.e. fatty acids, ATP, citrate, and oxaloacetate across their membranes. The role of transporters involved in the provision of energy-rich metabolites and in pathways supplying plastids with metabolic energy is summarized here.     

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.     

Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase

Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.     

New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking

The presence of sucrose (Suc) in plastids was questioned for several decades. Although it was reported some decades ago, neither Suc transporters nor Suc metabolizing enzymes were demonstrated to be active in those organelles. By biochemical, immunological, molecular and genetic approaches we show that alkaline/neutral invertases (A/N-Invs) are also localized in chloroplasts of spinach and Arabidopsis. A/N-Inv activity and polypeptide content were shown in protein extracts from intact chloroplasts. Moreover, we functionally characterized the Arabidopsis At-A/N-InvE gene coding for a chloroplast-targeted A/N-Inv. The At-A/N-InvE knockout plants displayed a lower total A/N-Inv activity in comparison with wild-type plants. Furthermore, neither A/N-Inv activity nor A/N-Inv polypeptides were detected in protein extracts prepared from chloroplasts of mutant plants. Also, the measurement of carbohydrate content, in leaves harvested either at the end of the day or at the end of the night period, revealed that the knockout plants showed a decrease in starch accumulation but no alteration in Suc levels. These are the first results demonstrating the presence of a functional A/N-Inv inside chloroplasts and its relation with carbon storage in Arabidopsis leaves. Taken together our data and recent reports, we conclude that the participation of A/N-Invs in the carbon flux between the cytosol and the plastids may be a general phenomenon in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis

Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.     

Passive nitrate transport by root plasma membrane vesicles exhibits an acidic optimal pH like the H(+)-ATPase

Previous work has indicated that sugar sensing may be important in the regulation of fructan biosynthesis in grasses. We used primary leaves of barley (Hordeum vulgare cv Baraka) to study the mechanisms involved. Excised leaf blades were supplied in the dark with various carbohydrates. Fructan pool sizes and two key enzymes of fructan biosynthesis, sucrose (Suc):Suc-1-fructosyltransferase (1-SST; EC 2. 4.1.99) and Suc:fructan-6-fructosyltransferase (6-SFT; EC 2.4.1.10) were analyzed. Upon supply of Suc, fructan pool sizes increased markedly. Within 24 h, 1-SST activity was stimulated by a factor of three and 6-SFT-activity by a factor of more than 20, compared with control leaves supplemented with mannitol (Mit). At the same time, the level of mRNA encoding 6-SFT increased conspicuously. These effects were increased in the presence of the invertase inhibitor 2, 5-dideoxy-2,5-imino-D-mannitol. Compared with equimolar solutions of Suc, glucose (Glu) and fructose stimulated 6-SFT activity to a lesser extent. Remarkably, trehalose (Tre; Glc-alpha-1 and 1-alpha-Glc) had stimulatory effects on 6-SFT activity and, to a somewhat lesser extent, on 6-SFT mRNA, even in the presence of validoxylamine A, a potent trehalase inhibitor. Tre by itself, however, in the presence or absence of validoxylamine A, did not stimulate fructan accumulation. Monosaccharides phosphorylated by hexokinase but not or weakly metabolized, such as mannose (Man) or 2-deoxy-Glc, had no stimulatory effects on fructan synthesis. When fructose or Man were supplied together with Tre, fructan and starch biosynthesis were strongly stimulated. Concomitantly, phospho-Man isomerase (EC 5.3.1.8) activity was detected. These results indicate that the regulation of fructan synthesis in barley leaves occurs independently of hexokinase and is probably based on the sensing of Suc, and also that the structurally related disaccharide Tre can replace Suc as a regulatory compound.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers

In the present paper we investigated the effect of the sucrose (Suc) analog palatinose on potato (Solanum tuberosum) tuber metabolism. In freshly cut discs of growing potato tubers, addition of 5 mM palatinose altered the metabolism of exogenously supplied [U-14C]Suc. There was slight inhibition of the rate of 14C-Suc uptake, a 1.5-fold increase in the rate at which 14C-Suc was subsequently metabolized, and a shift in the allocation of the metabolized label in favor of starch synthesis. The sum result of these changes was a 2-fold increase in the absolute rate of starch synthesis. The increased rate of starch synthesis was accompanied by a 3-fold increase in inorganic pyrophosphate, a 2-fold increase in UDP, decreased UTP/UDP, ATP/ADP, and ATP/AMP ratios, and decreased adenylate energy charge, whereas glycolytic and Krebs cycle intermediates were unchanged. In addition, feeding palatinose to potato discs also stimulated the metabolism of exogenous 14C-glucose in favor of starch synthesis. In vitro studies revealed that palatinose is not metabolized by Suc synthases or invertases within potato tuber extracts. Enzyme kinetics revealed different effects of palatinose on Suc synthase and invertase activities, implicating palatinose as an allosteric effector leading to an inhibition of Suc synthase and (surprisingly) to an activation of invertase in vitro. However, measurement of tissue palatinose levels revealed that these were too low to have significant effects on Suc degrading activities in vivo. These results suggest that supplying palatinose to potato tubers represents a novel way to increase starch synthesis.     

THE ULTRASTRUCTURAL MORPHOLOGY AND ONTOGENY OF OAT COLEOPTILE PLASTIDS

Structurally similar proplastids occur in the shoot, scutellum, and root of the oat embryo at the start of germination. These proplastids follow several pathways of differentiation, depending on their location within an organ and on previous exposure to light. During the first 24 hr of germination morphologically similar amyloplasts are formed from the preexisting proplastids in most of the cells of the seedling. After about 24 hr in the light, unique chloroplasts begin to develop in a subepidermal ring of small cortical parenchyma cells in the coleoptile and give the organ a pale green color. At 48 and 72 hr the coleoptile chloroplasts and etioplasts are conspicuously different from the corresponding leaf plastids in morphology and ontogeny but contain typical photosynthetic grana and prolamellar bodies. Study of the ontogeny of plastids in the epidermal and nongreening parenchymal regions of dark grown coleoptiles shows that these plastids undergo significant losses in starch content, and some increase of membranes within the plastid, related to the age of the cell. Light has little effect on the structure of these plastids. It is suggested that the ontogeny of all the plastid types of the oat seedling begins with a common precursor—a relatively simple proplastid that is present at the time of germination. Starch grains showing two distinct types of erosion, apparently enzymatic, were observed in oat coleoptile plastids. In one type (grooved appearance) the starch grains are consistently associated with plastid membranes, while in the other type (irregular, spiny appearance) the starch grains are associated with the plastid stroma only. We suggest that there are two enzyme systems for metabolizing starch in oat plastids—one membrane-bound and the other free in the stroma.     

RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation

Storage of potato tubers at low temperatures leads to the accumulation of glucose and fructose in a process called 'cold sweetening'. The aim of this work was to investigate the role of sucrose-phosphatase (SPP) in potato tuber carbohydrate metabolism at low temperature (4 degrees C). To this end, RNA interference (RNAi) was used to reduce SPP expression in transgenic potato tubers. Analysis of SPP specific small interfering RNAs (siRNAs), SPP protein accumulation and enzyme activity indicated that SPP silencing in transgenic tubers was stable during the cold treatment. Analysis of soluble carbohydrates showed that in transgenic tubers, cold-induced hexogenesis was inhibited while, despite strongly reduced SPP activity, sucrose levels exceeded wild-type (WT) values four- to fivefold after 34 d of cold treatment. This led to a drastic change in the hexose-to-sucrose ratio from 1.9 in WT tubers to 0.15 to 0.11 in transgenic tubers, while the total amount of soluble sugars was largely unchanged in both genotypes. Sucrose-6(F)-phosphate (Suc6P), the substrate of SPP, accumulated in transgenic tubers in the cold which most likely enables the residual enzyme to operate with maximal catalytic activity in vivo and thus, in the long term, counterbalances reduced SPP activity in the transformants. Northern analysis revealed that cold-induced expression of vacuolar invertase (VI) was blocked in SPP-silenced tubers explaining a reduced sucrose-to-hexose conversion. Suc6P levels were found to negatively correlate with VI expression. A possible role of Suc6P in regulating VI expression is discussed.     

Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

The effect of 3-Indolylacetic acid on the differentiation of plastids in callus culture ofCichorium intybus L.

Summary The effect of 3-indolylacetic acid (IAA) on structure of plastids in cells of callus developing on the phloem explants of chicory roots was investigated. In the absence of IAA the proplastids in the initial expiant developed into typical chloroplasts. The presence of increasing IAA concentrations in the medium resulted in a gradual reduction of the thylakoid system accompanied by an increasing starch content of the plastids. Depending on the IAA concentration used, various types of plastids from typical chloroplasts to typical amyloplasts were found. A possible relationship between auxins and sugar metabolism is indicated.     

Properties of fructan:fructan 1-fructosyltransferases from chicory and globe thistle,two Asteracean plants storing greatly different types of inulin

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.     

Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3-phosphate into MGDG by the enzymes associated with envelope membranes is not limited by the phosphatidate phosphatase. These results demonstrate that: (1) non-green plastids employ the same biosynthetic pathway as that previously established for chloroplasts (the formation of glycerolipids is a general property of all plastids, chloroplasts as well as non-green plastids), (2) the envelope membranes are the major structure responsible for the biosynthesis of phosphatidic acid, diacylglycerol and MGDG, and (3) the enzymes of the envelope Kornberg-Pricer pathway have the same properties in non-green starch-containing plastids as in mature chloroplasts from C16:3 and C18:3 plants.     

Preparation and characterization of envelope membranes from nongreen plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.