Imidazolinone-induced loss of acetohydroxyacid synthase activity in maize is not due to the enzyme degradation

Acetohydroxyacid synthase (AHAS), the first enzyme leading to the biosynthesis of valine, leucine, and isoleucine, is inhibited by different chemical classes of herbicides. There is a loss in the extractable AHAS activity in imidazolinone-treated plants. Immunological studies using a monoclonal antibody against AHAS revealed no degradation of AHAS protein in imidazolinone-treated maize (Zea mays) plants. Therefore, the loss in AHAS activity is not due to the loss of AHAS protein.     

Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase), which catalyses the first reaction common to the biosynthesis of the branched-chain amino acids, L-valine, L-leucine and L-isoleucine, and is the target of several classes of herbicides, has been studied in hydroponically-grown seedlings of wheat (Triticum aestivum L. cv. Vulcan). Enzyme activity was greater in leaves than roots, reaching a maximum between 4 and 6 days after germination. AHAS was associated with the chloroplasts after centrifugation in a density gradient. A preparation of the enzyme was obtained from wheat leaves which gave a single band after electrophoresis in native gels but was resolved by denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three polypeptide bands of molecular mass 58, 57 and 15 kDa. The native molecular mass was approximately 128 kDa. AHAS had optimum activity at pH 7 and did not require the addition of flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP) and MgCl₂ for activity. The enzyme did not display typical hyperbolic kinetics, in that the double reciprocal plot of activity against pyruvate concentration was non-linear. The concentration of pyruvate that gave half of the maximum activity was 4 mM. Sulfonylurea and imidazolinone herbicides were potent inhibitors of wheat leaf AHAS, with 50% inhibition being observed at concentrations of 0.6 and 0.3 μM for chlorsulfuron and metsulfuron methyl, respectively, and at 2.5, 5 and 10 μM for imazaquin, imazethapyr and imazapyr. Inhibition by both classes of compounds was reversed by removal of the inhibitor. Progress curves of product formation against time in the presence of the herbicides were non-linear, and based on the assumption that inhibition by the sulfonylureas was of the slow, tight-binding type, estimates of 0.17 and 0.1 nM were obtained for the dissociation constants of chlorsulfuron and metsulfuron methyl, respectively, from the steady-state enzyme-inhibitor complex.     

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.     

Purification and properties of glucoamylase from sugar beet cells in suspension culture

Glucoamylase and α-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with α-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the α-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of α-mannosidase and α-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained α-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M_r (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K_m value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some α-amylases and the debranching enzyme.     

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×10⁶/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R₁ plants were self-pollinated and immature R₂ embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R₂-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.Abbreviations BC backcross - BMS Black Mexican Sweetcorn - 2,4-D 2,4-dichlorophenoxyacetic acid - PWS protoplast wash solution (0.2 M mannitol, 80 mM CaCl₂) - FDA fluorescein diacetate - ABA abscisic acid     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

NADP-malic enzyme from maize leaf: purification and properties.

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C⁴ pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s_20,w) of 9.7 and molecular weight of 2.27 × 10⁵ in sucrose density gradient centrifugation, and molecular weight of 2.26 × 10⁵ calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO₃^? proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: K_m (malate), 0.4 mm; K_m (NADP), 17.6 μm; K_m (Mg²⁺), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol.     

Purification and properties of sucrose synthase from maize kernels

Sucrose synthase was purified from 22-day-old maize (Zea mays L.) kernels to homogeneity by the successive steps of ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and affinity chromatography on a UDP-hexanol-amino-agarose column. The degree of purification is 42-fold and the yield is over 80%. Polyacrylamide gel electrophoretic techniques, sedimentation velocity, and gel filtration studies revealed that the enzyme has identical subunits and could assume tetrameric, octameric, and other higher aggregated forms which are dependent on the ionic species and ionic strength of the solution. All of the enzyme forms exhibit catalytic activity but show differences in their specific activities. In most cases, the tetramer is the predominant form and has the highest specific activity. It is thus concluded that the tetramer could be the native form of the enzyme. The subunit protein has a molecular weight of 88,000 and a blocked NH₂ terminus which is not available to Edman degradation. Some general properties and the amino acid composition of the enzyme are also reported.     

Chain-length specificities of maize starch synthase I enzyme: studies of glucan affinity and catalytic properties

It is widely known that some of the starch synthases and starch-branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain-length specificities of the enzyme. Using affinity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of specificity in terms of its substrate-enzyme dissociation constant, but has a greatly elevated affinity for increasing chain lengths of alpha-1, 4 glucans. Deletion of the N-terminal arm of SSI did not affect the Kd value. Further small deletions of either N- or C-terminal domains resulted in a complete loss of any measurable affinity for its substrate, suggesting that the starch-affinity domain of SSI is not discrete from the catalytic domain. Greater affinity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest affinity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in affinity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several-fold using these glucans with extended chain lengths as substrates, and most of the label from [14C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high affinity for the longer glucan chains of dp approximately 20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B1 chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the specificities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.     

Purification and properties of a nitrate reductase inactivating factor from rice cells in suspension culture

The nitrate reductase inactivating factor in cultured rice cellswas purified 320-fold. The purification procedure involved precipitationwith (NH₄)₂SO₄, fractionation at pH 4.0, adsorption on CM-cellulose,and gel filtration on Sephadex G-200. The molecular weight wasestimated to be 200,000 from the Sephadex G-200 gel filtration. The inactivating factor shows maximal activity at pH 8.0 andappears to be located in the cytoplasm of the cultured ricecells. The inactivating factor was more stable to heat treatmentthan NADH nitrate reductase. The factor inactivated nitratereductase complex except for reduced methylviologen nitratereductase. It had no influence on the activity of nitrite reductase,glutamate dehydrogenase, and NADH diaphorase, but inactivatedxanthine oxidase. The inactivating factor had no protease activitywhen casein, bovine serum albumin, or nitrate reductase fractionwas used as the substrate. The type of inactivation of nitratereductase by the inactivating factor was noncompetitive. Inhibitionof the inactivating factor by o-phenanthroline, EDTA, and p-chloromercuribenzoicacid suggested the involvement of a metal and sulfhydryl groupat its active site. (Received January 28, 1977; )     

Characterization of maize endosperm-derived suspension cells throughout the culture cycle and enhancement of tissue friability

Batch suspension cultures derived from developing maize (Zea mays L.) endosperm were examined throughout the culture cycle to determine the interaction between the tissue and the medium in relation to sugar transport and the effect of subculturing procedures on growth and friability. The growth rate and friability were improved by increasing the frequency of subculture or by physical screening during transfer. An increase in the conductivity of the medium preceded a decrease in fresh weight associated with tissue senescence. Sucrose in the medium was rapidly hydrolysed, and fructose was depleted more rapidly than glucose. Tissue sucrose concentration expressed on a dry weight basis was higher during the middle of the growth cycle, but hexose, starch, zein, lipid, and soluble protein levels changed very little. The medium pH declined from 5.2 to about 4.5 within one day of subculture. Medium pH changed to 4.5 within one day regardless of initial pH (3.0 to 7.0), indicating regulation of external pH rather than passive acidification. Results are consistent with studies of sugar uptake by these cultures, and indicate that cell clump size can be manipulated without exogenous auxin. The characterization of this tissue line establishes its suitability as a model system for studies of sugar transport and other biochemical events in developing maize endosperm.     

Rearrangement of enzyme patterns in maize callus and suspension cultures

The development of enzyme patterns was followed in the course of: (a) the irreversible cell differentiation via division and expansion to maturity in the root tip and coleoptile of the intact seedlings, (b) the irreversible cell dedifferentation associated with induction and establishment of callus from the growing internodes, and (c) the growth cycle (proliferationstationary phase) in callus and cell-suspension cultures of maize (Zea mays L.). By measuring the activities of glycolytic, mitochondrial, microbody and hydrolytic enzymes cells proliferating in vivo and in vitro could be compared and changes related to cessation or resumption of cell division could be studied.Proliferating cells of callus and suspension cultures maintained by serial culture did not differ from those of the root meristem and coleoptile in the specific activities of hexokinase, phosphoglycerate kinase and phosphopyruvate hydratase. Proliferation in vitro resulted in an enormous increase in the ratio g glutamate-dehydrogenase/cytochrome-oxidase activity and in the level of acid-phosphatase activity, with concomitant drop in galactosidase and xylosidase activity. A 3-5-fold increase of alcohol-dehydrogenase, lactate-dehydrogenase and catalase activities was characteristic of dividing callus cells, while a ca. 100-fold increase in the fructofuranosidase-to-glucosidase activity ratio marked cell proliferation in suspension-cultured cells.Changing enzyme activities after cessation of proliferation were quite similar in root tips and coleoptiles, except those of alcohol dehydrogenase and catalase. The enzyme rearrangement during callus establishment and in the growth cycle of callus cultures was in most cases comparable to that in the intact tissues, while the changes from the dividing to the non-dividing cells in suspension cultures, in contrast, differed widely from those in the intact tissues and callus. Galactosidase and xylosidase were the only activities that showed a similar trend of changes in all the investigated, intact and in-vitro-grown cells.Thus, judged by the pattern of enzyme development, the cell suspension appears to be a unique system, virtually unrelated to the growing cells of the intact tissues. It is also very difficult to draw a definite distinction between the metabolic consequences of cell growth and enzyme modulations in cell suspensions as the cells adapt their metabolism to the environmental changes in liquid medium.     

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.     

Nickel in higher plants: further evidence for an essential role

Soybeans (Glycine max [L.] Merr.) grown in Ni-deficient nutrient solutions accumulated toxic urea concentrations which resulted in necrosis of their leaflet tips, a characteristic of Ni deficiency. Estimates of the Ni requirement of a plant were made by using seeds produced with different initial Ni contents. When compared to soybeans grown from seeds containing 2.5 nanograms Ni, plants grown from seeds containing 13 nanograms Ni had a significantly reduced incidence of leaflet tip necrosis. Plants grown from seeds containing 160 nanograms Ni produced leaves with almost no leaflet tip necrosis symptoms. Neither Al, Cd, Sn, nor V were able to substitute for Ni.

In other experiments, a small excess of EDTA was included in the nutrient solution in addition to that needed to chelate micronutrient metals. Under these conditions, nodulated nitrogen-fixing soybeans had a high incidence of leaflet tip necrosis, even when 1 micromolar NiEDTA was supplied. However, in nutrient solutions containing inorganic sources of N, 1 micromolar NiEDTA almost completely prevented leaflet tip necrosis, although no significant increase in leaf urease activity was observed. Cowpeas (Vigna unguiculata [L.] Walp) grown in Ni-deficient nutrient solutions containing NO₃ and NH₄ also developed leaflet tip necrosis, which was analogous to that produced in soybeans, and 1 micromolar NiEDTA additions prevented these symptoms.

These findings further support our contention that Ni is an essential element for higher plants.

     

UDPG: p-Coumarate glucosyltransferase activity in enzyme extracts from higher plants

Leaves of Coleus, Pilea, Cistus and Cestrum, and ripe tomatoes were all able to convert trans-cinnamic acid-[3-¹⁴C]into glucose esters of cinnamic acids. The pool sizes of these esters were measured by the radioisotopic dilution method, and they were found to be of the order of a few μg/g fresh plant material. 1-O-Caffeoyl-β-d-glucose in Cestrum leaves amounted to 70μg/g fresh plant material. Enzyme extracts from Cestrum leaves were able to convert trans-p-coumaric acid-[3-¹⁴C] to 1-O-p-coumaroyl-β-d- glucose, using UDPG as a source of glucose. This enzyme activity could be measured only by trapping techniques, due to the presence of considerable hydrolase activity in crude enzyme extracts.     

The trafficking of the cellulose synthase complex in higher plants

Background

Cellulose is an important constituent of plant cell walls in a biological context, and is also a material commonly utilized by mankind in the pulp and paper, timber, textile and biofuel industries. The biosynthesis of cellulose in higher plants is a function of the cellulose synthase complex (CSC). The CSC, a large transmembrane complex containing multiple cellulose synthase proteins, is believed to be assembled in the Golgi apparatus, but is thought only to synthesize cellulose when it is localized at the plasma membrane, where CSCs synthesize and extrude cellulose directly into the plant cell wall. Therefore, the delivery and endocytosis of CSCs to and from the plasma membrane are important aspects for the regulation of cellulose biosynthesis.

Scope

Recent progress in the visualization of CSC dynamics in living plant cells has begun to reveal some of the routes and factors involved in CSC trafficking. This review highlights the most recent major findings related to CSC trafficking, provides novel perspectives on how CSC trafficking can influence the cell wall, and proposes potential avenues for future exploration.     

On the variation in enzyme activities during the growth of burkitts lymphoma cells in suspension culture

• 1.1. The growth characteristics of Burkitt's lymphoma cells in suspension culture have been studied, and a mean population doubling time of 20–21 hr established for this cell line under a range of nutritional and physical conditions; data which have provided a basis for the assessment of the reproducibility of the culture techniques and conditions which were employed in the subsequent studies.
• 2.2. Activities of lactate dehydrogenase (LDH), aldolase and esterase, as well as the cellular content of total soluble protein, and the isoenzyme pattern of LDH, were monitored in randomly growing Raji cells for the duration of a complete growth cycle.
• 3.3. In this period, the temporal pattern of variation in the levels of total soluble protein were seen to reflect alterations in LDH activity during a single growth cycle.
• 4.4. The fluctuations observed in LDH activity were greater than those observed for either aldolase or esterase activity, and, from the data considered, the maximum degree of variation appeared to be confined to the initial stages of growth.
• 5.5. Extracellular levels of LDH activity remained relatively constant throughout the growth cycle. so that the large fluctuations in intracellular LDH activity could not be attributed to either secretion or leakage of the enzyme into the culture medium.
• 6.6. No gross changes in the pattern of LDH isoenzymes in these Raji cells were detected during the course of a single growth cycle.

     

Purification and some properties of chalcone synthase from a carrot suspension culture induced for anthocyanin synthesis and preparation of its specific antiserum

Chalcone synthase was purified to homogeneity by polyacrylamide gel electrophoresis from cell suspension cultures of carrot in which anthocyanin synthesis was induced by transferring the cells from a medium containing 2,4-dichlorophenoxy-acetic acid (2,4-D) to one lacking it. A molecular weight of 80,000-85,000 for the enzyme was determined by gel filtration and disc-gel polyacrylamide electrophoresis, and one of about 40,600 for the subunit by SDS slab-gel electrophoresis. The primary reaction product was chalcone and the pH optimum of the reaction was 8.0. The Km values for 4-coumaroyl-CoA and malonyl-CoA were 5.7 microM and 18 microM, respectively. These properties of carrot chalcone synthase were discussed in comparison to those of that from cell cultures of parsley reported previously. Antiserum against chalcone synthase from carrot was obtained from mice bred under specific pathogen free conditions. Crossreactivity was examined by Western-blotting, and the high specificity of the antiserum against chalcone synthase was demonstrated.     

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP‐galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.