High resolution NMR microscopy of plants and fungi.

Nuclear magnetic resonance (NMR) microscopy is a completely noninvasive technique that can be used to acquire images with high spatial resolution through opaque objects such as plant organs and tissue parts. The image contrast can be chosen to represent the anatomical details or to visualize the spatial distribution of a range of physico-chemical parameters such as the apparent diffusion constant of water or the velocity of water flow within plants in vivo. In addition, images can be generated which show the spatial distribution of metabolites. Furthermore, it is possible to detect chemical compounds labelled with the stable isotope (13)C and to generate images showing the spatial distribution of the (13)C label in the intact plant. The ability to monitor water flow and transport of (13)C-labelled tracer in intact plants with NMR microscopy favours the use of this technique in the investigation of long-distance transport processes in plants. A short introduction into the technical principles of NMR microscopy is provided and the problems associated with applications to plants are summarized. The potential of the technique is explained with applications to Zinnia elegans plants, wheat grains and Brassica napus siliques.

Efficient regeneration systems for two closely related Moricandia species possessing a C3 or C3-C4 intermediate photosynthetic character.

The C(3)-C(4) intermediate species Moricandia arvensis ( Brassicaceae) and its closest C(3) relative, Moricandia moricandioides, represent model species for studying the C(3)-C(4) photosynthetic character relative to the C(3) phenotype. In order to enable transgenic analyses in these two species, optimal regeneration systems based on leaf and/or stem internode segments were developed, and genotypes suitable for in vitro tissue culture were identified. Evaluation of the regeneration capability of 30 M. arvensis genotypes and 12 M. moricandioides genotypes revealed that all could form callus. However, shoots were only produced by 40% of the M. arvensis genotypes and 8% of the M. moricandioides genotypes. The two Moricandia species showed significant genotypic differences with respect to callus formation and shoot regeneration. For the 12 regenerative M. arvensis genotypes, 29-100% of the explants developed shoots, while 71% of the explants from the single regenerable M. moricandioides genotype formed shoots. The genotype used, the choice of stem or leaf explants and the composition of the medium (i.e. concentrations of different hormones and salts) significantly affected plant regeneration (chi-square analyses, P<0.05). Whole plants could be obtained in the greenhouse after 3-3.5 months for M. arvensis genotypes and after 4-4.5 months for M. moricandioides.

Fatty acid synthesis in pea root plastids is inhibited by the action of long-chain acyl- coenzyme as on metabolite transporters.

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1--2 microM). The IC(50) (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nM; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mM) and CoASH (coenzyme A; 0.3 mM), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 microM) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA.

Inhibition of the glucose-6-phosphate transporter in oilseed rape (Brassica napus L.) plastids by acyl-CoA thioesters reduces fatty acid synthesis.

Addition of oleoyl-CoA (1 microM), or other acyl-CoA thioesters with a chain length of C(16) or greater, to oilseed rape plastids (Brassica napus L.) inhibited the rate of D-glucose 6-phosphate (Glc6P) uptake by 70% after 2 min. The IC(50) value for oleoyl-CoA inhibition of the transporter was approx. 0.2-0.3 microM. Inhibition was alleviated by the addition of acyl-CoA binding protein (ACBP) or BSA at slightly higher concentrations. Oleic acid (5-25 microM), Tween 40 (10 microM), Triton-X 100 (10 microM) and palmitoylcarnitine (5 microM) had no effect on Glc6P uptake. The uptake of [1-(14)C]Glc6P occurred in exchange for P(i), 3-phosphoglycerate or Glc6P at a typical rate of 30 nmol Glc6P/min per unit of glyceraldehyde-3-phosphate dehydrogenase (NADP(+)). The K(m(app)) of the Glc6P transporter for Glc6P was 100 microM. Neither CoA (0.3 mM) nor ATP (3 mM) inhibited Glc6P uptake, but the transporter was inhibited by 72% when ATP and CoA were added together. This inhibition was attributable to the synthesis of acyl-CoA thioesters, predominantly oleoyl-CoA and palmitoyl-CoA, by long-chain fatty acid-CoA ligase (EC 6.2.1.3) from endogenous fatty acids in the plastid preparations. Acyl-CoA thioesters did not inhibit the uptake of [2-(14)C]pyruvate or D-[1-(14)C]glucose into plastids. In vivo quantities of oleoyl-CoA and other long-chain acyl-CoA thioesters were lower than those for ACBP in early cotyledonary embryos, 0.7+/-0.2 pmol/embryo and 2.2+/-0.2 pmol/embryo respectively, but in late cotyledonary embryos quantities of long-chain acyl-CoA thioesters were greater than ACBP, 3+/-0.4 pmol/embryo and 1.9+/-0.2 pmol/embryo respectively.

Inhibition by long-chain acyl-CoAs of glucose 6-phosphate metabolism in plastids isolated from developing embryos of oilseed rape (Brassica napus L.).

The effects of long-chain acyl-CoA (lcACoA) esters (both added exogenously and synthesized de novo) and acyl-CoA binding protein (ACBP) on plastidial glucose 6-phosphate (Glc6P) and pyruvate metabolism were examined using isolated plastids. The binding of lcACoA esters by ACBP stimulated the utilization of Glc6P for fatty acid synthesis, starch synthesis and reductant supply via the oxidative pentose phosphate (OPP) pathway. Stimulation occurred at low (1-10 microM) concentrations of ACBP. Pyruvate-dependent fatty acid synthesis was not directly affected by ACBP. However, addition of ACBP did increase the Glc6P-dependent stimulation of pyruvate utilization mediated through the OPP pathway. On the basis of these experiments, we conclude that lcACoA esters may inhibit Glc6P uptake into plastids, and that this inhibition is relieved by ACBP. We also suggest that utilization of other substrates for fatty acid synthesis may be affected by lcACoA/ACBP via their effects on the OPP pathway.

The rug3 locus of pea encodes plastidial phosphoglucomutase.

Two cDNA clones were isolated from pea (Pisum sativum L.) and their deduced amino acid sequences shown to have significant homology to phosphoglucomutases from eukaryotic and prokaryotic sources. The longer cDNA contained a putative transit-peptide-encoding sequence, supporting the hypothesis that the isolated clones represent the cytosolic and plastidial isoforms of phosphoglucomutase in pea. Plastid protein import assays confirmed that the putative plastidial isoform was targeted to the plastid stroma where it was proteolytically processed. Expression, co-segregation, linkage, and molecular analyses have confirmed that the rug3 locus of pea encodes plastidial phosphoglucomutase. Mutations at this locus result in a near-starchless phenotype of the plant.

Coordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos.

Measurements of metabolic fluxes in whole embryos and isolated plastids have revealed major changes in the pathways of carbon utilization during cotyledon filling by oilseed rape (Brassica napus L.) embryos. In the early cotyledon stage (stage A), embryos used sucrose (Suc) predominantly for starch synthesis. Plastids isolated from these embryos imported glucose-6-phosphate (Glc-6-P) and partitioned it to starch and fatty acids synthesis and to the oxidative pentose phosphate pathway in the ratio of 2:1:1 on a hexose basis. Of the substrates tested, Glc-6-P gave the highest rates of fatty acid synthesis by the plastids and pyruvate was used weakly. By the mid- to late-cotyledon stage (stage C), oil accumulation by the embryos was rapid, as was their utilization of Suc for oil synthesis in vitro. Plastids from C-stage embryos differed markedly from those of stage-A embryos: (a) pyruvate uptake and utilization for fatty acid synthesis increased by respectively 18- and 25-fold; (b) Glc-6-P partitioning was predominantly to the oxidative pentose phosphate pathway (respective ratios of 1:1:3); and (c) the rate of plastidial fatty acid synthesis more than doubled. This increased rate of fatty synthesis was dependent upon the increase in pyruvate uptake and was mediated through the induction of a saturable transporter activity.

Effects of manipulating expression of acetyl-CoA carboxylase I in Brassica napus L. embryos.

Acetyl-CoA carboxylase I (ACCase I) in developing oilseed rape embryos is predominantly cystosolic, based upon measurement of its propionyl-CoA carboxylase activity. Reduction of ACCase I by antisense expression reduces seed lipid content and affects carbohydrate metabolism.

The role of plastidial transporters in developing embryos of oilseed rape (Brassica napus L.) for fatty acid synthesis.

The phosphoenolpyruvate transporter (PPT) is one of several important transporters for channelling carbon intermediates utilized for fatty acid synthesis and other plastidial pathways from the cytosol into the plastid. In this paper we show results on how the activity of the PPT changes between two important, physiologically different developmental stages of oilseed rape embryos.

Carbon supply for storage-product synthesis in developing seeds of oilseed rape.

The aim of this work was to find out how the sugars in the endosperm of oilseed rape contribute to the flux of oil synthesis. While the hexose content of the liquid endosperm decreased during development the sucrose content increased. It is important to understand the relative rates of use of the endosperm sugars for two reasons. Firstly we need to know which sugars are used, and at what stages in development, in order to understand the roles of enzymes involved in their metabolism. Secondly, changes in sugar concentration have been implicated in the regulation of expression of genes determining storage-product synthesis [see Weber, Borisjuk and Wobus (1997) Trends Plant Sci. 2, 169-174, for review]. The rate of consumption of sugar is one factor governing its concentration. We present data showing both the concentration-dependence of conversion of sugar to oil, and the in vivo concentrations of sugars; we relate these data sets to each other and discuss the effects of the intracellular pool of sucrose. Glucose, fructose and sucrose are all substrates for oil synthesis, but the rates of their use (particularly sucrose) are underestimated because of dilution by sucrose from the intracellular pool.

Role of acyl-CoAs and acyl-CoA-binding protein in regulation of carbon supply for fatty acid biosynthesis.

Acyl-CoA esters inhibit the plastidial glucose 6-phosphate (Glc-6-P) transporter and the adenylate transporter; the IC(50) values for the inhibition by oleoyl-CoA (18:1-CoA) are 200-400 nM and 1-2 microM respectively. The inhibition of either of these processes significantly reduces the flux of carbon from Glc-6-P or from acetate into long-chain fatty acids. The effect is dependent on the acyl chain length, e.g. lauryl-CoA is less inhibitory than oleoyl-CoA, causing 34 and 68% inhibition respectively of Glc-6-P uptake after 30 s. The inhibition of Glc-6-P and ATP transport is alleviated by addition of an equivalent concentration of acyl-CoA-binding protein (ACBP) or BSA. Acyl-CoAs do not inhibit pyruvate or glucose transporters. The endogenous concentrations of acyl-CoAs and ACBP are similar during embryo maturation.

Fatty acid breakdown in developing embryos of Brassica napus L.

Developing Brassica napus embryos are primarily concerned with the accumulation of storage products, namely oil, starch and protein. The presence of fatty acid catabolic pathways in the background of this biosynthetic activity was investigated. Enzymes involved in the process of lipid mobilization, such as malate synthase and isocitrate lyase, are detectable towards the late stages of embryo development. [(14)C]Acetate feeding experiments also reveal that fatty acid catabolism becomes increasingly functional as the embryo matures.

Developmental and environmental effects on the expression of the C3-C4 intermediate phenotype in moricandia arvensis

Cellular anatomy and expression of glycine decarboxylase (GDC) protein were studied during leaf development of the C3-C4 intermediate species Moricandia arvensis. Leaf anatomy was initially C3-like and the number and profile area of mitochondria in the bundle-sheath cells were the same as those in adjacent mesophyll cells. Between a leaf length of 6 and 12 mm there was a bundle-sheath-specific, 4-fold increase in the number of mitochondrial profiles, followed by a doubling of their individual profile areas as the leaves expanded further. Subunits of GDC were present in whole-leaf extracts before the anatomical development of bundle-sheath cells. Whereas the GDC H-protein content of leaves increased steadily throughout development, the increase in GDC P-protein was synchronous with the development of mitochondria in the bundle sheath. The P-protein was confined to bundle-sheath mitochondria throughout leaf development, and its content in individual mitochondria increased before the anatomical development of the bundle sheath. Anatomical and biochemical attributes of the C3-C4 character were present in the cotyledons and sepals but not in other photosynthetic organs/tissues. In leaves and cotyledons that developed in the dark, the expression of the P-protein and the organellar development were reduced but the bundle-sheath cell specificity was retained.

Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm.

In this study we examined the processes by which malate and pyruvate are taken up across the leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.

Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis.

In the oilseed rape Brassica napus there are two forms of acetyl-CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were clones, two of which contained the entire coding region. The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences. We also report the cloning of a second type II ACCase component from oilseed rape, the beta-carboxyltransferase subunit (betaCT), which is chloroplast-encoded. Northern analysis showed that although the relative levels of BCCP and betaCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development. At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera. With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast. This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis.

In order to compare the dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxylase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49,753 +/- 5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.

Cloning and characterization of a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex from Arabidopsis thaliana.

A cDNA encoding a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex has been isolated from Arabidopsis thaliana. A cell culture cDNA expression library was screened with a monoclonal antibody (JIM 63) raised against nuclear matrix proteins, and four clones were isolated. One of these was 2175 base pairs in length, and it contained an open reading frame with an amino acid sequence and domain structure with strong similarity to the E2s of other eukaryotic and prokaryotic organisms. The organization and number of functional domains within the Arabidopsis protein are identical to those of the human E2, although the amino acid sequences within these domains are equally similar to those of the yeast and human proteins. The predicted amino acid sequence reveals the presence of a putative amino-terminal leader sequence with characteristics similar to those of other proteins, which are targeted to the plant mitochondrial matrix. The cross-reactivities of plant mitochondrial matrix proteins with JIM 63 and antibodies raised against the E2 and protein X components of eukaryotic pyruvate dehydrogenase complexes are consistent with the clone encoding a mitochondrial form of E2 and not the smaller protein X. The E2 mRNA of 2.2 kilobases was expressed in a range of Arabidopsis and Brassica napus tissues.

T-protein of the glycine decarboxylase multienzyme complex: evidence for partial similarity to formyltetrahydrofolate synthetase.

We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum. The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins. Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes. This suggests that the T-protein sequence is not as unique as previously thought.

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum).

Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.

Identification and localization of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform.

Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.