Stephen Rawsthorne

Starch metabolism in developing embryos of oilseed rape

Abstract.The aim of this work was to characterise the metabolism of starch in developing embryos of oilseed rape (Brassica napus L. cv. Topaz). The accumulation of starch in embryos in siliques which were darkened or had been exposed to the light was similar, suggesting that the starch is synthesised from imported sucrose rather than via photosynthesis in the embryo. Starch content and the activities of plastidial enzymes required for synthesis of starch from glucose 6-phosphate (Glc6P) both peaked during the early-mid stage of cotyledon development (i.e. during the early part of oil accumulation) and then declined. The mature embryo contained almost no starch. The starch-degrading enzymes α-(EC 3.2.1.1) and β-amylase (EC 3.2.1.2) and phosphorylase (EC 2.4.1.1) were present throughout development. Most of the activity of these three enzymes was extraplastidial and therefore unlikely to be involved in starch degradation, but there were distinct plastidial and extraplastidial isoforms of all three enzymes. Activity gels indicated that distinct plastidial isoforms increase during the change from net synthesis to net degradation of starch. Plastids isolated from embryos at stages both before and after the maximum starch content could convert Glc6P to starch although the rate was lower at the later stage. The results are consistent with the idea that starch synthesis and degradation occur simultaneously during embryo development. The possible roles of transient starch accumulation during embryo development are discussed.

Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

The aim of this work was to investigate the partitioning of imported glucose 6-phosphate (Glc6P) to starch and fatty acids, and to CO2 via the oxidative pentose phosphate pathway (OPPP) in plastids isolated from developing embryos of oilseed rape (Brassica napus L.). The ability of the isolated plastids to utilize concurrently supplied substrates and the effects of these substrate combinations on the Glc6P partitioning were also assessed. The relative fluxes of carbon from Glc6P to starch, fatty acids, and to CO2 via the OPPP were close to 2∶1∶1 when Glc6P was supplied alone. Under these conditions NADPH generated via the OPPP was greater than that required by the concurrent rate of fatty acid synthesis. Fatty acid synthesis was unaffected by the presence or absence of exogenous NADH and/or NADPH and the requirement of fatty acid synthesis for reducing power is therefore met entirely by intraplastidial metabolism. When Glc6P was supplied in the presence of either pyruvate or pyruvate and acetate, the total flux from these metabolites to fatty acids was up to threefold greater than that from either Glc6P or pyruvate when they were supplied singly. In these experiments there was little competition between Glc6P and pyruvate in fatty acid synthesis and the flux to starch was unchanged. This implies that the starch and fatty acid biosynthesis pathways did not compete for the exogenously supplied ATP on which they were strongly dependent. When Glc6P and pyruvate were provided together, the NADPH generated by the OPPP pathway was less than that required by the concurrent rate of fatty acid synthesis. This suggests that the metabolism of exogenous Glc6P via the OPPP can contribute to the NADPH demand created during fatty acid synthesis but it also indicates that other intraplastidial sources of reducing power must be available under the in-vitro conditions used.

CO2 Assimilation in C3-C4 Intermediate Plants

The mode of CO2 assimilation known as C3-C4 intermediate photosynthesis provides intriguing insight into a novel mechanism for reducing photorespiratory CO2 loss and possible evolutionary pathways to C4 photosynthesis. Plants with the C3-C4 pathway have been reported from twenty-five species in nine genera representing six families, and they are principally associated with warm or hot habitats. The ecological distribution of C3-C4 plants is consistent with an adaptive role for re-assimilation of photorespired CO2 at warm temperatures. Photorespired CO2 is re-assimilated through the differential partitioning of photorespiratory organelles between mesophyll cells and bundle-sheath cells in C3-C4 intermediates, including total isolation of glycine decarboxylase activity to the bundle-sheath. Thus, glycine diffuses to the bundle sheath tissue, where photorespired CO2 is released and assimilated by surrounding chloroplasts before it can escape the leaf. Models of this ‘glycine shuttle’ reveal possible advantages in terms of CO2 assimilation rate, water-use efficiency, and nitrogen-use efficiency, though there are strong constraints on the fraction of RuBP carboxylation capacity that can be allocated to the bundle sheath to assimilate the photorespired CO2. The models further predict distinctive gas-exchange patterns with respect to the CO2 compensation point and discrimination against 13CO2. These predictions have been validated with gas-exchange measurements. One group of C3-C4 species (those belonging to the genus Flaveria) exhibit evidence of functional C4 biochemistry and assimilation of at least some atmospheric CO2 through the C4 pathway. In the Flaveria intermediates, photorespiration rates are reduced as in C3-C4 intermediates from other genera, and in several Flaveria species O2 inhibition of photosynthesis is reduced, suggesting the presence of a CO2-concentrating mechanism. It is not understood how photorespiration rates are reduced in these ‘biochemical intermediates,’ though there is evidence that at least part of it is due to the same glycine shuttle found in other C3-C4 intermediates. The glycine shuttle may represent an initial step in the evolutionary path to C4 photosynthesis.

Coordination of the cell-specific distribution of the four subunits of glycine decarboxylase and of serine hydroxymethyltransferase in leaves of C3-C4 intermediate species from different genera

The cell-specific distribution of the four subunit proteins (P, L, T and H) of glycine decarboxylase (GDC) and of serine hydroxymethyltransferase (SHMT) has been studied in the leaves of C3-C4 intermediate and C4 species of three genera (Flaveria, Moricandia and Panicum) using immunogold localization. Antibodies raised against these proteins from pea leaf mitochondria were used to probe Western blots of total leaf proteins of F. linearis Lag., M. arvensis (L.) DC and P. milioides Nees ex Trin. (C3-C4), and F. trinervia (Spring.) Mohr and P. miliaceum (L.) (C4). For all species, each antibody recognised specifically a protein of similar molecular weight to that in pea leaves. In leaves of M. arvensis the P protein was present in the mitochondria of the bundle-sheath cells but was undetectable in those of the mesophyll, whereas the L, T and H proteins and SHMT were present in both cell types. The density of immunogold labelling of SHMT on the mitochondria of mesophyll cells was less than that on those of the bundle-sheath cells, which correlates with the relative activities of SHMT in these cell types. These data reveal that the lack of functional GDC in the mesophyll cells of M. arvensis, which is the principal biochemical reason for reduced photorespiration in this species, is due to the loss of a single subunit protein. This lack of coordinate expression of the subunit proteins of GDC within a photosynthetic cell represents a clear difference between M. arvensis and other C3 and C3-C4 species. None of the GDC proteins was detectable in the mesophyll cells of the C3-C4 and C4Flaveria and Panicum species but all were present in the bundle-sheath cells. The differences in the distribution of the GDC proteins in leaves of the C3-C4 species studied are discussed in relation to the evolution of photosynthetic mechanisms.

Initial Events during the Evolution of C4 Photosynthesis in C3 Species of Flaveria

The initial stages of C4 photosynthetic evolution in Flaveria species is apparent in the C3 species Flaveria pringlei and Flaveria robusta, which show increases in organelle number and size in bundle sheath cells and a redistribution of mitochondria to the inner region of the bundle sheath. The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C2 photosynthesis. How C3 species evolved C2 metabolism is critical to understanding the initial phases of C4 plant evolution. To evaluate early events in C4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C3 and C2 species of Flaveria, a model genus for C4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C3 species that are most closely related to the C2 Flaveria species, would show rudimentary characteristics of C2 physiology. Compared with less-related C3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO2 compensation point of photosynthesis was slightly lower than in the less-related C3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C2 photosynthesis.

The activity of acetyl-CoA carboxylase is not correlated with the rate of lipid synthesis during development of oilseed rape (Brassica napus L.) embryos

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) activity has been determined in seed tissues of oilseed rape (Brassica napus L.), pea (Pisum sativum L.) and castor bean (Ricinus communis L.). A new method is described which leads to significantly higher measurable activities of the enzyme in tissue homogenates than previously reported. This method does not involve either Triton X-100 or centrifugation treatments which have been used previously in the study of the enzyme. In the case of oilseed rape the activity was also increased by removal of the testa from the seed. The activity of ACCase was determined throughout the development of oilseed rape embryos. Enzyme activity increased 3.5-fold as the embryo fresh weight increased from 0.3 to 2.0 mg and then reached a plateau at 1.1 nmol malonyl-CoA-min−1 · embryo−1. The main period of lipid accumulation commenced at an embryo fresh weight of 2.3 mg, which was after the plateau in ACCase activity had been reached. Activity of the enzyme declined after an embryo fresh weight of 3.5 mg, which was before lipid accumulation in the embryo had been completed. Comparison of the activity of ACCase and the apparent in-vivo rate of lipid synthesis on an embryo-fresh-weight basis (i.e. nmol malonyl-CoA formed or utilized·min−1·mg−1 fresh weight) revealed that ACCase activity declines relative to the rate of lipid synthesis throughout development. The negative correlation between these two rates is discussed in relation to the role of ACCase in the regulation of accumulation of storage lipid during embryo development.

Storage lipid formation in seeds

The mechanisms and regulation of storage lipid formation in seeds are reviewed. Seed storage lipids are ultimately derived from simple carbon compounds, such as sucrose, which are imported into seeds from source tissues, such as leaves or pods. The partitioning of this important carbon towards storage lipid, carbohydrate or protein is regulated by a complex interaction between genetically-determined endogenous factors and external environmental influences. Storage lipids are synthesized from fatty acids, derived from acetyl-CoA, and glycerol 3-phosphate. Fatty acid biosynthesis and probably acetyl-CoA formation, occurs within the plastids to produce oleoyl-CoA. Further elaboration of oleoyl-CoA to produce polyunsaturates, hydroxylates or very long chain acyl-CoAs occurs on the endoplasmic reticulum, as does the esterification of acyl-CoAs to glycerol-3-phosphate to produce the final triacylglycerol storage oil. The temporal and hormonal regulation of storage lipid accumulation in seeds is discussed.

The relationship between the post-illumination CO2 burst and glycine metabolism in leaves of C3 and C3-C4 intermediate species of Moricandia

The free-pool sizes of amino acids involved in photorespiratory metabolism have been determined in leaves of Moricandia species during the post-illumination CO2 burst. The kinetics of the burst and the time to attainment of steady-state rates of dark respiration were much slower in the C3-C4 intermediate species Moricandia arvensis (L.) DC than in the C3 species Moricandia moricandioides (Boiss.) Heywood. When plants were equilibrated at a high photon flux density (PFD; 1200 μmol · m−2 · s−1 PAR) the glycine and serine pool sizes in leaves of M. arvensis were 1.9 and 1.4 μmol · mg−1 phaeophytin, respectively, values which were twice those in leaves of M. moricandioides. Amounts of glycine and serine were smaller at a lower PFD (150 μmol · m−2 · s−1) but were still twice as large in M. arvensis. Amounts of other amino acids involved in photorespiration or background cell metabolism (glutamate/glutamine, alanine, valine and threonine) were comparable in both species and did not respond to irradiance or change markedly during the dark burst. In contrast, during the first minute of the post-illumination burst the glycine pool in the leaves of both species had declined by at least 60%. It continued to decline, reaching 6–7 % of the level in the light by the time steady-state rates of dark respiration had been established. The rate of disappearance of glycine was comparable in both species and therefore depletion to steady-state dark levels took longer in M. arvensis than in M. moricandioides (8.4 and 4.6 min, respectively). These data indicate that almost all of the glycine pool in the leaves of C3 and C3-C4Moricandia species is a consequence of photorespiratory metabolism. The significance of a large but readily metabolised pool of glycine in the leaves of M. arvensis is discussed.

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.

Carbon flux to fatty acids in plastids

This article considers our understanding of the pathways involved in the provision of precursors for de novo fatty acid synthesis in plastids. The characteristics of the plastidial enyzmes required for synthesis of acetyland malonyl-CoA are reviewed. The role of transporters in determining the metabolic routes by which carbon is imported into the plastids and the extent to which the different transporters are utilized for fatty acid synthesis are discussed. Interactions between metabolic pathways in plastids are illustrated by considering the partitioning of imported glucose 6-phosphate to starch, fatty acids and to CO2 via the oxidative pentose phosphate pathway in plastids isolated from developing rapeseed embryos.