Contrapuntal networks of gene expression during Arabidopsis seed filling

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, approximately 35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid-regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO(2) refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, <1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.     

Sunflower (Helianthus annuus) long‐chain acyl‐coenzyme A synthetases expressed at high levels in developing seeds

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl‐CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.     

Lipids in developing and mature rice grain

The neutral fraction of nonstarch lipids in developing brown rice (Oryza sativa L., cv IR42) was accumulated up to 16 days after flowering (DAF), but phospholipids and glycolipids increased only up to 8 DAF. Fatty acids accumulated in nonstarch lipids until 12 DAF. However, the proportion of linolenic acid in the lipid fraction decreased and that of oleic acid increased during this period. Accumulation of fat-by-hydrolysis in the brown rice occurred until 20 DAF and followed closely that of starch. The proportion of linolenic acid decreased and that of linoleic acid increased until 16 DAF. The fatty acid composition of fat-by-hydrolysis and starch lipids were identical and fat-by-hydrolysis accounted for 48% by weight of starch lipids. Nonstarch lipids were mainly composed of triglycerides and were located in the bran and embryo of mature brown rice. Starch lipids were mainly composed of lysophosphatidyl choline, free fatty acids and lysophosphatidyl ethanolamine, and were located in the endosperm.     

Storage oil breakdown during embryo development of Brassica napus (L.)

In this study it is shown that at least 10% of the major storage product of developing embryos of Brassica napus (L.), triacylglycerol, is lost during the desiccation phase of seed development. The metabolism of this lipid was studied by measurements of the fate of label from [1-(14)C]decanoate supplied to isolated embryos, and by measurements of the activities of enzymes of fatty acid catabolism. Measurements on desiccating embryos have been compared with those made on embryos during lipid accumulation and on germinating seedlings. Enzymes of beta-oxidation and the glyoxylate cycle, and phosphoenolpyruvate carboxykinase were present in embryos during oil accumulation, and increased in activity and abundance as the seeds matured and became desiccated. Although the activities were less than those measured during germination, they were at least comparable to the in vivo rate of fatty acid synthesis in the embryo during development. The pattern of labelling, following metabolism of decanoate by isolated embryos, indicated a much greater involvement of the glyoxylate cycle during desiccation than earlier in oil accumulation, and showed that much of the (14)C-label from decanoate was released as CO(2) at both stages. Sucrose was not a product of decanoate metabolism during embryo development, and therefore lipid degradation was not associated with net gluconeogenic activity. These observations are discussed in the context of seed development, oil yield, and the synthesis of novel fatty acids in plants.     

Acyl-acyl carrier protein thioesterase activity from sunflower (Helianthus annuus L.) seeds

During sunflower (Helianthus annuus L.) seed formation there was an active period of lipid biosynthesis between 12 and 28 days after flowering (DAF). The maximum in-vitro acyl-acyl carrier protein (ACP) thioesterase activities (EC 3.1.2.14) were found at 15 DAF, preceding the largest accumulation of lipid in the seed. Data from the apparent kinetic parameters, V _max and K _m, from seeds of 15 and 30 DAF, showed that changes in acyl-ACP thioesterase activity are not only quantitative, but also qualitative, since, although the preferred substrate was always oleoyl-ACP, the affinity for palmitoyl-ACP decreased, whereas that for stearoyl-ACP increased with seed maturation. Bisubstrate assays carried out at 30 DAF seemed to indicate that the total activity found in mature seeds is due to a single enzyme with 100/75/15 affinity for oleoyl-ACP/stearoyl-ACP/palmitoyl-ACP. In contrast, at 15 DAF, enzymatic data together with partial sequences from cDNAs indicated the presence of at least two enzymes with different properties, a FatA-like thioesterase, with a high affinity for oleoyl-ACP, plus a FatB-like enzyme, with preference for long-chain saturated fatty acids, both being expressed during the active lipid biosynthesis period. Competition assays carried out with CAS-5, a mutant with a higher content of palmitic acid in the seed oil, indicated that a modified FatA-type thioesterase is involved in the mutant phenotype. Received: 17 December 1999 / Accepted: 25 February 2000     

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.     

Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica

Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%–50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%–60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68 mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild‐type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine‐diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20 days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86 mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.     

Acyl-ACP thioesterases from castor (Ricinus communis L.): An enzymatic system appropriate for high rates of oil synthesis and accumulation

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.     

Lipid biosynthesis in seeds of mustard (Brassica juncea) influenced by zinc and sulphur deficiency

In developing seeds of mustard ( Brassica juncea L. cv. RLM 198) the period between 20 and 30 days after fertilization (DAF) was identified as the period of active lipid biosynthesis, although dry matter continued to accumulate until maturity. The period of lipid synthesis was associated with a decrease in starch, soluble sugars and protein, thus, giving rise to precursors for the biosynthesis of lipids. Besides decreasing the dry matter content (on both % and seed basis), Zn and S deficiency caused a significant ( P > 0.05) reduction in oil content. As compared to control, the decrease in oil content was 11, 12 and 18% at 30 DAF and 4, 9 and 16% at maturity in Zn, S and (Zn+S) deficient treatments, respectively. Throughout the period of seed development, a significant decrease in starch and protein with a slight accumulation of soluble sugars was observed due to deficiency of Zn or S. The rate of [l-¹⁴C]-acetate incorporation into total lipids, which was maximal at 30 DAF, also displayed a significant decrease due to the abovementioned mineral deficiencies. Addition of Zn or S in vitro, enhanced the lipid synthesis at all stages of seed development. Under Zn and S deficiency, the phospholipids increased from 10 to 30 DAF and then declined until maturity. However, the proportion of glycolipids and free fatty acids increased, with a corresponding decrease in total glycerides. Further, in deficiency treatments, there was an increase in 22:1 with a corresponding decrease in 18:1, 18:2 and 18:3 in developing and mature mustard seeds.     

Lipids in grain tissues of oat (Avena sativa): differences in content, time of deposition, and fatty acid composition

Oat (Avena sativa) is unusual in comparison with other cereals since there are varieties with up to 18% oil content. The lipid content and fatty acid composition in different parts of the grain during seed development were characterized in cultivars Freja (6% oil) and Matilda (10% oil), using thin-layer and gas chromatography, and light and electron microscopy. The majority of lipids (86-90%) were found in the endosperm. Ninety-five per cent of the higher oil content of cv. Matilda compared with cv. Freja was due to increased oil content of the endosperm. Up to 84% of the lipids were deposited during the first half of seed development, when seeds where still green with a milky endosperm. Microscopy studies revealed that whereas oil bodies of the embryo and scutellum still contained a discrete shape upon grain maturation, oil bodies of the endosperms fused upon maturation and formed smears of oil.     

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.     

The modulating effect of the perisperm-endosperm envelope on ABA-inhibition of seed germination in cucumber

Abscisic acid (ABA) markedly reduced the germination of developing seeds at much lower concentrations (ABA50=0.1 mM) compared with that of mature seeds (ABA50=1.6 mM) in cucumber (Cucumis sativus L. cv. Green long). The perisperm-endosperm (PE) envelope in developing seeds showed partly differentiated lipid and callose layers, considerable ABA biosynthetic activity in endosperm cells, and appreciable permeability to applied ABA. The decrease in the sensitivity of seeds to applied ABA was coincident with the complete development of lipid and callose layers, diminished ABA biosynthetic activity in endosperm cells in imbibed mature seeds, and moderate permeability of the PE envelope to applied ABA. Decoated seeds pretreated with chloroform showed decreased germination (ABA50=0.4 mM) in response to applied ABA and increased ABA permeation through the PE envelope. ABA thus allowed to permeate into embryonic tissues substantially reduced the pregerminative activity of beta-glucanase in the radicles. The structure and biophysical/biochemical properties of the PE envelope seem to modulate the effect of ABA on the germination of developing and mature cucumber seeds.