Inhibition by cerulenin of lipid synthesis in Crambe abyssinica tissues

Cerulenin is a potent inhibitor of fatty acid synthesis from ¹⁴C-labelled acetate in leaves and developing seeds of Crambe abyssinica. The antibiotic is equally inhibitory on the elongation of [1-¹⁴C]-oleic acid to erucie acid which is the major fatty acid of the seed. There is no significant inhibition of fatty acid desaturation in either tissue. Acylation of lipids is not a primary target of cerulenin's action.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Prediction of desiccation sensitivity in seeds of woody species: a probabilistic model based on two seed traits and 104 species

BACKGROUND AND AIMS: Seed desiccation sensitivity limits the ex situ conservation of up to 47 % of plant species, dependent on habitat. Whilst desirable, empirically determining desiccation tolerance levels in seeds of all species is unrealistic. A probabilistic model for the rapid identification of woody species at high risk of displaying seed desiccation sensitivity is presented. METHODS: The model was developed using binary logistic regression on seed trait data [seed mass, moisture content, seed coat ratio (SCR) and rainfall in the month of seed dispersal] for 104 species from 37 families from a semi-deciduous tropical forest in Panamá. KEY RESULTS: For the Panamanian species, only seed mass and SCR were significantly related to the response to desiccation, with the desiccation-sensitive seeds being large and having a relatively low SCR (i.e. thin 'seed' coats). Application of this model to a further 38 species, of known seed storage behaviour, from two additional continents and differing vegetation types (dryland Africa and temperate Europe) correctly predicted the response to desiccation in all cases, and resolved conflicting published data for two species (Acer pseudoplatanus and Azadirachta indica). CONCLUSIONS: This model may have application as a decision-making tool in the handling of species of unknown seed storage behaviour in species from three disparate habitats.     

The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.     

Antisense expression of 3-oxoacyl-ACP reductase affects whole plant productivity and causes collateral changes in activity of fatty acid synthase components

Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.     

Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles

Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed‐specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. Here we show by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of the oilseed crop Camelina sativa, indicating tissue‐specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over‐expression of an acyl–acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in Camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

Role of Endogenous Growth Regulators in Seed Dormancy of Avena fatua: I. Short Chain Fatty Acids

The hypothesis that endogenous short chain fatty acids (C 6-C 10) are important in maintaining seeds of wild oat (Avena fatua L.) in the dormant state by acting as natural germination inhibitors (Berrie, Buller, Don, Parker, 1979 Plant Physiol 63: 758-764) was investigated. When germination of nondormant seeds was inhibited by treatment with short chain fatty acids, the seeds did not revert to a similar biochemical and physiological state as exhibited by dormant seeds. First, nonanoic acid-induced inhibition of seed germination was not reversed by hormone treatments which normally break dormancy in wild oat seeds. Second, nondormant seeds treated with short chain fatty acids maintained similar relative proportions of the pentose phosphate pathway and the Embden-Meyerhoff-Parnas pathway for respiratory glucose metabolism as that found in the nondormant controls. Seeds imbibed in the presence of nonanoic acid lost more amino acids and proteins into the imbibition solution than did the untreated controls, suggesting membrane damage had occurred. Inasmuch as increasing concentrations of nonanoic acid also progressively reduced the growth of the coleoptile and roots of intact seedlings until all growth ceased and no germination occurred, the inhibition of seed germination could be due to a nonspecific inhibition of growth of the embryo, perhaps because of disruption of membrane structure and function. Finally, no correlation between endogenous levels of short chain fatty acids in seeds or isolated embryonic axes and seed dormancy could be demonstrated.     

Regulated expression of the rat medium chain hydrolase gene in transgenic rape seed

Medium chain hydrolase (MCH) is an enzyme which regulates the chain length of fatty acid synthesis specifically in the mammary gland of the rat. During lactation, MCH interacts with fatty acid synthase (FAS) to cause premature release of acyl chains, thus providing medium chain fatty acids for synthesis of milk fat. In this study we have investigated the ability of rat MCH to interact with the phylogenetically more distant FAS structure present in plant systems and to cause a perturbation of fatty acid synthesis. Inin vitro experiments, addition of purified MCH to rapeseed homogenates was found to cause a significant perturbation of fatty acid synthesis towards medium chain length products. The rat MCH gene was expressed in transgenic oilseed rape using a seed specific rape acyl carrier protein (ACP) promoter and a rape ACP plastid targeting sequence. Western analysis showed MCH protein to be present in transgenic seed and for its expression to be developmentally regulated in concert with storage lipid synthesis. The chimaeric preprotein was correctly processed and immunogold labelling studies confirmed MCH to be localized within plastid organelles. However, fatty acid analysis of oil from MCH-expressing rape seed showed no significant differences to that from control seed.     

Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects

A bacterial phytoene synthase (crtB) gene was overexpressed in a seed-specific manner and the protein product targeted to the plastid in Brassica napus (canola). The resultant embryos from these transgenic plants were visibly orange and the mature seed contained up to a 50-fold increase in carotenoids. The predominant carotenoids accumulating in the seeds of the transgenic plants were alpha and beta-carotene. Other precursors such as phytoene were also detected. Lutein, the predominant carotenoid in control seeds, was not substantially increased in the transgenics. The total amount of carotenoids in these seeds is now equivalent to or greater than those seen in the mesocarp of oil palm. Other metabolites in the isoprenoid pathway were examined in these seeds. Sterol levels remained essentially the same, while tocopherol levels decreased significantly as compared to non-transgenic controls. Chlorophyll levels were also reduced in developing transgenic seed. Additionally, the fatty acyl composition was altered with the transgenic seeds having a relatively higher percentage of the 18 : 1 (oleic acid) component and a decreased percentage of the 18 : 2 (linoleic acid) and 18 : 3 (linolenic acid) components. This dramatic increase in flux through the carotenoid pathway and the other metabolic effects are discussed.     

Dormancy and Germination of Abscisic Acid-Deficient Tomato Seeds : Studies with the sitiens Mutant

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.     

Successful seed germination of the nickel hyperaccumulator Stackhousia tryonii

* BACKGROUND AND AIMS: Stackhousia tryonii, a rare nickel hyperaccumulating herb, is endemic to ultramafic (serpentine) soils of central Queensland, Australia. The effects of eight dormancy-relieving treatments on germination of stored seeds of Stackhousia tryonii were investigated under controlled light and temperature conditions. * METHODS: The treatments were: untreated (control i), leached and dehydrated (primed control ii), treating with gibberellic acid (150 and 300 microM), smoke extract (5 and 10 %, v/v) and potassium cyanide (40 and 80 mM). * KEY RESULTS: Freshly harvested seeds did not germinate. Germination percentage increased with time of storage for up to 18 months (38.3 %). Gibberellin, smoke extract and cyanide treatments did not significantly improve germination. Light did not affect seed germination and there was no interaction between dormancy-relieving treatments and light. A significant inhibition of germination occurred in seeds treated with 5 % (but not 10 %) aqueous smoke extract. Saturated fatty acids, predominantly tridecanoic (C13:0), constituted about 90 % of the total fatty acids in the oil of freshly harvested seeds. In contrast, there was increased accumulation (>75 %) of mono-unsaturated (oleic, c18:1) and poly-unsaturated (linoleic, c18:2; linolenic, c18:3) fatty acids in the oil of stored seeds. * CONCLUSIONS: Seeds of S. tryonii require an after-ripening period for germination.     

种子顽拗性的形成机理及其保存技术

根据种子的脱水行为将种子分为正常性种子、顽拗性种子和中间性种子。顽拗性种子在发育的末期不经历成熟脱水．脱落时有相对高的含水量,并且对低温和脱水干燥非常敏感。在自然界,顽拗性种子存在一个连续群,即低度、中度和高度顽拗性种子,其差异在于对脱水伤害的敏感程度。影响种子顽拗性的因素,既有种子本身的生理生化物质基础,也有种子在母株上发育过程中所经受的外界环境的影响。目前,对种子脱水耐性的分子机制及其保存技术研究得较多。本文综述了有关顽拗性种子研究的近期进展。     

Seed storage at elevated partial pressure of oxygen, a fast method for analysing seed ageing under dry conditions

Background and Aims

Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen.

Methods

Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 45 °C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed.

Key Results

The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under long-term dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 °C after equilibration at 85 % RH.

Conclusions

Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice.     

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.