Oleate desaturation and acyl turnover in sunflower (Helianthus annuus L.) seed lipids during rapid temperature adaptation

In-vivo experiments with developing sunflower (Helianthus annuus L.) seeds demonstrated that oleate desaturase activity was stimulated by low temperature (10 °C), repressed by high temperature (30 °C) and rapidly restored by returning the seeds to low temperature. Within time periods of 2–4 h, in which the de-novo fatty acid synthesis was negligible, the percentages of oleate (18:1) and linoleate (18:2) were modified in the seed lipids as a consequence of temperature adaptation. When the seeds were transferred to low temperature, the 18:2 content increased in all lipids from both microsomal membranes and oil bodies. After shifting to high temperature, the overall 18:2 content remained constant, but the 18:2 content decreased in diacylglycerols, phosphatidylcholine (PC) and other polar lipids of the two fractions and also in triacylglycerols (TAGs) of the microsomes but increased in TAGs of the oil bodies. The results indicate that the mechanism for the rapid adaptation of sunflower seeds to temperature changes involves (i) the synthesis or activation of oleate desaturase at low temperature and the reversible inhibition of this enzyme at high temperature and (ii) the exchange of 18:1 and 18:2 between TAGs and PC. Under both low and high temperature, 18:1 is transferred from reserve TAGs to PC and 18:2 is transferred from PC to reserve TAGs. At low temperature, 18:1 is desaturated to 18:2 thus allowing the enrichment of membrane lipids with 18:2, the excess being stored in reserve TAGs. At high temperature, however, and provided that oleate desaturase is repressed, the membrane lipids become enriched in 18:1 and the oil-body TAGs become enriched in 18:2. Received: 11 August 1997 / Accepted: 10 November 1997     

Oleate desaturation in young winter wheat root tissue

[1,2-¹⁴C]Acetate was incorporated into the lipids of young wheat (Triticum aestivum L. cv Kharkov 22 MC) root tissue, but predominantly into sterols. [1-¹⁴C]Ammonium oleate was initially incorporated mainly into phosphatidylcholine (PC), and later into triglycerides (TGs). Diglycerides (DGs) contained 16% of the lipid ¹⁴C after 5 minutes and 8% after 40 minutes. The proportion of the label of each lipid group incorporated into linoleate during an 80-minute incubation increased at similar rates for each group, and was always highest in PC. Radioactivity was detected in PC-linoleate earlier than in linoleate of the other groups. During a prolonged incubation after a 15-minute pulse labeling, the percentage of the lipid ¹⁴C incorporated into PC and DGs was high at the end of the pulse but decreased later, while that in TGs increased to 64% after 4 hours. The proportion of the label of each group recovered in linoleic acid peaked in all groups after 4 hours, except for the TGs where it increased slowly throughout the experiment. Only traces of radioactivity were detected in linolenate. The data are compatible with a pathway in which oleate is incorporated into PC, is desaturated to linoleate on PC, and where the linoleate-enriched DGs are transferred from PC to TGs.     

Oleate from triacylglycerols is desaturated in cold-induced developing sunflower (Helianthus annuus L.) seeds

For the first time, an active fatty-acid metabolism is indicated for triacylglycerols (TAG) of developing sunflower (Helianthus annuus L.) seeds. When the developing seeds were transferred to low temperature, the total amount of oleate found in TAG decreased as that of linoleate increased, while the contents of total lipids and TAG remained unchanged. These results suggest that oleate from TAG was used for desaturation. This occurred first in microsomal TAG, but after a long cold period it was observed mainly in the oil-body fraction. Thesn-2 position of TAG was preferentially enriched in linoleate. Apparently, more linoleate than necesary for the maintenance of membrane fluidity was synthesized at the expense of TAG oleate.     

Temperature and oxygen regulation of oleate desaturation in developing sunflower (Helianthus annuus) seeds

The effect of low (10°C) and high (30°C) temperature on in vivo oleate desaturation has been studied in developing sunflower ( Helianthus annuus L.) seeds under conditions of different oxygen availability (capitulum, detached achenes or peeled seeds). In seeds remaining in the capitulum, only a part of the oleate newly synthesized at high temperature was desaturated to linoleate, whereas more oleate than that synthesized de novo was desaturated at low temperature. Achenes were only able to significantly desaturate oleate at low temperatures. In contrast, oleate desaturation was detected in peeled seeds incubated at low and high temperatures, showing the highest rate at 20°C. Hull removing dramatically increased the activity of the microsomal oleate desaturase (FAD2, EC 1.3.1.35) at all studied temperatures, although a long-term inactivation of the enzyme was observed at high temperatures. Low oxygen concentration (1–2%) obtained by respiration of peeled seeds incubated in sealed vials, brought about the inactivation of the enzyme. All these data suggest that temperature regulates oleate desaturation controlling the amount of oleate and the FAD2 activity. In addition, this enzyme seems to be also regulated by the availability of oxygen, which is affected inside the achene by its diffusion through the hull, and the competition with respiration, both factors being temperature-dependent.     

Photochemical response to drought acclimation in two sunflower genotypes

The effects of drought acclimation on CO₂ assimilation and light utilization were investigated in two sunflower genotypes ( Helianthus annuus L., T32 and Viki) in relation to water deficit and/or high light conditions. Drought interaction with PSII efficiency was observed in the genotype T32 with a sustained decrease in the potential photochemical efficiency of PSII, F_n/F_m. In response to drought acclimation, T32 displayed some tendency to accumulate closed PSII traps (higher value of 1-qp) without an enhancement of thermal deactivation (Stem-Volmer non-photochemical quenching, NPQ). Irrespective of the growth conditions (growth chamber or greenhouse), only Viki was responsive to drought acclimation, with (1) increased net photosynthesis in well-watered plants, (2) higher maintenance of photochemical electron transfer under water deficit and/or high light, (3) limited PSII inactivation (lower value of 1-qp) through increased non-photochemical energy dissipation (Stern-Volmer NPQ) which was readily reversible even at low leaf water potentials, and (4) higher F_v/F_m recovery after high light treatment. Additionally, drought acclimation delayed turgor loss during subsequent water stress in Viki. Thus, the response to drought acclimation, with an adjustment of water relations and of energy utilization by PSII, was observed under both growth conditions and was mainly genotype dependent.     

Alternaria metabolites in sunflower seeds

The alternariol and alternariol monomethyl ether contamination in sunflower seeds was determined. The levels of alternariol found ranged between 35 to 792 µg/kg and 90 to 630 µg/kg for alternariol monomethyl ether. The fungicides showed different effect on the mycotoxin production depending on the substrate, strains and toxin analysed. Mercury phenyl acetate, Octave, Metiram, Thiram and Orthene inhibited alternariol monomethyl ether production while for alternariol production only Thiram, Metiram and Octave were effective.     

Diacylglycerol acyltransferase in maturing sunflower seeds

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-(14)C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent K(m) was 60 microM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.     

Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.)

Lack of a genotype-independent transformation protocol for sunflower is a major bottleneck in improving this important oilseed crop. An efficient Agrobacterium-mediated transformation protocol is described, which was adaptable across a broad range of sunflower genotypes. The improved transformation approach used cotyledons from mature seeds vertically split through the embryo axis. The LBA 4404 Agrobacterium strain was used, which carried pCAMBIA 2301 plasmid containing UidA as the reporter gene and nptII as the selectable marker for transformants on kanamycin. Bacterial titer, cotyledon type, acetosyringone concentration and vacuum application enhanced the transformation efficiency. Wounding, enzyme pretreatment and sonication significantly reduced the transformation frequency. Putative transgenic shoots were obtained through both axillary proliferation and adventitious shoot regeneration. Following two and three cycles of selection on kanamycin for axillary and adventitious shoots, respectively, putative transformed shoots were obtained at an average frequency of 3.0?%. Reporter gene histochemical assay and molecular characterization of primary and T₁ transgenic plants revealed stable transgene integration, expression and monogenic inheritance. The standardized procedure was tested on 28 genotypes comprising sets of inbred, maintainer, restorer and hybrid lines. Transformation was successful in all genotypes albeit with variable frequency in all except the hybrid lines wherein it was stable around 4.0?%. The procedure opens possibilities of directly improving any commercial genotypes of sunflower.     

Ageing-induced changes in glutathione system of sunflower seeds

The glutathione system is thought to be involved in defence mechanisms present in plant tissues. The efficacy of this system was evaluated in large seeds of sunflower ( Helianthus annuus L. cv. Peredovik) in response to accelerated ageing (43°C/75% relative humidity from 1 to 11 days). Differences between the embryo axis and cotyledons in relation to the glutathione system were also investigated. Additionally, lipid peroxidation was determined by measuring the malondialdehyde (MDA) content. All assays were performed using dry seeds and seeds subsequently hydrated by imbibition in distilled water for 12 h at 25°C. Accelerated ageing caused a marked decrease in seed viability, accompanied by an increase in mean germination time. There were no changes in total glutathione in dry seeds. However, the distribution in its reduced (GSH) and oxidized (GSSG) forms revealed that ageing produced a slow conversion from GSH to GSSG. As the ageing period increased, this effect was accompanied by a decrease in glutathione reductase (GR, EC 1.6.4.2) activity. The results also indicated that the GSH system exerts a different response in the embryo axis as compared with the cotyledon: (1) the GSH levels decreased less in the cotyledons than in axes of aged seeds, and (2) the GSSG level in cotyledons was independent of ageing, while its amount increased in aged embryo axes. These different responses, in conjunction with the lower MDA levels in large as compared with small seeds, indicate a possible protective role of the reserve lipids. The efficacy of the GSH system in aged seeds was associated with seed viability, as revealed by multiple regression analysis. Upon imbibition, aged seeds were able to restore their GSH levels, reaching values approximating those of unaged seeds.     

Genetic analysis of phytosterol content in sunflower seeds

Interest in phytosterol contents due to their potential benefits for human health has been largely documented in several crop species. Studies were focused mainly on total sterol content and their concentration or distribution in seed. This study aimed at providing new insight into the genetic control of total and individual sterol contents in sunflower seed through QTL analyses in a RIL population characterized over 2?years showing contrasted rainfall during seed filling. Results indicated that 13 regions on 9 linkage groups were involved in different phytosterol traits. Most of the QTL mapped were stable across years in spite of contrasted growing conditions. Some of them explained up to 30?% of phenotypic variation. Two QTL, located on LG10, near b1, and on LG14, were found to co-localize with QTL for oil content, indicating that likely, a part of the genetic variation for sterol content is only the result of genetic variation for oil content. However, three other QTL, stable over the 2?years, were found on LG1, LG4 and LG7 each associated with a particular class of sterols, suggesting that some enzymes known to be involved in the sterol metabolic pathway may determine the specificity of sterol profiles in sunflower seeds. These results suggest that it may be possible to introduce these traits as criteria in breeding programmes for quality in sunflower. The molecular markers linked to genetic factors controlling phytosterol contents could help selection during breeding programs.     

Identification of sunflower genotypes using microsatellite markers

Polymorphism of some DNA microsatellite sequences of sunflower breeding genotypes (inbred lines, hybrids) was investigated. Allelic composition on 8 SSR loci is unique characteristic of every analysed genotype allowing their identification. Microsatellite markers are proposed to be used for definition of line genetic purity and of F1 seeds hybridity range.     

Inhibitors of fatty acid biosynthesis in sunflower seeds

During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds.     

Analysis of globulin maturation in developing sunflower seeds

The synthesis of major storage globulin polypeptides has been examined in developing seeds of sunflower(Helianthus annuus L.). Analyses of total proteins and purified globulins, also called helianthinin, by gel electrophoresis and immunoelectrophoresis have shown that a burst of protein synthesis and accumulation occurs around 10 d after flowering. There is no mature globulin before that time and only small amounts of precursor forms can be detected. Thus, 10–12 d after flowering appears to be a transition period during which genetic information for the globulin becomes actively expressed. Immunoelectrophoresis has confirmed that globulin is the main storage protein, at seed maturation, accounting up to 70 % of total proteins per kernel. Pulse chase experiments have shown that synthesis initially involves the formation of high molecular mass precursors and that storage proteins are post-translationally processed. Intermediary products, with molecular mass higher than early translational products, can be detected, together with mature globulin polypeptides.     

Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

Leaf water characteristics and drought acclimation in sunflower genotypes

The responses of leaf water parameters to drought were examined using three sunflower (Helianthus annuus L.) genotypes. Osmotic potential at full water saturation (π¹⁰⁰), apoplastic water fraction (AWF) and bulk elastic modulus (BEM) were determined by pressure-volume curve analysis on well watered or on water-stressed plants (−1.0 MPa Ψ₁ < −1.5 MPa) previously drought-pretreated or not. The drought-pretreated plants were subjected to a 7-day drought period (predawn leaf water potential reached −0.9 MPa) followed by 8 days of rewatering. In well watered plants, all genotypes in response to drought acclimation displayed a significantly decreased π¹⁰⁰ associated with a decrease in the leaf water potential at the turgor-loss point (decrease in Ψ_tlp was between 0.15 and 0.21 MPa, depending on the genotype). In two genotypes, drought acclimation affected the partitioning of water between the apoplastic and symplastic fractions without any effect on the total amount of water in the leaves. As a third genotype displayed no modification of AWF and BEM after drought acclimation, the decreased π¹⁰⁰ was only due to the net accumulation of solutes and was consistent with the adjustment of the photochemical efficiency observed previously in this genotype in response to drought acclimation. In water-stressed plants, the osmotic adjustment (OA) can increase further beyond that observed in response to the drought pretreatment. However, the maintenance of photosynthetic rate and stomatal conductance at low leaf water potentials not only depends on the extent of osmotic adjustment, but also on the interaction between OA and AWF or BEM. Adaptative responses of leaf water parameters to drought are thus quite contrasted in sunflower genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and characterization of alpha-galactosidase from sunflower seeds

From 100 g sunflower seeds, 1.2 mg purified -galactosidase was obtained with an overall yield of 51%. The -galactosidase acted on both terminal -galactosyl residues and side-chain -galactosyl residues of the galactomanno-oligosaccharides and galactomannans. The cDNA coding for sunflower -galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40263. Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence. The deduced amino acid sequences of mature enzyme and -galactosidases from coffee, guar and Mortierella vinacea -galactosidase II showed over 81%, 77%, and 47% homology, respectively. These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups.     

Autoproteolytic processing of aspartic proteinase from sunflower seeds

The autoproteolytic processing of mature aspartic proteinase from sunflower seeds was investigated. The mature aspartic proteinase (48 kDa) was processed at N65s-D66s in the plant-specific region of the enzyme to form 34-kDa and 14-kDa subunits. The next step was the hydrolysis of the A25s-Q26s and N97s-E98s bonds to form a 39-kDa enzyme that consisted of 29-kDa and 9-kDa disulfide-bonded subunits. Finally, bonds including V1s-M2s, M2s-S3s, C100s-D101s, and D101s-R102s were cleaved to form non-covalently bound subunits (29 kDa and 9 kDa) by eliminating the disulfide bonds in the plant-specific region of the protein.     

Temperature-related non-homogeneous fatty acid desaturation in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) seeds

The fatty acid compositions of half-seeds and whole seeds of the temperature-dependent high-stearic-acid sunflower (Helianthus annuus L.) mutant CAS-14 were unexpectedly different. We found that there is a longitudinal gradient starting from the embryo up to the end of the cotyledon. The stearic acid content varied from 9.7 to 34.6% in seeds produced in a growth chamber (39/24 degrees C; day/night), and from 14.0 to 34.4% in seeds produced in the field during the summer season (35-40 degrees C in daylight and 20-25 degrees C at night). The gradient occurs throughout seed formation, and is due to a spatial and non-temporal regulation of stearic acid desaturation. A similar temperature-regulated behaviour, but for oleic and linoleic acid contents, was found in normal sunflower seeds. Since the deposition of oil bodies was homogeneous during seed formation, seeds showed the gradient throughout their development. This non-homogeneous distribution must be due to differences in the enzymatic pathway of de-novo fatty acid desaturation along the seed, resembling a morphogen gradient. Other high-stearic-acid mutant lines, such as CAS-3, did not show any gradient. This is the first time that a gradient and an inheritable maternal control of the fatty acid composition have been found in oilseeds.