Relations between heterosis and enzymatic polymorphism in populations of cultivated sunflowers (Helianthus annuus L.)

Nine polymorphic isoenzymatic systems were studied in 39 cultivated sunflower populations originating from ten countries. Analysis of combining abilities with four tester lines was also performed on these populations for seed yield, seed moisture and seed oil content. The MDH, PGI, PGD and GOT systems appeared to provide the best discrimination of specific combining ability effects with the four testers. The MDH and GOT systems provided a between-population structure that was consistent with the country of origin.Abbreviations MDH Malate dehydrogenase - PGD phosphogluconate dehydrogenase - PGI phosphoglucoisomerase - PGM phosphoglucomutase - ACO aconitase hydratase - ADH2 alcohol dehydrogenase - GOT glutamate oxaloacetate transaminase - LAP leucine amino peptidase - EST esterases     

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.     

Prolyl iminopeptidase from seeds of sunflower (Helianthus annuus L.)

Prolyl iminopeptidase from sunflower seed (Helianthus annuus L.) was purified to molecular homogeneity. It is a 105-kDa heterodimer consisting of two subunits: 53 and 55 kDa. It has pI of 6.2 and optimal activity at pH 8.0–8.5 and 45–50°C. The inhibitory analysis was inconclusive about its catalytic machinery, as a significant degree of modification was not observed with any of the used diagnostic inhibitors. Its specificity is restricted to removal of N-terminal prolyl residues.     

Temperature regulation of oleate desaturase in sunflower (Helianthus annuus L.) seeds

The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-¹⁴C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10 C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.Abbreviations DAF days after flowering - ODS oleoyl phosphatidylcholine desaturase - PC phosphatidylcholine - PE phosphatidylethanolamine - TAG triacylglycerol - 181 oleic acid - 182 linoleic acid To whom correspondence should be addressedThanks are due to M.C. Ruiz for skillful technical assistance. This work was supported by a grant from Junta de Andalucia, Spain.     

A species-selective allelopathic substance from germinating sunflower (Helianthus annuus L.) seeds

From the exudate of germinating sunflower (Helianthus annuus L.) seeds was isolated a stereoisomer of diversifolide, 4, 15-dinor-3-hydroxy-1(5)-xanthene-12,8-olide (designated sundiversifolide) as determined by analysis of its IR, APCI-, ESI- and HR-MS and 13C and 1H NMR spectra. This substance inhibited shoot and root growth of cat's-eyes by about 50% at a concentration of 30 ppm. It also showed species-selective activity on the shoot and root growth of tested plants. When cat's-eyes seeds were incubated together with sunflower seeds, the cat's-eyes growth was inhibited. Furthermore, it was detected from an extract of river sand when sunflower seeds were incubated on the sand. These results indicate that sundiversifolide has an allelopathic function in sunflower 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     

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds

The glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.1.15) from sunflower (Helianthus annuus L.) microsomes has been characterised and partially purified. The in vitro determination of activity was optimized, and the maximum value for GPAT activity identified between 15 and 20 days after flowering. The apparent Michaelis–Menten K_m for the glycerol 3-phosphate was 354 μM. The preferred substrates were palmitoyl-CoA = linoleoyl-CoA > oleoyl-CoA with the lowest activity using stearoyl-CoA. High solubilisation was achieved using 0.75% Tween80 and the solubilised GPAT was partially purified by ion-exchange chromatography using a Hi-Trap DEAE FF column, followed by gel filtration chromatography using a Superose 12 HR column. The fraction containing the GPAT activity was analysed by SDS-PAGE and contained a major band of 60.1 kDa. Finally, evidence is provided which shows the role of GPAT in the asymmetrical distribution, between positions sn-1 and sn-3, of saturated fatty acids in highly saturated sunflower triacylglycerols. This work provides background information on the sunflower endoplasmic reticulum GPAT which may prove valuable for future modification of oil deposition in this important crop.     

Vascular connections between the receptacle and empty achenes in sunflower (Helianthus annuus L.)

Empty achenes in sunflower, particularly in the centre of the capitulum, may be caused by poor vascularization. This hypothesis was tested by microscopic examination and translocation experiments. Phloem and xylem were identified by fluorescence of aniline-blue-stained callose and autofluorescence, respectively. Vascular strands that extended from the receptacle into empty achenes were regularly found in longitudinal sections. The phloem-mobile probe, carboxyfluorescein, was translocated from the receptacle to the pericarp and the testa of empty achenes. Similarly, (14)CO(2)-derived (14)C-photoassimilates moved into empty achenes. The observations suggest that empty achenes are both structurally and functionally connected with the vascular system of the receptacle. Hence, deficient vascular connections do not prevent seed filling in sunflower.     

Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos

The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.     

Systemic acquired resistance in sunflower (Helianthus annuus L.)

Systemic acquired resistance (SAR) to infection by Botrytis cinerea in the leaves of sunflower (Helianthus annuus L.) plants was induced following cotyledon inoculation with B. cinerea or treatment with abiotic inducers. Salicylic acid (SA), benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA) or EDTA protected sunflower plants against Botrytis infection, that was revealed by a reduction in the number and area of the necrotic lesions in upper leaves after challenge inoculation with the pathogen. SA and BTH were more potent inducers than INA, EDTA or pre-inoculation with the fungus. In addition to resistance to B. cinerea, the upper leaves have also developed resistance to maceration by a mixture of cell wall-degrading enzymes. Calcium nitrate inhibited both the protective effect and the resistance of leaf discs to cell-wall degrading enzymes. All the tested chemicals increased the synthesis and excretion of sunflower phytoalexins--coumarins scopoletin and ayapin and induced the PR-proteins chitinase and 1,3-beta-glucanase, being the inducer effect of each activator correlated with the level of protection against B. cinerea (BTH > SA > INA > EDTA). Thus, SAR induction is mediated by general increase of plant defence responses. This is the first report on SAR in sunflower.     

Genetics of species differences in the wild annual sunflowers, Helianthus annuus and H. petiolaris

Much of our knowledge of speciation genetics stems from quantitative trait locus (QTL) studies. However, interpretations of the size and distribution of QTL underlying species differences are complicated by differences in the way QTL magnitudes are estimated. Also, many studies fail to exploit information about QTL directions or to compare inter- and intraspecific QTL variation. Here, we comprehensively analyze an extensive QTL data set for an interspecific backcross between two wild annual sunflowers, Helianthus annuus and H. petiolaris, interpret different estimates of QTL magnitudes, identify trait groups that have diverged through selection, and compare inter- and intraspecific QTL magnitudes. Our results indicate that even minor QTL (in terms of backcross variance) may be surprisingly large compared to levels of standing variation in the parental species or phenotypic differences between them. Morphological traits, particularly flower morphology, were more strongly or consistently selected than life history or physiological traits. Also, intraspecific QTL were generally smaller than interspecific ones, consistent with the prediction that larger QTL are more likely to spread to fixation across a subdivided population. Our results inform the genetics of species differences in Helianthus and suggest an approach for the simultaneous mapping of inter- and intraspecific QTL.     

Changes in mass and dimensions of sunflower (Helianthus annuus L.) Achenes and seeds due to carbonization

When analyzing sunflower (Helianthus annuus L.) remains, which are often carbonized, archaeobotanists commonly differentiate between wild and domesticated achenes and seeds based on the measured length (L) and width (W) or the calculated index L*W. Carbonization reduces the dimensions. To compensate for these reductions, archaeobotanists use a single correction factor proposed by Richard Yarnell (1978) for all cases. The use of a single correction factor can bias the reconstructed dimensions as carbonization is a highly variable process. The current study determines the relationship between carbonization and the dimensions of length and width. Measurements established that a decrease of 2.5-22.5% in achene length and 10-29% in achene width can occur, depending on temperature, heating rate, and variety. For seeds, temperature is of most importance, and shrinkage ranges from 0-27% for the length and from 0-20% for the width. These ranges make the use of a single correction factor problematic. A method is developed in which reflectance (an optical property applied in coal technology to determine coal rank) is used to measure the carbonization temperature, and in turn the shrinkage can be calculated. Subsequently, correction factors are calculated to reconstruct the original length and width. When applied to an assemblage of carbonized sunflower achenes, the newly developed method shows that the Yarnell single correction factor may bias the dimensions towards classifications of “wild” or “ruderal” forms of sunflower     

Unsaturated fatty acid synthesis in sunflower (Helianthus annuus L.) seeds in response to night temperature

The incorporation of ¹⁴CO₂ into unsaturated fatty acids during seed development was measured in sunflowers grown in controlled environments with day temperatures of 28°C and night temperatures of 15°C or 22°C. While the average temperatures to which the plants were exposed did not differ greatly, the ratio of linoleic acid to oleic acid synthesized was much greater at a night temperature of 15°C than at 22°C. These results support the proposal (Harris et al. 1978) that the mean minimum temperature experienced during seed development is the major environmental factor influencing the unsaturated fatty acid composition of sunflower seed oil.     

Penicillium yugoslavicum sp. nov., isolated from sunflower seeds (Helianthus annuus L.) in Yugoslavia

A new species of Penicillium Link ex Fries is described and illustrated. It was recovered from sunflower seed (Helianthus annuus L.) in Yugoslavia. It clearly differs from all species of the genus described so far and is, therefore, proposed as a new taxon: Penicillium yugoslavicum sp. nov.     

Novel proteinase inhibitors in seeds of sunflower (Helianthus annuus L.): polymorphism, inheritance and properties

A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the M _r of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in M _r from about 1,500 to 6,000. Received: 7 March 1999 / Accepted: 18 March 1999     

Cavitation fatigue and its reversal in sunflower (Helianthus annuus L.)

"Cavitation fatigue" is the increased susceptibility of a xylem conduit to cavitation as a result of its prior cavitation. It was investigated whether cavitation fatigue induced in vivo could be repaired in intact plants. Sunflowers (Helianthus annuus L.) were subjected to soil drought in the greenhouse. Native embolism and vulnerability to cavitation was measured in well-watered controls and after 5 d and 10 d of controlled drought. A dramatic cavitation fatigue was observed where droughted xylem that was refilled in the laboratory developed up to 60 PLC (percentage loss of hydraulic conductivity) at -1 MPa versus only 5.2 PLC in non-droughted controls. Rewatered plants showed the complete reversal of cavitation fatigue over 4 d. Reversal of fatigue was correlated with the refilling of embolized vessels in the intact plants (r(2)=0.91, P<0.01), suggesting that xylem transport to fatigued vessels was required for their repair. The in vivo reversal of fatigue was partially duplicated in excised stem segments by perfusing them with root exudates from droughted (DR) and well-watered (WW) plants. The DR exudate had a greater effect, and this was associated with a greater pH in the DR versus WW saps, but there was no difference in total cation concentration. Perfusions with 2 mM CaCl(2) and KCl solutions also partially reversed cavitation fatigue as opposed to no effect with deionized water, suggesting a role of ions in addition to a pH effect. It is suspected that fatigue is caused by stretching and partial disruption of linkages between cellulose microfibrils in inter-conduit pit membranes during air seeding, and that the reversal of fatigue involves restoring these linkages by ingredients in xylem sap.     

Production of stable transgenic sunflowers (Helianthus annuus L.) from wounded immature embryos by particle bombardment and co-cultivation with Agrobacterium tumefaciens

Split embryonic axes of 21-day old immature sunflower (Helianthus annuus L.) embryos were bombarded by microparticles and then co-cultured with disarmed Agrobacterium tumefaciens strain EHA105 bearing a binary vector carrying nptII and uidA genes. Apical shoot bud development and organogenesis induced on the explants led T₀ transgenic plants. Southern blot analysis revealed complex integration patterns in T₀ plants. The uidA gene segregated as a dominant trait and single-insertion events were observed in T₁ plants. Patterns similar to those of T₁ plants were observed in T₂ progeny.     

The role of lysophosphatidylcholine in lipid synthesis by developing sunflower (Helianthus annuus L.) seed microsomes

The incorporation of oleate from oleoyl-CoA into lipids by microsomes from developing sunflower (Helianthus annuus L.) seeds has been investigated. Oleate was incorporated mainly into position 2 of phosphatidylcholine or released as free fatty acid. The addition of exogenous 1-acyl-lysophosphatidylcholine increased the incorporation of oleate into position 2 of phosphatidylcholine and decreased the release of free oleate. In the absence of exogenous lysophosphatidylcholine, the incorporation of oleate into phosphatidylcholine was limited by the amount of endogenous acceptor present. DH-990, an inhibitor of acyl-CoA:lysophosphatidylcholine acyltransferase, almost completely inhibited the incorporation of oleate from oleoyl-CoA into phosphatidylcholine at a concentration of 2.5 mM. These results indicate that the incorporation of oleate from oleoyl-CoA into microsomal phosphatidylcholine occurs mainly by the acylation of a 1-acyl-lysophosphatidylcholine acceptor rather than by acyl exchange between oleoyl-CoA and phosphatidylcholine. While the incorporation of oleoyl-CoA was completed within 2 to 5 min, exogenous 1-acyl-lysophosphatidylcholine was incorporated into phosphatidylcholine for up to 30 min. Addition of oleoyl-CoA resulted in an increase in both the rate and magnitude of lysophosphatidylcholine incorporation, which could not be accounted for by a stoichiometric reaction between the two substrates. Evidence is provided that free CoA had an independent stimulatory effect on the incorporation of lysophosphatidylcholine. The implications of this finding are discussed.