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     

Enzymatic characterisation of high-palmitic acid sunflower (Helianthus annuus L.) mutants

Two high-palmitic acid sunflower (Helianthus annuus L.) mutants, CAS-5 and CAS-12, have been biochemically characterised. The enzymatic activities found to be responsible for the mutant characteristics are β-keto-acyl-acyl carrier protein synthetase II (KASII; EC 2.3.1.41) and acyl-acyl carrier protein thioesterase (EC 3.1.2.14). Our data suggest that the high-palmitic acid phenotype observed in both mutant lines is due to the combined effect of a lower KASII activity and a higher thioesterase activity with respect to palmitoyl-acyl carrier protein (16:0-ACP). The level of the latter enzyme appeared to be insufficient to hydrolyse the produced 16:0-ACP completely. As a consequence of this, three new fatty acids appear: palmitoleic acid (16:1 Δ9), asclepic acid (18:1 Δ11), and palmitolinoleic acid (16:2 Δ9 Δ12). These fatty acids should be synthesised from palmitoyl-ACP or a derivative by the action of the stearoyl-ACP desaturase, fatty acid synthetase II and oleoyl-phosphatidylcholine desaturase, respectively. Received: 11 July 1998 / Accepted: 10 October 1998     

Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.)

A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).     

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.     

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.     

Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production

Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10^–2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - IBA indole-3-butanoic acid - IAA indole acetic acid - MES 2-N-morpholinoethane sulfonic acid - NAA 1-naphthalene acetic acid - 2,4D 2,4 dichlorophenoxyacetic acid     

Transformation of sunflower (Helianthus annuus L.): a reliable protocol

Summary A reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A minority of plants was shown to be chimaeric for expression of ß-glucuronidase activity while most appeared to be uniformly transformed. Genetic segregation was 31 for both ß-glucuronidase and neomycine phospho transferase in some plants, indicating that the respective mother plants were uniformly transformed. Integration of the foreign genes was also shown by Southern analysis.Abbreviations BAP benzyl amino purine - EDTA ethylene diamine tetraacetic acid - GUS ß-glucuronidase - npt II neomycine phospho-transferase II     

Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.)

Summary Sunflower hypocotyl protoplasts (Helianthus annuus L.) from 5 PIONEER genotypes (PT024, SMF3, EMIL, HA300*PT024, VK5F) and 1 public line (RHa 274) formed colonies at frequencies of up to 60% when plated in 0.25ml agarose beads in a modified L4 medium (Lenée and Chupeau 1986) containing 3mg/l NAA, 1mg/l BA and 0.1mg/l 2,4-D, and 1000mg/l casamino acids. Protoplast-derived colonies grew slowly into calli. Organogenesis was obtained from callus of PT024 on a MS medium containing NAA and BA at 1mg/l and GA at 0.1mg/l. Freshly excised shoots were induced to root by an IAA treatment. Regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.Abbreviations BA 6-benzylaminopurine - NAA -naphtaleneacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog mineral elements - B5 Gamborg mineral elements     

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.     

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

Summary Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FeNaEDTA ethylenediamine tetraacetic acid ferric sodium salt - IAA indole acetic acid - MES morpholinoethane sulfonic acid - NAA 1-naphtalene acetic acid     

The effects of methyl jasmonate on sunflower (Helianthus annuus L.) seed germination and seedling development

Some effects of methyl jasmonate (Me-Ja) on sunflower (Helianthus annuus L.) seed germination and seedling development are described and compared with those of ABA. Both growth regulators have very similar action. They inhibit germination, but high concentrations of O₂ in the atmosphere suppress this inhibitory action. Depending on the concentration, Me-Ja inhibits root and hypcotyl growth, however the root is more sensitive to Me-Ja than to ABA. Me-Ja also strongly reduces oxygen uptake during germination and inhibits chlorophyll biosynthesis in isolated cotyledons.     

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.     

Kinetic properties of phenylalanine ammonia-lyase from sunflower (Helianthus annuus L.) hypocotyls.

Phenylalanine ammonia-lyase (PAL) from sunflower hypocotyls has been partially purified by selective precipitation with ammonium sulfate and molecular gel filtration on Sephacryl S-300. Kinetic assays carried out with this partially purified PAL preparation revealed that the enzyme did not show a homogeneous kinetic behaviour. The observed kinetic pattern and parameters (Km and Vmax) depended on the assay conditions used and the protein concentration added to the assay mixture. PAL displayed Michaelian or negative cooperativity kinetics. Such behaviour can be explained by the existence of an association-dissociation process of PAL-protein subunits. The presence of mono-, tri- and tetrameric forms of PAL has been assessed by molecular gel filtration on Sephacryl S-200, using different elution conditions.     

Isolation of HMW DNA from sunflower (Helianthus annuus L.) for BAC cloning

The cultivated sunflower (Helianthus annuus L.) is one of the most important oil crops in the world. The importance of sunflower oil in human nutrition and in the chemical industry makes the sunflower a major research interest. An essential element for genomic libraries is the preparation of high molecular weight (HMW) DNA. We developed 2 methods for isolating HMW sunflower DNA. We prepared the DNA from nuclei and from protoplasts isolated from mesophyll tissue with the enzymes cellulase RS and pectolyase Y23. The HMW DNA was digested with restriction endonucleases. The ethidium bromide-stained gel suggested the DNA to be completely digested. These results were confirmed by Southern analysis using a radiolabeled RFLP marker. Both methods made it possible to generate sufficient quantities of megabase-size sunflower DNA suitable for bacterial artificial chromosome (BAC) cloning.     

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.     

Screening of plant growth promoting Rhizobacteria isolated from sunflower (Helianthus annuus L.)

Background and Aims

This study was aimed at assessing the diversity of putatively diazotrophic rhizobacteria associated with sunflower (Helianthus annuus L.) cropped in the south of Brazil, and to examine key plant growth promotion (PGP) characteristics of the isolates for the purposes of increasing plant productivity.

Methods

299 strains were isolated from the roots and rhizosphere of sunflower cultivated in five different areas using N-free media. 16S rDNA PCR-RFLP and 16S rRNA partial sequencing were used for identification and the Shannon index was used to evaluate bacterial diversity. Production of siderophores and indolic compounds (ICs), as well phosphate solubilization activities of each isolate were also evaluated in vitro. On the basis of multiple PGP activities, eight isolates were selected and tested for their N-fixation ability, and their capacity as potential PGPR on sunflower plants was also assessed.

Results

All except three Gram-positive strains (phylum Actinobacteria) belonged to the Gram-negative Proteobacteria subgroups [Gamma (167), Beta (78), and Alpha (50)] and the family Flavobacteriaceae (1)]. Shannon indexes ranged from 0.96 to 2.13 between the five sampling sites. Enterobacter and Burkholderia were the predominant genera isolated from roots and rhizosphere, respectively. Producers of siderophores and ICs were widely found amongst the isolates, but only 19.8% of them solubilized phosphate. About 8% of the isolates exhibited all three PGP traits, and these mostly belonged to the genus Burkholderia. Four isolates were able to stimulate the growth of sunflower plants under gnotobiotic conditions.

Conclusions

Enterobacter and Burkholderia were the dominant rhizospheric bacterial genera associated with sunflower plants. Inoculation with isolates belonging to the genera Achromobacter, Chryseobacterium, Azospirillum, and Burkholderia had a stimulatory effect on plant growth.     

EDTA-enhanced lead phytoremediation in sunflower (Helianthus annuus L.) hydroponic culture

The aim of the present study was to investigate the capability of Sunflower (Helianthus annuus L.) to tolerate and accumulate high amount of lead (Pb) and propose it for soil phytoremediation. To this regard, plants were grown in hydroponics and treated with different Pb concentrations (10 to 160 ??M) and a fixed concentration (500 ??M) EDTA (ethylene diamine tetra acetic acid) for 14 and 28 days (d). Effects on total biomass production, photosynthetic pigments and protein contents as well as the quantities of non protein thiols (NP-SH), glutathione (GSH), phytochelatins (PCs) and activity of glutathione reductase (GR) were estimated. Results revealed that roots (575 ??g g^?1 DW) and shoots (135 ??g g^?1 DW) accumulated Pb after 28 d of exposure, however, addition of EDTA enhanced the Pb accumulation in roots (645 ??g g^?1 DW) and shoots (255 ??g g^?1 DW ). Exposure of Pb (28 d) registered a significant (P?<?0.05) reduction in growth parameters and induction of phytochelatins (P?<?0.05; r?=?0.26) plus some of the important antioxidants (P?<?0.05; r?=?0.42), which were positively correlated to metal accumulation. Sunflower exposed at 40 ??M of Pb for 28 d synthesized higher quantity of PC₂ (18.5 fold) and PC₃ (10.5 fold), as compared to control. However, the results showed that addition of EDTA resulted in low toxicity compared to Pb alone. These data support the capability of H. annuus L. to accumulate and tolerate significant quantity of Pb and its utility for phytoremediation. This is because of the plant has the capacity to combat metal induced oxidative stress via significant synthesis of NP-SH, GSH and high activity of GR, as it would provide sufficient GSH not only for PCs synthesis but also for antioxidant function.     

A composite map of expressed sequences and phenotypic traits of the sunflower (Helianthus annuus L.) genome

 A map of the sunflower genome, based on expressed sequences and consisting of 273 loci, was constructed. The map incorporates data from seven F₂ populations, for a total of 1115 individuals. Two hundred and fourty five loci corresponding to 170 anonymous cDNA markers and four loci for morphological markers were mapped. We also mapped 18 loci corresponding to previously described genes or to sequences obtained through homology cloning. The unit maps vary from 774 cM to 1060 cM, with an average value of 14 major linkage groups. The integrated map is arranged in 17 major linkage groups including 238 loci, plus four small segments with 2–5 marker loci; and covers 1573 cM with an overall average marker interval of 7 cM. Thirty five percent of the markers were dominant in nature and 30% showed inter-linkage group duplication without any indication of homoeologous linkage groups. Evidence is provided for the independence of two distinct fertility restoration genes, for the presence of two loosely linked branching loci, and for marker tightly linked to the Rf1 restoration locus. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection. Received: 27 August 1998 / Accepted: 28 December 1998     

Effect of source–sink ratio on seed set and filling in sunflower (Helianthus annuus L.)

Poor seed development in sunflower may result from insufficient assimilate supply (source limitation). To test this hypothesis, the effects of changed source–sink ratio on seed set (measured as percentage of empty achenes) and seed filling (measured as dry mass per filled achene) in individual plants were investigated. Source–sink ratio, defined as leaf area per floret (LAF), was experimentally altered using invasive (floret removal, defoliation) and non‐invasive (pulse of chilling, short days or shading during leaf or floret initiation) treatments. Shading at floret initiation proved the most effective non‐invasive method. Generally, an increase, or decrease, in LAF improved, or impaired, both seed set and filling. Increasing LAF by 2.0 cm²[95% confidence interval (1.5, 2.5)] decreased the percentage of empty achenes by 36.9%‐points (?41.9, ?30.9) and increased dry mass per filled achene by 20.1 mg (13.6, 26.7) in the capitulum centre. The effect of source–sink ratio on seed set was always strongest in the centre, whereas peripheral whorls were not affected. Achene mass was affected in all parts of the capitulum. It is concluded that source limitation is a major cause for empty achenes in sunflower plants grown under non‐stress conditions.