A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Exon shuffling in anther-specific genes from sunflower

We describe here the nucleotide sequence of an anther-specific gene (sf18) from sunflower, encoding a proline- and glycine-rich polypeptide with a hydrophobic amino-terminus (signal peptide). The gene is split by a 211 by intron and is partially related to another anther-specific gene (sf2) from sunflower with which it shares important sequence stretches in the 5 coding and upstream regions. We propose that the two genes have originated via exon shuffling, during which a copy of a DNA segment including the promoter region as well as a signal peptide coding sequence has been transferred into the upstream region of two different potential coding sequences, generating two novel genes which display the same specificity of expression and which both encode an extracellular protein. While the 5 region of the intron is highly conserved as part of the transferred region and may play a role in the selection of the 5 splice site, a common octanucleotide at the 3 end of the intron of the two genes might be involved in 3 splice site selection.     

Characterization of a sunflower (Helianthus annuus L.) mutant,deficient in carotenoid synthesis and abscisic-acid content,induced by in-vitro tissue culture

Genetic variation induced by tissue culture has been characterized in many species. The present study was conducted to genetically and phenotypically characterize an albino mutant in sunflower induced by in-vitro culture. A single recessive gene defective in carotenoid biosynthesis eventually leads to a chlorophyll loss due to photobleaching, absence of seed dormancy, and a low level of endogenous abscisic acid (ABA) in cotyledons and leaves. Further characterization has shown that the endogenous level of the hormone does not increase after drought stress and that the mutation prevents anthocyanin synthesis.     

Relationship between polyamines and paraquat toxicity in sunflower leaf discs

Polyamines have been reported as efficient antioxidantcompounds in plants. Sunflower leaf discs, treatedwith 100 µM paraquat (PQ), a well known oxidativestress inducer, showed decreased levels of putrescine(Put), spermidine (Spd) and spermine (Spm) (between33% and 80% with respect to the controls). Argininedecarboxylase (ADC) and ornithine decarboxylase (ODC)activities decreased 42% and 33% respectively. Amongthe markers of oxidative stress measured after PQtreatment, chlorophyll and glutathione content werereduced (30% and 49% respectively) andthiobarbituric acid reactive substances (TBARS)content increased (60%). Superoxide dismutase (SOD)activity declined 60% with respect to the control andlipoxygenase (LOX) increased 25% when leaf-discs weretreated with the herbicide. Pretreatment withexogenous polyamines (1 mM) reversed paraquat toxicityto different degrees according to the polyamine and/orthe tested parameter. Spermidine was able to inhibitchlorophyll loss, while Spm reverted the effect of PQon the level of TBARS almost completely and alsorestored SOD activity close to control values.Putrescine was the least effective as an oxidantprotectant. These results provide support for theargument that polyamines are effective antioxidantsthrough their ability to act as radical scavengers.     

Light-induced inhibition of elongation growth in sunflower hypocotyls

Summary The long-term effects of white light (WL) on epidermal cell elongation and the mechanical properties and ultrastructure of cell walls were investigated in the subapical regions of hypocotyls of sunflower seedlings (Helianthus annuus L.) that were grown in darkness. Upon transition to WL a drastic inhibition of epidermal cell elongation was observed. However, the mechanical properties of the inner tissues (cortex, vascular bundles, and pith) were unaffected by WL. Thus, the light-induced decrease in cell wall plasticity measured on entire stems occurs exclusively in the peripheral tissues (epidermis and 2 to 3 subepidermal cell layers).An electronmicroscopic investigation of the epidermal cell walls showed that they are of the helicoidal type with the direction of microfibrils monotonously changing during deposition. This cell wall type was identified by the appearance of arced patterns of microfibrils in cell walls sectioned oblique to the plane of their synthesis. WL irradiation did not change the periodicity of this pattern nor the thickness of the lamellae. Thus, the inhibition of cell elongation was not caused or accompanied by a shift in the direction of microfibril deposition in the growth-limiting outer tissues. However, cell wall thickness, the number of lamellae and hence the amount of cellulose oriented parallel and transverse to the longitudinal cell axis increased in WL. This may account for the effect of WL on the reduction of cell wall plasticity and growth.Abbreviations D darkness - PATAg periodic acid-thiocarbohydracide-silver protein - WL white light     

Leaf area estimation of sunflower leaves from simple linear measurements

Simple, accurate, and non-destructive methods for determining leaf area (LA) of plants are important for many experimental comparisons. Determining the individual LA of sunflower (Helianthus annuus L.) involves measurements of leaf parameters such as length (L) and width (W), or some combinations of these parameters. Two field experiments were carried out during 2003 and 2004 to compare predictive equations of sunflower LAs using simple linear measurements. Regression analyses of LA vs. L and W revealed several equations that could be used for estimating the area of individual sunflower leaves. A linear equation having W² as the independent variable provided the most accurate estimate (r ² = 0.98, MSE = 985) of sunflower LA. Validation of the equation having W² of leaves measured in the 2004 experiment showed that the correlation between calculated and measured areas was very high.     

High frequency embryogenesis in immature zygotic embryos of sunflower

In the present investigation, nutritional requirements for induction of a high frequency of well formed somatic embryos (SEs) from zygotic embryos (ZEs) of sunflower were assessed. Variables like genotype, embryo size (0.5–10 mm), sucrose concentration (30–240 g l⁻¹), carbohydrate source (sucrose, glucose, maltose), agar strength (0.2–1.0%), basal media (MS, Gamborg, Nitsch, White), photoperiod (light/dark) and temperature (20–36°C) were tested. All these variables except photoperiod had significant effect on the frequency of embryogenesis. Highest frequency of embryogenesis was facilitated by Gamborg basal salt media, 120–210 g l⁻¹ sucrose, 0.8–1.0% agar, smaller sized embryos (0.5–2 mm) and incubation temperature of 28–32°C. In addition to these, growth regulator combinations (2,4-D, 2,4-D+kinetin, BA+NAA) in varying concentrations were tried. Media supplemented with 2,4-D promoted direct embryogenesis, BA+NAA facilitated formation of single/multiple shoots while there was no response on 2,4-D+kinetin supplemented media. Zygotic embryos with well differentiated embryos were transferred to growth regulator free half strength MS medium for whole plantlet development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variation in form and size of Plasmopara halstedii (sunflower downy mildew) zoosporangia

Zoosporangia form and size were studied on a collection of 94 strains of Plasmopara halstedii (sunflower downy mildew). Both oval and round forms were present in all strains analysed. The proportion of two forms varied significantly according to strain and plant age but more especially to host plant genotype. Whatever the strain or host genotype, oval zoosporangia were larger than round ones, but there was no relation between the proportion of the oval form and mean zoosporangia size. There was no relation between zoosporangia form or size and race virulence profiles or aggressiveness criteria, with the possible exception of zoosporangia size and sporulation density. It is concluded that, for this obligate parasite, although form and size of zoosporangia depend on pathogen strain, these characters also vary according to growth conditions of Plasmopara halstedii, in particular to the genotype of the plant host.     

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

Summary A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (–)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.Abbreviations PAA phenylalkylamine - DHP dihydropyridine - FDA fluorescein diacetate     

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     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

Reduction of hyperhydricity in sunflower tissue culture

In order to reduce the occurrence of hyperhydrated shoots, the response of three sunflower inbred lines was examined on regeneration media containing various concentrations of kinetin, silver nitrate, and casein hydrolysate, calcium nitrate and cobalt nitrate. There were differences among the inbred lines for all the parameters taken into account to outline the in vitro efficiency. Percentage of hyperhydrated primordia, average number of shoots and of primordia per total explants, and percentage of hyperhydrated shoot traits differed among all media. The genotype × culture media interaction was significant for average number of shoots and primordia per total explants, regeneration percentage and percentage of hyperhydrated primordia. Among all media tested, those containing silver nitrate significantly reduced hyperhydricity in a dose-dependent way. The addition of silver nitrate showed to be useful in improving the quality of sunflower micropropagated plants by reducing this undesirable phenomenon.