Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings

Removal of the main root system of sunflower ( Helianthus annuus ) initiates adventitious root development on the lower portion of the hypocotyl. The first cytological changes (enlarged nuclei in the interfascicular parenchymatous cells adjacent to the phloem and some cell divisions) are observed 24 h after root excision. On the basis of experiments in which (a) roots, apical buds and various amounts of cotyledonary tissue were removed, (b) cuttings were subjected to various light regimes, (c) benzyladenine oas applied to cotyledons to create an artificial sink, it was concluded that the roots normally produce factors inhibiting to adventitious rooting and might be a sink for stimulatory substances produced in the shoots. The cotyledons seem to be the major source of these stimulators. Application of aqueous and ethanolic extracts of cotyledons and hypocotyls to cuttings promoted adventitious rooting.     

Transformation of sunflower (Helianthus annuus L.) following wounding with glass beads

A procedure was developed for transformation of Helianthus annuus (sunflower) using Agrobacterium tumefaciens. Cotyledons were removed from young seedlings, and the remaining tissue was uniformly wounded by shaking with glass beads. The wounded tissue was then co-cultivated with a hypervirulent strain of Agrobacterium tumefaciens harboring the binary plasmid pCNL56. Minimal use of defined medium was required, and no callus was observed. The polymerase chain reaction (PCR) followed by DNA hybridization demonstrated the presence of gusA DNA from pCNL56 in total leaf DNA of 6 primary transformants and 2 progeny plants. No Agrobacterium DNA was detected in total DNA from transformed sunflower leaves that was amplified with primers specific to the miaA chromosomal gene of Agrobacterium. Foreign DNA was also detected in the next generation. -Glucuronidase (GUS) activity was demonstrated for 5 of the T₂ transgenic plants. Grafting was used to increase the number of seeds present on plants that had undergone tissue culture manipulations.Abbreviations PCR polymerase chain reaction - EMBL European Molecular Biology Laboratory - M U 7-hydroxy-4-methylumbelliferone - GUS -Glucuronidase Disclaimer: Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Response of sunflower (Helianthus annuus L) genotypes to PEG-mediated water stress

Drought tolerance of two sunflower (Helianthus annuus L.) genotypes, cultivated cultivar 1114 and interspecific line H. annuus × H. mollis, was studied under laboratory conditions using PEG-6000. Four levels of osmotic stress (?0.4, ?0.6, ?0.8 and ?1.0 MPa) were created and performances were monitored against a control. Physiological and biochemical stress determining parameters such as malondialdechyde (MDA), proline content, and hydrogen peroxide (H₂O₂) were compared between seedlings of both genotypes. The results indicated that both genotypes have similar responses at four osmotic potentials for all traits studied. All seedling growth parameters such as germination percentage, root length, shoot length, root and shoot dry weight decreased with increasing osmotic stress. MDA, proline, and H₂O₂ were found to be increased at different osmotic gradients in comparison to control. Cultivar 1114 was less affected than the interspecific line under these stress conditions. The data observed in the experiments revealed that perennial wild H. mollis can hardly be considered to be an excellent candidate of drought tolerance genes.     

The domestication of Helianthus annuus L. (sunflower)

All modern domesticated sunflowers can be traced to a single center of domestication in the interior mid-latitudes of eastern North America. The sunflower achenes and kernels recovered from six eastern North American sites predating 3000 b.p. that document the early history of this important crop plant are reanalyzed, and two major difficulties in the interpretation of archaeological sunflower specimens are addressed. First, achenes and kernels obtained from a modern wild sunflower population included in a prior genetic study because of its minimal likelihood for crop-wild gene flow, and its close genetic relationship to domesticated sunflowers, provide a new and more tightly drawn basis of comparison for distinguishing between wild and domesticated achene and kernel specimens recovered from archaeological contexts. Second, achenes and kernels from this modern wild baseline population were carbonized, allowing a direct comparison between carbonized archaeological specimens and a carbonized modern wild reference class, thereby avoiding the need for the various problematic shrinkage correction conversion formulas that have been employed over the past half century. The need for further research on museum collections is underscored, and new research directions are identified.     

Composition of lipids from sunflower pollen (Helianthus annuus)

The contents of the pollen lipids of the sunflower Helianthus annuus are described. The major component is the seco-triterpene helianyl octanoate, followed by new beta-diketones as second major group of compounds. They exhibit a shorter chain length and often other positions of the functional group compared to already known beta-diketones. Of particular note are the 1-phenyl-beta-diketones, not previously reported from nature. Further lipid classes present are related hydroxyketones and diols. Interestingly, new beta-dioxoalkanoic acids are present in the extracts, which most likely are biogenetic precursors of the diketones. Additionally, we investigated the composition of the pollen coat which resembles the total extract, but lacks the dioxoalkanoic acids and certain estolides.     

Differences in the response of sunflower (Helianthus annuus) subjected to flooding and drought stress

Wample, R. L. and Thornton, R. K. 1984. Differences in the response of sunflower ( Helianthus annuus ) subjected to flooding and drought stress.
Comparison of drought- and flood-stressed sunflower plants ( Helianthus annuus L. hybrid 894) showed some similarities in response but differences in the mechanisms responsible for the responses to stress. Drought–stressed plants showed typical reductions in leaf water potential with increasing stress accompanied by increased leaf resistance. Photosynthesis declined while photorespiration increased after 48 and 96 h of drought stress. A primary reason for reduced photosynthesis in drought-stressed plants was increased stomatal resistance. No significant 0change in leaf water potential or in leaf resistance in flooded plants was found in this study. However, photosynthesis declined in a manner similar to that in drought-stressed plants and photorespiration showed only a transient increase at 48 h. Dark respiration was significantly higher at 48 and 96 h but the magnitude of the increase cannot account for the reduction in photosynthesis. Since the photosynthetic rate of flooded plants declined while stomata remained open, an effect at a more fundamental level is suggested and is thought to be related to disruption of carbohydrate transport.     

Physiology and proteomics of drought stress acclimation in sunflower (Helianthus annuus L.)

An easy and manageable in vitro screening system for drought tolerance of sunflower seedlings based on MS media supplemented with polyethylene glycol 6000 was evaluated. Morphological and physiological parameters were compared between control (-0.05 MPa) and drought-stressed (-0.6 MPa) seedlings of Helianthus annuus L. cv. Peredovick. There was a significant growth deficit in drought-stressed plants compared to control plants in terms of hypocotyl length, and shoot and root fresh mass. Shoot growth was more restricted than root growth, resulting in an increased root/shoot ratio of drought-stressed plants. Accumulation of osmolytes such as inositol (65-fold), glucose (58-fold), proline (55-fold), fructose (11-fold) and sucrose (eightfold), in leaves of drought-stressed plants could be demonstrated by gas-liquid chromatography. Soluble protein patterns of leaves were analysed with two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry. A set of 46 protein spots allowed identification of 19 marker proteins. Quantitative changes in protein expression of drought-stressed versus control plants were detected. In leaves of drought-stressed sunflower seedlings six proteins were significantly up-regulated more than twofold: a putative caffeoyl-CoA 3-O-methyltransferase (4.5-fold), a fructokinase 3 (3.3-fold), a vegetative storage protein (2.5-fold), a glycine-rich RNA binding protein (2.2-fold), a CuZn-superoxide dismutase (2.1-fold) and an unknown low molecular weight protein (2.3-fold). These proteins represent general stress proteins induced under drought conditions or proteins contributing to basic carbon metabolism. The up-regulated proteins are interesting candidates for further physiological and molecular investigations regarding drought tolerance in sunflower.     

The mechanics of anchorage in seedlings of sunflower, Helianthus annuus L.

Forces applied to plants will subject many of the roots to tension, which must be transferred to the soil via shear if uprooting is to be prevented. The stress distribution will depend on the relative stiffnesses of the earth and root, and the mode of failure will depend on the relative strength of the soil and of the root soil bond. This study of the anchorage of sunflower radicles combined uprooting tests performed by a tensile testing machine with mechanical tests on the roots and soil.
The maximum extraction force increased with length to an asymptotic value and was reached at a very low displacement. Root hairs and soil particles covered the tapered top 20 mm of extracted root, but the lower cylindrical region was bare. The soil was stiffer than the root, so shear stress was initially concentrated at the top of the root, soil strength over the top 20 mm resisting uprooting. Lower regions of the root were stressed later, their sparser root hairs being sheared off, and resist uprooting only by friction. In a further lest upper and lower regions of radicles were uprooted separately. As predicted, the upper region generated much greater resistance to uprooting per unit length, and at much lower displacements than the lower region.
The top of the radicle is well adapted for anchorage, the profuse root hairs and mucigel it produces glueing the root to the soil. The lower regions are thus protected from damage.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. III. The role of ethylene

While ethylene is suspected to be one of the many factors that play a role in rooting, some studies have found that ethylene can promote rooting, while others show it to inhibit this process or to have no effect. Using seedlings of sunflower ( Helianthus annuus L. cv. Dahlgren 131) we carried out observations on the rates of ethylene production and the levels of the ethylene precursor, 1-aminocyclopropane-l-car-boxylic acid (ACC), and ACC conjugate, l-(malonylamino)cyclopropane-l-car-boxylic acid (MACC), during the process of root initiation. The changes in these substances in the base of the hypocotyls (the portion that produces roots) were compared to the changes that occurred in the top of the hypocotyls (non-rooting portion). We also supplied a number of presumptive inhibitors of ethylene biosynthesis and inhibitors of ethylene action for short periods during the early and critical stages of root formation. Their effects on ethylene action, synthesis and rooting were examined. We conclude that the wound-induced increase in ethylene, seen within 3 h of production of the cuttings, is a key stimulatory factor in the formation of root primordia. When this increase in ethylene is localized in the lower portion of the hypocotyl, there is a promotion of rooting. On the other hand, higher concentrations in the top of the hypocotyls (as compared to the bottom) may inhibit rooting.     

Genetic control of organogenesis in cotyledons of sunflower (Helianthus annuus)

Genetic variability for regeneration ability was evaluated by studying direct organogenesis from cotyledons of thirteen genotypes including three cytoplasmic male sterile, three maintenor, three restorer inbred lines, and four F1 hybrids obtained by crosses between some of these inbred lines. The experimental design was a complete randomized block with three replications. A high genetic variability for organogenesis parameters between genotypes was observed in this study. Evidence of cytoplasmic effect and nucleo-cytoplasmic interaction for some of regeneration parameters was observed. The data also showed the importance of additive genetic control for organogenesis parameters in most genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration

High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO(2) -Tyr) studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin-NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.     

Salicylic acid-induced adaptive response to copper stress in sunflower (Helianthus annuus L.)

The ameliorative effect of salicylic acid (SA: 0.5 mM) on sunflower (Helianthus annuus L.) under Cu stress (5 mg l⁻¹) was studied. Excess Cu reduced the fresh and dry weights of different organs (roots, stems and leaves) and photosynthetic pigments (chlorophyll a, b and carotenoids) in four-week-old plants. There was a considerable increase in Chl a/b ratio and lipid peroxidation in both the roots and leaves of plants under excess Cu. Soluble sugars and free amino acids in the roots also decreased under Cu stress. However, soluble sugars in the leaves, free amino acids in the stems and leaves, and proline content in all plant organs increased in response to Cu toxicity. Salicylic acid (SA) significantly reduced the Chl a/b ratio and the level of lipid peroxidation in Cu-stressed plants. Under excess Cu, a higher accumulation of soluble sugars, soluble proteins and free amino acids including proline occurred in plants treated with 0.5 mM SA. Exogenous application of SA appeared to induce an adaptive response to Cu toxicity including the accumulation of organic solutes leading to protective reactions to the photosynthetic pigments and a reduction in membrane damage in sunflower.     

Reevaluation of biosecurity of Ophraella communa against sunflower (Helianthus annuus)

Ophraella communa (Coleoptera: Chrysomelidae), originally from North America, has been used for biological control of common ragweed, Ambrosia artemisiifolia, in China since 2007. However, there is still a debate on whether O. communa can attack sunflowers under field conditions. To re-evaluate the biosecurity of O. communa against sunflower (Helianthus annuus), we investigated the population density of O. communa on three sunflower varieties that were intercropped with or planted in circumambience of A. artemisiifolia under field conditions. Our results showed that only very few O. communa eggs (<0.5 eggs/plant) were found on sunflower plants at the last two surveys when sunflowers were planted in circumambience of common ragweed. O. communa eggs were not found on sunflower plants at each survey when sunflowers were intercropped with common ragweed. The first–second instar larvae, third instar larvae, pupae and adults of O. communa were occasionally found on sunflower plants, but their densities were very low under either case of planting patterns. Based on these results, we conclude that sunflower is not a potential host plant for O. communa and the beetle is an effective host-specific biological control agent of common ragweed.     

Induction of leaf senescence by low nitrogen nutrition in sunflower (Helianthus annuus) plants

Different parameters which vary during the leaf development in sunflower plants grown with nitrate (2 or 20 mM) for a 42‐day period have been determined. The plants grown with 20 mM nitrate (N+) showed greater leaf area and specific leaf mass than the plants grown with 2 mM nitrate (N?). The total chlorophyll content decreased with leaf senescence, like the photosynthetic rate. This decline of photosynthetic activity was greater in plants grown with low nitrogen level (N?), showing more pronounced senescence symptoms than with high nitrogen (N+). In both treatments, soluble sugars increased with aging, while starch content decreased. A significant increase of hexose to sucrose ratio was observed at the beginning of senescence, and this raise was higher in N? plants than in N+ plants. These results show that sugar senescence regulation is dependent on nitrogen, supporting the hypothesis that leaf senescence is regulated by the C/N balance. In N+ and N? plants, ammonium and free amino acid concentrations were high in young leaves and decreased progressively in the senescent leaves. In both treatments, asparagine, and in a lower extent glutamine, increased after senescence start. The drop in the (Glu+Asp)/(Gln+Asn) ratio associated with the leaf development level suggests a greater nitrogen mobilization. Besides, the decline in this ratio occurred earlier and more rapidly in N? plants than in N+ plants, suggesting that the N? remobilization rate correlates with leaf senescence severity. In both N+ and N? plants, an important oxidative stress was generated in vivo during sunflower leaf senescence, as revealed by lipid peroxidation and hydrogen peroxide accumulation. In senescent leaves, the increase in hydrogen peroxide levels occurred in parallel with a decline in the activity of antioxidant enzymes. In N+ plants, the activities of catalase and ascorbate peroxidase (APX) increased to reach their highest values at 28 days, and later decreased during senescence, whereas in N? plants these activities started to decrease earlier, APX after 16 days and catalase after 22 days, suggesting that senescence is accelerated in N‐leaves. It is probable that systemic signals, such as a deficit in amino acids or other metabolites associated with the nitrogen metabolism produced in plants grown with low nitrogen, lead to an early senescence and a higher oxidation state of the cells of these plant leaves.     

Microsatellite isolation and characterization in sunflower (Helianthus annuus L.).

Development of microsatellite markers for sunflower (Helianthus annuus L.) was performed to estimate their frequency, nature (structure), levels of polymorphism, usefulness for genotype identification, and calculation of genetic relationships between inbred lines representing the species diversity. Isolation was performed from a small-insert genomic library followed by hybridization screening using oligonucleotide probes containing different nucleotide arrays. In this work, 503 unique microsatellite clones were sequenced and 271 PCR primer sequences bordering the microsatellite repeat were designed. For polymorphism assessment, 16 H. annuus germplasm accessions were checked and 170 of the primers tested were shown to be polymorphic for the selected lines. The polymorphic microsatellites produced an average of 3.5 alleles/locus and an average polymorphism information content (PIC) of 0.55. The most frequently found motifs within polymorphic simple-sequence repeats (SSRs) were: (GA)n, (GT)n, (AT)n, followed by trinucleotides (ATT)n, (TGG)n, and (ATC)n, and the tetranucleotide (CATA)n. Most of the 170 SSRs obtained showed important differences in the 16 reference inbred lines used for their characterization. In this work, 20 of the most informative SSRs destined to sunflower genotyping and legal fingerprinting purposes are fully described.     

Genetic characterization and molecular mapping of a chlorophyll deficiency gene in sunflower (Helianthus annuus)

A major gene controlling chlorophyll deficiency (phenotyped by yellow leaf color, yl) in sunflower was identified and mapped in an F(2) population derived from a cross between two breeding lines. Greenness degree was scored by a hand-held chlorophyll meter in the F(2) population. Leaf tissue from the parents, F(1) hybrids, and some F(2) progenies were also sampled to determine the chlorophyll content. All F(1) plants had normal green leaf color and the segregation of the plants in the F(2) population fits the monogenic ratio (chi((3:1))(2)=0.03, p>0.9), indicating that leaf color is a monogenic trait with normal green dominant over yellow leaf color in this population. The contents of chlorophyll a, chlorophyll b, and total chlorophyll in the yellow-leafed lines were reduced by 41.6%, 53.5%, and 44.3%, respectively, in comparison with those in the green-leafed lines. Genetic mapping with molecular markers positioned the gene, yl, to linkage group 10 of sunflower. An SSR marker, ORS 595, cosegregated with yl, and a TRAP marker, B26P17ga5-300, was linked to yl with a genetic distance of 4.2cM. The molecular marker tightly linked to the chlorophyll deficiency gene will provide insight into the process of chlorophyll metabolism in sunflower.