Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L

In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg^?1). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.     

Productivity-independent initial growth of individual leaves in sunflower (Helianthus annuus L.)

Growth rates of individual leaves attached to sunflower (Helianthus annuus) plants were measured experimentally under different levels of environmental productivity, modified by irradiance and nutrient conditions. The unfolding rate and final area of an individual leaf increased with increasing environmental productivity. The final area of an individual leaf also varied according to differences in leaf order. The declining pattern of relative leaf area growth rate (RLGR) varied with environmental productivity; leaves in a productive environment had a longer period of high sustained initial RLGR than leaves in a less productive environment. However, maximum RLGR was hardly influenced by leaf order or environmental factors such as irradiance and mineral nutrition. This article is dedicated to Prof. Emeritus Toshiro Saeki in recognition of his fruitful career in plant ecology.     

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.     

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.     

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.     

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.     

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     

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.     

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     

Control of leaf expansion in sunflower (Helianthus annuus L.) by nitrogen nutrition

Seedlings of Helianthus annuus L. were grown at an initiallyhigh relative nitrate supply rate (0.27 mol N mol N^–1d^–1). The supply was subsequently reduced to a low rate(0.04 mol N mol N^–1 d^–1). The response of leaf areadevelopment to this abrupt decrease in nitrate availabilitywas characterized by following the expansion of the primaryand secondary leaf pairs. The timing of the drop in nitratesupply was when cell division in the epidermis of the primaryleaf pair was largely complete. Reducing the availability ofnitrate had a strong effect on leaf area expansion. The finalleaf size of the primary leaf pair was affected indicating aneffect of nitrate availability on cell expansion. By the endof the experiment the secondary leaf pair was only one-thirdthe area of that on control seedlings. The role of epidermalcell turgor pressure in this growth response was assessed bydirect measurements with a miniature cell pressure probe. Noreduction in cell turgor pressure following the decrease innitrate availability was detected. It is concluded that a reductionin turgor pressure was not responsible for the reduction inleaf area expansion and it is suggested that reduced cell expansionwas due to changes in cell wall properties. Concentrations ofleaf and root abscisic acid increased following the reductionin nitrate availability. Key words: Abscisic acid, cell size, cell turgor pressure, nitrate, nitrogen, relative rate of nitrate supply     

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.     

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     

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.     

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.     

Influence of analogue-resistant mutants ofAzotobacter chroococcum solubilizing phosphate on yield and quality of sunflower (Helianthus annuus)

Two phosphate-solubilizing analogue-resistant mutants ofAzotobacter chroococcum showed a positive interactive effect on sunflower (Helianthus annuus). Plant height, leaf area, seed mass, seed yield, protein and oil content increased significantly by inoculation withA. chroococcum mutants. Both levels of fertilizers applied (80N+30P₂O₅ and 80N+60P₂O₅ kg/hm² showed increased seed yield. Likewise, inoculation resulted in a significantly higher yield. Application ofP-solubilizingA. chroococcum strains with the lower fertilizer level showed no significant difference on plant growth parameters when compared with the higher fertilizer level, indicating that inoculation of P-solubilizingAzotobacter strains can reduce the necessary levels of fertilization from 60P₂O₅ to 30P₂O₅ kg/hm².     

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     

Effect of atmospheric pressure on sunflower (Helianthus annuus L.) protoplast division

Summary Sunflower protoplasts were cultured in liquid medium under high atmospheric pressure (0.2 to 0.6 MPa) and the plating efficiency, cell wall synthesis and microtubule organization were assessed. In 7-day-old cultures under a pressure of 0.4 MPa and above, the division rate was strongly reduced by more than 60% as compared to the control. Although most of the protoplasts had begun to regenerate a new cell wall they were unable to complete this process. Pressure also had an inhibitory effect on microtubule synthesis. The percentage of protoplasts showing a disassembled cortical network of microtubules was significantly increased up to 60% of the population. These effects were reversible: when protoplasts were transferred to normal pressure most of them rapidly recovered their capacity to divide and afterwards developed normally. Culturing protoplasts under a pressurized atmosphere revealed to be a good model system for studying cortical microtubule dynamics.Abbreviations BSA bovine serum albumin - PBS phosphate buffered saline - TBS tris buffer saline - MT(s) microtubule(s)     

Comparative genetic analysis of quantitative traits in sunflower (Helianthus annuus L.)

One hundred and fifty F₂–F₃ families from a cross between two inbred sunflower lines FU and PAZ2 were used to map quantitative trait loci (QTL) for resistance to white rot (Sclerotinia sclerotiorum) attacks of terminal buds and capitula, and black stem (Phoma macdonaldii). A genetic linkage map of 18 linkage groups with 216 molecular markers spanning 1,937 cM was constructed. Disease resistances were measured in field experiments for S. sclerotiorum and under controlled conditions for P. macdonaldii. For resistance to S. sclerotiorum terminal bud attack, seven QTL were identified, each explaining less than 10% of phenotypic variance. For capitulum attack by this parasite, there were four QTL (each explaining up to 20% of variation) and for P. macdonaldii resistance, four QTL were identified, each having effects of up to 16%. The S. sclerotiorum capitulum resistance QTL were compared with those reported previously and it was concluded that resistance to this disease is governed by a considerable number of QTL, located on almost all the sunflower linkage groups.     

Accumulation of radioiodine from aqueous solution by hydroponically cultivated sunflower (Helianthus annuus L.)

The suitability of sunflower plants (Helianthus annuus L.) for the phytoremediation of soils and waters contaminated with radioactive iodine was tested following the ¹²⁵I uptake from a hydroponic medium and translocation during 32-day cultivation. The plants accumulated about 26% of the applied activity in case of combination of ¹²⁵I (1.3 MBq) and 0.1 mM K¹²⁷I (“carrier ¹²⁵I”) and 47% when only ¹²⁵I (1.3 MBq, “non-carrier ¹²⁵I”) was added. When hydroponic medium was changed for the fresh one every 4 days, the plants accumulated up to 59% of starting activity of non-carrier ¹²⁵I. The ¹²⁵I distribution within the plant was followed using autoradiography. At low iodine level (non-carrier ¹²⁵I) the radionuclide was localized mainly in the roots. At high iodine concentrations (carrier ¹²⁵I) it was found mainly in the upper part of sunflower plants. All iodine removed from the liquid medium was found in the plant body. Volatilization of iodine (in the form of I⁰ or volatile organic compounds) apparently did not occur during accumulation and translocation. The achieved results indicate that sunflower can be used for phytoremediation of radioactive iodine, even if it is not its hyperaccumulator.