Simultaneous hyperaccumulation of multiple heavy metals by Helianthus annuus grown in a contaminated sandy-loam soil

Phytoremediation is a promising means for the treatment of contamination arising from heavy metal spills. Although several species have been identified as hyperaccumulators, most of the studies were performed with only one heavy metal. Experiments were conducted with two cultivars of H. annuus exposed to different combinations of metal contamination (30 mg/kg Cd, Cr, Ni, As, and/or Fe). Cultivar efficiency was based on total metal uptake, as well as translocation and selectivity of each metal. The results for each cultivar were also compared after 0.1 g/kg or 0.3 g/kg EDTA was added to enhance metal bioavailability. The key finding was that H. annuus achieved hyperaccumulator status for multiple metals simultaneously: Cd, Cr, and As.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Inhibition of anthocyanin synthesis in the first internode of Sorghum bicolor by cobaltous ions. The site of action of cobalt

Cobaltous ions (Co²⁺) inhibited light-mediated anthocyanin synthesis and phenylalanine ammonia-lyase (PAL; EC 4. 3. 1. 5) activity in excised first internodes of Sorghum bicolor (L.) Moench. Ethephon (an exogenous source of ethylene) restored anthocyanin synthesis and PAL activity. Sulfhydryl-containing compounds like cysteine and glutathione completely restored anthocyanin synthesis and PAL activity. N-ethylmaleimide (NEM), a sulfhydryl reagent, inhibited anthocyanin synthesis and PAL activity; cysteine or glutathione reversed the effect of NEM. EDTA and other organic acids like citric, malic, oxalic and tartaric acid were also effective in bringing about recovery. It is suggested that the site of action of Co²⁺ might be at sulfhydryl groups or two adjacent carboxylic groups of a macromolecule that is involved either in anthocyanin or ethylene biosynthesis     

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.     

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.     

Induction of transfer-cell formation by iron deficiency in the root epidermis of Helianthus annuus L.

Helianthus annuus L. responds to iron deficiency by forming a thickened cortex and abundant root hairs in a zone near the root apex that corresponds to the primary developmental stage. Cytological investigations revealed that within 24 to 48 h of iron deficiency most of the peripheral cells differentiate into transfer cells. The wall labyrinth is always situated on the peripheral walls that face the external medium. The cytoplasm of these cells is characterized by numerous mitochondria, extensive rough endoplasmic reticulum, and large leucoplasts containing protein bodies. These observations are discussed in relation to the fact that Helianthus, as an iron efficient plant, responds physiologically to iron deficiency by extrusion of H⁺, production of reducing substances, and a steep increase in the uptake efficiency of Fe.     

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence.

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.     

Spontaneous and Nitrosomethylurea-Induced Reversions in Plastome Chlorophyll Mutants of Sunflower Helianthus annuus L.

Spontaneous and induced revertants obtained from plastome chlorophyll mutants of sunflower were subjected to genetic analysis. The mechanism of reversion (nuclear suppression, plastid suppression, true reversion, or mitochondrial suppression) of the plastid mutation was shown to affect the degree of restoration of morphological and physiological traits of sunflower plants.     

Fractionation by centrifugation of leaf proteins from varieties and cultivars of Helianthus annuus and Helianthus debilis as a function of pH and temperature

In order to select plant material for obtaining a high proportion of chlorophyll-free protein from Helianthus species by heat fractionation, leaf extracts from 11 field grown cultivars of H. annuus and H. debilis were investigated. In addition, press juices from H. annuus cv. Kinesisk were supplied with salts or urea before heat treatment in order to increase the proportion of chlorophyll-free protein during heat fractionation. The extracts were adjusted to pH 5.0, 6.0 and 8.0; then left for 20 min at 20°C, 50°C or 60°C and centrifuged at 2,500 g. The highest percentage chlorophyll-free protein of the total protein in the extract, more than 10%, was obtained for H. debilis cv. fl.pl. Sun Gold, H. annuus cv. Giganta, H. annuus cv. uniflorus and H. debilis cv. Stella, at pH 6.0 and 50°C. The low percentage chlorophyll-free protein obtained could be explained by the fact that a considerable part of the chlorophyll-free protein sedimented at original pH, low temperature and low centrifugation speed. This sedimentation at low temperature was probably due to interactions of phenolics. Besides, if the chlorophyll-associated membranes were highly fragmented during the disintegration of the plant material, the high temperature required to precipitate them completely led to further aggregation and thus to loss of chlorophyll-free proteins. Salts or urea present during heat treatment (pH 6.0/55°C for 20 min) did not considerably increase the proportion of chlorophyll-free proteins obtained.     

Heterochromatin and repetitive DNA frequency variation in regenerated plants of Helianthus annuus L.

Plant regeneration from cotyledons of seeds of a single progeny of a pure line of Helianthus annuus was studied in respect of the nuclear DNA contents of control and regenerated plants. Control plants were divided into two groups: those developed from seeds at the periphery of the inflorescence (showing a high basic 4C DNA content) and those from seeds developed in the middle of the inflorescence (showing a low basic 4C DNA content). It was observed that plants from peripheral seeds have a higher morphogenetic potential than those from central seeds. Cytophotometric analyses indicated that plants regenerated from cotyledons of both peripheral and central seeds show the same basic 4C DNA amount, which is higher that that observed in vivo in peripheral seeds. Molecular analysis by slot blotting and hybridization with different DNA families showed that the difference in nuclear DNA content between plants from peripheral and central seeds in vivo are mainly related to differences in the frequency of highly repeated, slow medium repeated (MR2), and ribosomal DNA families; by contrast, the increase in DNA amount in regenerated plants is mainly due to fast medium repeated sequences (MR1). Moreover, the frequency of kinetically isolated unique sequences was higher in peripheral seeds than in central ones and still higher in regenerated plants. Optical-density measurements of interphase nuclei showed an increase of heterochromatin in regenerated plants, suggesting that, whatever DNA is amplified in these plants, it remains condensed and probably inactive.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 2069     

Enhanced phytoextraction: II. Effect of EDTA and citric acid on heavy metal uptake by Helianthus annuus from a calcareous soil

High biomass producing plant species, such as Helianthus annuus, have potential for removing large amounts of trace metals by harvesting the aboveground biomass if sufficient metal concentrations in their biomass can be achieved However, the low bioavailability of heavy metals in soils and the limited translocation of heavy metals to the shoots by most high biomass producing plant species limit the efficiency of the phytoextraction process. Amendment of a contaminated soil with ethylene diamine tetraacetic acid (EDTA) or citric acid increases soluble heavy metal concentrations, potentially rendering them more available for plant uptake. This article discusses the effects of EDTA and citric acid on the uptake of heavy metals and translocation to aboveground harvestable plant parts in Helianthus annuus. EDTA was included in the research for comparison purposes in our quest for less persistent alternatives, suitable for enhanced phytoextraction. Plants were grown in a calcareous soil moderately contaminated with Cu, Pb, Zn, and Cd and treated with increasing concentrations of EDTA (0.1, 1, 3, 5, 7, and 10 mmol kg(-1) soil) or citric acid (0.01, 0.05, 0.25, 0.442, and 0.5 mol kg(-1) soil). Heavy metal concentrations in harvested shoots increased with EDTA concentration but the actual amount of phytoextracted heavy metals decreased at high EDTA concentrations, due to severe growth depression. Helianthus annuus suffered heavy metal stress due to the significantly increased bioavailable metal fraction in the soil. The rapid mineralization of citric acid and the high buffering capacity of the soil made citric acid inefficient in increasing the phytoextracted amounts of heavy metals. Treatments that did not exceed the buffering capacity of the soil (< 0.442 mol kg(-1) soil) did not result in any significant increase in shoot heavy metal concentrations. Treatments with high concentrations resulted in a dissolution of the carbonates and compaction of the soil. These physicochemical changes caused growth depression of Helianthus annuus. EDTA and citric acid added before sowing of Helianthus annuus did not appear to be efficient amendments when phytoextraction of heavy metals from calcareous soils is considered.     

Wide hybridization: pollination of Zea mays L. by Sorghum bicolor (L.) Moench

Summary Foreign pollen tubes in the stigma of Zea mays can be prevented from reaching the ovary cavity by the unusual length of the pollen tube pathway. A simple and rapid procedure is described for overcoming this difficulty by pollinating the basal parts of the stigmas without removing the ensheathing bracts (husks). The method maintains high humidity in the vicinity of the ovaries, and by conserving photosynthetic tissues probably also ensures a more normal O₂ /CO₂ balance in the neighbourhood of the stigmas than do bagging procedures. It is shown that Sorghum pollen tubes readily reach the ovary after pollination by the method. Their presence induces some of the characteristic post-pollination effects caused by Zea pollen tubes, but they frequently also stimulate premature enlargement of the nucellus and lysis of nucellar cells. Although Sorghum tubes have been traced across the inner ovary wall, they have not been seen to enter the micropyle, and hybrid embryos have not yet been obtained.     

Influence of some trace metals and stimulants on citric acid production from brewery wastes by Aspergillus niger

The addition of increasing levels of Mn²⁺, Fe³⁺, Zn²⁺, Co²⁺, Cu²⁺, Ca²⁺, sodium monofluoracetate and methanol during citric acid surface fermentation of spent grain liquor by Aspergillus niger (ATCC 9142) was investigated. For spent grain liquor the addition of 51 ppb Mn²⁺, 5 ppb Fe³⁺, 75 ppb Zn²⁺ and 4% (v/va) methanol caused a 4.9, 1.9, 10.9 and 16.8% increase in citric acid yield respectively. In all other fermentations the yield of citric acid was decreased whereas the biomass production in some cases was increased.     

Surface chemistry of Sorghum bicolor and its importance in feeding by Locusta migratoria

ABSTRACT. Seedling Sorghum bicolor cv.65D was rejected at palpation by nymphs of Locusta migratoria L. unless the surface wax was removed. The wax by itself was unpalatable, and when fractionated chromatographically into constituent chemicals its deterrent effects were attributable to the p -hydroxybenzal-dehyde, n -alkane, and ester fractions. Analysis of these last two fractions showed that they were unusual in that the carbon chain lengths of the molecules were shorter than expected from previous literature, and it was these smaller molecules (tested as pure chemicals) that were repellent to L.migratoria. The short-chain esters are more volatile than the ineffective longer-chain ones, and it is proposed that the insect may detect these chemicals by short-range olfaction and hence reject the plant before biting, thus imparting a more effective defence to the plant.     

Hairy roots of Helianthus annuus: a model system to study phytoremediation of tetracycline and oxytetracycline

The release of antibiotics to the environment has to be controlled because of serious threats to human health. Hairy root cultures of Helianthus annuus (sunflower), along with their inherent rhizospheric activity, provide a fast growing, microbe-free environment for understanding plant-pollutant interactions. The root system catalyzes rapid disappearance of tetracycline (TC) and oxytetracycline (OTC) from aqueous media, which suggests roots have potential for phytoremediation of the two antibiotics in vivo. In addition, in vitro modifications of the two antibiotics by filtered, cell- and microbe-free root exudates suggest involvement of root-secreted compounds. The modification is confirmed from changes observed in UV spectra of exudate-treated OTC. Modification appears to be more dominant at the BCD chromophore of the antibiotic molecule. Kinetic analyses dismiss direct enzyme catalysis; the modification rates decrease with increasing OTC concentrations. The rates increase with increasing age of cultures from which root exudates are prepared. The decrease in modification rates upon addition of the antioxidant ascorbic acid (AA) suggests involvement of reactive oxygen species (ROS) in the antibiotic modification process.     

Ultrastructural and physiological changes in root cells of Sorghum plants (Sorghum bicolor S. sudanensis cv. Sweet Sioux) induced by NaCl

The xerophytic, but salt-sensitive Sorghum cultivar ‘SweetSioux’ is known as an ion excluder with a high K/Na selectivityat the plasmalemma and tonoplast of epidermal root cells. Theaim of this study is the correlation of salt-effected changesin physiological parameters with structural and ultrastructuralchanges in root cells. The investigation was carried out withroot cells because these cells are most directly exposed tothe growth medium. Sorghum bicolor S. sudanensis cv. Sweet Sioux plants weregrown under steady-state conditions on nutrient solutions withor without 40 mol m^–3 NaCl. Sorghum sustained this treatmentbut showed several salt-induced structural and physiologicalchanges which were studied in various cell types of the roottip. (1) NaCl salinity led to a shorter growth region and to salt-inducedalterations in the chemical and physical properties of the cellwalls in the root tips. (2) Salt treatment also increased the membrane surface in rootcells: root cells showed an increase in the quantity of vesiclesin the epidermis and in the middle cortex cells. Additionally,some of the epidermis cells of salt-treated plants revealeda characteristic build-up of transfer cells, suggesting an increasein membrane surfaces to increase the uptake and storage of substances. (3) The number of mitochondria increased in the epidermal andin the cortex cells after salt stress thus indicating an additionalsupply of energy for osmotic adaptation and for selective uptakeand transport processes. (4) In the epidermal cytoplasm NaCl stress led to a significantdecrease of the P, K, Ca, and S concentrations accompanied byan increase of Na concentration. Electron micrographs show anincrease in electron optical contrast within the cytosol andin the matrix of the mitochondria. These results are discussedwith regard to the possibility of influence on the part of metabolicfunctions. (5) The NaCl concentrations were seen to increase and the Kconcentrations to decrease during salt stress in the vacuolesof the epidermis and cortex cells. The salt-induced increasein vacuolar NaCl concentrations of epidermis and cortex cellsare in the region 2 cm behind the root tip, which is sufficientfor an osmotic balance towards the growth medium. Additionalsolutes are necessary 0.5 mm behind the root tip to facilitateosmotic adaptation. The results show ultrastructural changes caused by an Na-avoidingmechanism characterized by a high level of energy consumption.The exclusion of Na from the symplast seems to lead additionallyto a decrease in cytoplasmic concentrations of such essentialelements as Mg, P, S, and Ca and is thus responsible directly(via energy supply in mitochondria, homeostasis, selectivityof K over Na) or indirectly (via enzyme conformation, cytoplasmichydration) for the ultra-structural degradation indicated. Thesalinity-induced multiplicity of structural and functional changeswithin cell compartments constitutes a group of indicators forthe limited NaCl tolerance of Sorghum. Key words: Sorghum bicolor S. sudanensis, ultrastructure, salt tolerance, NaCl, Ca-deficiency     

Removal of Heavy Metals from Soil Polluted with Effluents from a Paint Industry Using Helianthus annuus L. and Tithonia diversifolia (Hemsl.) as Influenced by Fertilizer Applications

Contamination of soils by effluents from industries is on the increase. There is the possibility of remediating these contaminated soils through the use of certain plants. This work investigated the remediating ability of Helianthus annuus and Tithonia diversifolia on the soil polluted with effluents from a paint industry in Ibadan, Nigeria. The experiment consisted of three treatments (H. annuus, T. diversifolia, and control) each replicated three times in a factorial combination of four different fertility managements, viz mineral fertilizer (MF); Grade A organomineral fertilizer (OMF); control₁, plants without fertilizer application; and control₂, where no fertilizer and no crop was planted using randomized complete block design. A total of 12 plots of 2 × 4 m² each per phytoplant were obtained. Each plot was planted with the viable seeds of the phytoplant at a spacing of 60 × 30 cm² and at the seed rate of four seeds per hole. The seedlings were thinned to two stands per hole 2 weeks after planting (WAP) and also weeded two times (2 and 5 WAP). After in situ second successive cultivation, percentage removal of heavy metals by Helianthus annuus with MF and OMF, respectively, were Cu 32.5 and 41.6; Pb 30.3 and 42.8; and Cd 44.5 and 56.7. Tithonia diversifolia, similarly, removed, respectively, Cu 16.9 and 23.4; Pb 36.9 and 43.7; and Cd 20.1 and 35.1. Lower percentages were removed in the controls where no fertilizer was applied. In the shoot of H. annuus with OMF, significantly (p< .05) higher values of 0.27, 1.72, and 0.11 mg kg^?1 of Cu, Pb, and Cd, respectively, were removed and stored at second cultivation as against 0.21, 3.39 and 0.08 mg kg^?1 in the shoot of T diversifolia. Lower values of Cu, Pb, and Cd were removed with MF, and also at first cultivation with OMF and MF. This study therefore recommends the use of sunflower plants, whether hybrids or wild-types along with the application of OMF for the effective remediation of soils contaminated with heavy metals, particularly in tropical climate.     

Glutamate induces series of action potentials and a decrease in circumnutation rate in Helianthus annuus

Reports concerning the function of glutamate (Glu) in the electrical and movement phenomena in plants are scarce. Using the method of extracellular measurement, we recorded electrical potential changes in the stem of 3‐week‐old Helianthus annuus L. plants after injection of Glu solution. Simultaneously, circumnutation movements of the stem were measured with the use of time‐lapse images. Injection of Glu solution at millimolar (200, 50, 5 mM) concentrations in the basal part of the stem evoked a series of action potentials (APs). The APs appeared in the site of injection and in different parts of the stem and were propagated acropetally and/or basipetally along the stem. Glu injection also resulted in a transient, approximately 5‐h‐long decrease in the stem circumnutation rate. The APs initiated and propagating in the sunflower stem after Glu injection testify the existence of a Glu perception system in vascular plants and suggest its involvement in electrical, long‐distance signaling. Our experiments also demonstrated that Glu is a factor affecting circumnutation movements.